The C-terminal domain of the MERS coronavirus M protein contains a trans-Golgi network localization signal
Identifieur interne : 000D84 ( Pmc/Corpus ); précédent : 000D83; suivant : 000D85The C-terminal domain of the MERS coronavirus M protein contains a trans-Golgi network localization signal
Auteurs : Anabelle Perrier ; Ariane Bonnin ; Lowiese Desmarets ; Adeline Danneels ; Anne Goffard ; Yves Rouillé ; Jean Dubuisson ; Sandrine BelouzardSource :
- The Journal of Biological Chemistry [ 0021-9258 ] ; 2019.
Abstract
Coronavirus M proteins represent the major protein component of the viral
envelope. They play an essential role during viral assembly by interacting with
all of the other structural proteins. Coronaviruses bud into the endoplasmic
reticulum (ER)–Golgi intermediate compartment (ERGIC), but the mechanisms
by which M proteins are transported from their site of synthesis, the ER, to the
budding site remain poorly understood. Here, we investigated the intracellular
trafficking of the Middle East respiratory syndrome coronavirus (MERS-CoV) M
protein. Subcellular localization analyses revealed that the MERS-CoV M protein
is retained intracellularly in the
Url:
DOI: 10.1074/jbc.RA119.008964
PubMed: 31399512
PubMed Central: 6768645
Links to Exploration step
PMC:6768645Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">The C-terminal domain of the MERS coronavirus M protein contains a
<italic>trans</italic>-Golgi network localization signal</title><author><name sortKey="Perrier, Anabelle" sort="Perrier, Anabelle" uniqKey="Perrier A" first="Anabelle" last="Perrier">Anabelle Perrier</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author><author><name sortKey="Bonnin, Ariane" sort="Bonnin, Ariane" uniqKey="Bonnin A" first="Ariane" last="Bonnin">Ariane Bonnin</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author><author><name sortKey="Desmarets, Lowiese" sort="Desmarets, Lowiese" uniqKey="Desmarets L" first="Lowiese" last="Desmarets">Lowiese Desmarets</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author><author><name sortKey="Danneels, Adeline" sort="Danneels, Adeline" uniqKey="Danneels A" first="Adeline" last="Danneels">Adeline Danneels</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author><author><name sortKey="Goffard, Anne" sort="Goffard, Anne" uniqKey="Goffard A" first="Anne" last="Goffard">Anne Goffard</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author><author><name sortKey="Rouille, Yves" sort="Rouille, Yves" uniqKey="Rouille Y" first="Yves" last="Rouillé">Yves Rouillé</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author><author><name sortKey="Dubuisson, Jean" sort="Dubuisson, Jean" uniqKey="Dubuisson J" first="Jean" last="Dubuisson">Jean Dubuisson</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author><author><name sortKey="Belouzard, Sandrine" sort="Belouzard, Sandrine" uniqKey="Belouzard S" first="Sandrine" last="Belouzard">Sandrine Belouzard</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31399512</idno><idno type="pmc">6768645</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6768645</idno><idno type="RBID">PMC:6768645</idno><idno type="doi">10.1074/jbc.RA119.008964</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000D84</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000D84</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">The C-terminal domain of the MERS coronavirus M protein contains a
<italic>trans</italic>-Golgi network localization signal</title><author><name sortKey="Perrier, Anabelle" sort="Perrier, Anabelle" uniqKey="Perrier A" first="Anabelle" last="Perrier">Anabelle Perrier</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author><author><name sortKey="Bonnin, Ariane" sort="Bonnin, Ariane" uniqKey="Bonnin A" first="Ariane" last="Bonnin">Ariane Bonnin</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author><author><name sortKey="Desmarets, Lowiese" sort="Desmarets, Lowiese" uniqKey="Desmarets L" first="Lowiese" last="Desmarets">Lowiese Desmarets</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author><author><name sortKey="Danneels, Adeline" sort="Danneels, Adeline" uniqKey="Danneels A" first="Adeline" last="Danneels">Adeline Danneels</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author><author><name sortKey="Goffard, Anne" sort="Goffard, Anne" uniqKey="Goffard A" first="Anne" last="Goffard">Anne Goffard</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author><author><name sortKey="Rouille, Yves" sort="Rouille, Yves" uniqKey="Rouille Y" first="Yves" last="Rouillé">Yves Rouillé</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author><author><name sortKey="Dubuisson, Jean" sort="Dubuisson, Jean" uniqKey="Dubuisson J" first="Jean" last="Dubuisson">Jean Dubuisson</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author><author><name sortKey="Belouzard, Sandrine" sort="Belouzard, Sandrine" uniqKey="Belouzard S" first="Sandrine" last="Belouzard">Sandrine Belouzard</name><affiliation><nlm:aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of Biological Chemistry</title><idno type="ISSN">0021-9258</idno><idno type="eISSN">1083-351X</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Coronavirus M proteins represent the major protein component of the viral
envelope. They play an essential role during viral assembly by interacting with
all of the other structural proteins. Coronaviruses bud into the endoplasmic
reticulum (ER)–Golgi intermediate compartment (ERGIC), but the mechanisms
by which M proteins are transported from their site of synthesis, the ER, to the
budding site remain poorly understood. Here, we investigated the intracellular
trafficking of the Middle East respiratory syndrome coronavirus (MERS-CoV) M
protein. Subcellular localization analyses revealed that the MERS-CoV M protein
is retained intracellularly in the <italic>trans</italic>-Golgi network (TGN),
and we identified two motifs in the distal part of the C-terminal domain as
being important for this specific localization. We identified the first motif as
a functional diacidic DxE ER export signal, because substituting Asp-211 and
Glu-213 with alanine induced retention of the MERS-CoV M in the ER. The second
motif, <sup>199</sup>KxGxYR<sup>204</sup>, was responsible for retaining the M
protein in the TGN. Substitution of this motif resulted in MERS-CoV M leakage
toward the plasma membrane. We further confirmed the role of
<sup>199</sup>KxGxYR<sup>204</sup> as a TGN retention signal by using
chimeras between MERS-CoV M and the M protein of infectious bronchitis virus
(IBV). Our results indicated that the C-terminal domains of both proteins
determine their specific localization, namely TGN and
ERGIC/<italic>cis</italic>-Golgi for MERS-M and IBV-M, respectively. Our
findings indicate that MERS-CoV M protein localizes to the TGN because of the
combined presence of an ER export signal and a TGN retention motif.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Masters, P X0a S" uniqKey="Masters P">P.
S. Masters</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Perlman, S" uniqKey="Perlman S">S. Perlman</name></author><author><name sortKey="Netland, J" uniqKey="Netland J">J. Netland</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Corman, V X0a M" uniqKey="Corman V">V.
M. Corman</name></author><author><name sortKey="Muth, D" uniqKey="Muth D">D. Muth</name></author><author><name sortKey="Niemeyer, D" uniqKey="Niemeyer D">D. Niemeyer</name></author><author><name sortKey="Drosten, C" uniqKey="Drosten C">C. Drosten</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Belouzard, S" uniqKey="Belouzard S">S. Belouzard</name></author><author><name sortKey="Millet, J X0a K" uniqKey="Millet J">J.
K. Millet</name></author><author><name sortKey="Licitra, B X0a N" uniqKey="Licitra B">B.
N. Licitra</name></author><author><name sortKey="Whittaker, G X0a R" uniqKey="Whittaker G">G.
R. Whittaker</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Heald Sargent, T" uniqKey="Heald Sargent T">T. Heald-Sargent</name></author><author><name sortKey="Gallagher, T" uniqKey="Gallagher T">T. Gallagher</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ruch, T X0a R" uniqKey="Ruch T">T.
R. Ruch</name></author><author><name sortKey="Machamer, C X0a E" uniqKey="Machamer C">C.
E. Machamer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nieto Torres, J X0a L" uniqKey="Nieto Torres J">J.
L. Nieto-Torres</name></author><author><name sortKey="Verdia Baguena, C" uniqKey="Verdia Baguena C">C. Verdiá-Báguena</name></author><author><name sortKey="Casta O Rodriguez, C" uniqKey="Casta O Rodriguez C">C. Castaño-Rodriguez</name></author><author><name sortKey="Aguilella, V X0a M" uniqKey="Aguilella V">V.
M. Aguilella</name></author><author><name sortKey="Enjuanes, L" uniqKey="Enjuanes L">L. Enjuanes</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Westerbeck, J X0a W" uniqKey="Westerbeck J">J.
W. Westerbeck</name></author><author><name sortKey="Machamer, C X0a E" uniqKey="Machamer C">C.
E. Machamer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Westerbeck, J X0a W" uniqKey="Westerbeck J">J.
W. Westerbeck</name></author><author><name sortKey="Machamer, C X0a E" uniqKey="Machamer C">C.
E. Machamer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De Haan, C A X0a M" uniqKey="De Haan C">C. A.
M. de Haan</name></author><author><name sortKey="Rottier, P J X0a M" uniqKey="Rottier P">P. J.
M. Rottier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De Haan, C X0a A" uniqKey="De Haan C">C.
A. de Haan</name></author><author><name sortKey="Kuo, L" uniqKey="Kuo L">L. Kuo</name></author><author><name sortKey="Masters, P X0a S" uniqKey="Masters P">P.
S. Masters</name></author><author><name sortKey="Vennema, H" uniqKey="Vennema H">H. Vennema</name></author><author><name sortKey="Rottier, P X0a J" uniqKey="Rottier P">P.
J. Rottier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Opstelten, D X0a J" uniqKey="Opstelten D">D.
J. Opstelten</name></author><author><name sortKey="Raamsman, M X0a J" uniqKey="Raamsman M">M.
J. Raamsman</name></author><author><name sortKey="Wolfs, K" uniqKey="Wolfs K">K. Wolfs</name></author><author><name sortKey="Horzinek, M X0a C" uniqKey="Horzinek M">M.
C. Horzinek</name></author><author><name sortKey="Rottier, P X0a J" uniqKey="Rottier P">P.
J. Rottier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De Haan, C X0a A" uniqKey="De Haan C">C.
A. de Haan</name></author><author><name sortKey="Smeets, M" uniqKey="Smeets M">M. Smeets</name></author><author><name sortKey="Vernooij, F" uniqKey="Vernooij F">F. Vernooij</name></author><author><name sortKey="Vennema, H" uniqKey="Vennema H">H. Vennema</name></author><author><name sortKey="Rottier, P X0a J" uniqKey="Rottier P">P.
J. Rottier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Arndt, A X0a L" uniqKey="Arndt A">A.
L. Arndt</name></author><author><name sortKey="Larson, B J" uniqKey="Larson B">B. J. Larson</name></author><author><name sortKey="Hogue, B X0a G" uniqKey="Hogue B">B.
G. Hogue</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tseng, Y T" uniqKey="Tseng Y">Y.-T. Tseng</name></author><author><name sortKey="Wang, S M" uniqKey="Wang S">S.-M. Wang</name></author><author><name sortKey="Huang, K J" uniqKey="Huang K">K.-J. Huang</name></author><author><name sortKey="Lee, A X0a I R" uniqKey="Lee A">A.
I.-R. Lee</name></author><author><name sortKey="Chiang, C C" uniqKey="Chiang C">C.-C. Chiang</name></author><author><name sortKey="Wang, C T" uniqKey="Wang C">C.-T. Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De Haan, C A X0a M" uniqKey="De Haan C">C. A.
M. de Haan</name></author><author><name sortKey="Vennema, H" uniqKey="Vennema H">H. Vennema</name></author><author><name sortKey="Rottier, P J X0a M" uniqKey="Rottier P">P. J.
M. Rottier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baudoux, P" uniqKey="Baudoux P">P. Baudoux</name></author><author><name sortKey="Carrat, C" uniqKey="Carrat C">C. Carrat</name></author><author><name sortKey="Besnardeau, L" uniqKey="Besnardeau L">L. Besnardeau</name></author><author><name sortKey="Charley, B" uniqKey="Charley B">B. Charley</name></author><author><name sortKey="Laude, H" uniqKey="Laude H">H. Laude</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vennema, H" uniqKey="Vennema H">H. Vennema</name></author><author><name sortKey="Godeke, G X0a J" uniqKey="Godeke G">G.
J. Godeke</name></author><author><name sortKey="Rossen, J X0a W" uniqKey="Rossen J">J.
W. Rossen</name></author><author><name sortKey="Voorhout, W X0a F" uniqKey="Voorhout W">W.
F. Voorhout</name></author><author><name sortKey="Horzinek, M X0a C" uniqKey="Horzinek M">M.
C. Horzinek</name></author><author><name sortKey="Opstelten, D X0a J" uniqKey="Opstelten D">D.
J. Opstelten</name></author><author><name sortKey="Rottier, P X0a J" uniqKey="Rottier P">P.
J. Rottier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lim, K X0a P" uniqKey="Lim K">K.
P. Lim</name></author><author><name sortKey="Liu, D X" uniqKey="Liu D">D. X. Liu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Siu, Y X0a L" uniqKey="Siu Y">Y.
L. Siu</name></author><author><name sortKey="Teoh, K T" uniqKey="Teoh K">K. T. Teoh</name></author><author><name sortKey="Lo, J" uniqKey="Lo J">J. Lo</name></author><author><name sortKey="Chan, C X0a M" uniqKey="Chan C">C.
M. Chan</name></author><author><name sortKey="Kien, F" uniqKey="Kien F">F. Kien</name></author><author><name sortKey="Escriou, N" uniqKey="Escriou N">N. Escriou</name></author><author><name sortKey="Tsao, S X0a W" uniqKey="Tsao S">S.
W. Tsao</name></author><author><name sortKey="Nicholls, J X0a M" uniqKey="Nicholls J">J.
M. Nicholls</name></author><author><name sortKey="Altmeyer, R" uniqKey="Altmeyer R">R. Altmeyer</name></author><author><name sortKey="Peiris, J S X0a M" uniqKey="Peiris J">J. S.
M. Peiris</name></author><author><name sortKey="Bruzzone, R" uniqKey="Bruzzone R">R. Bruzzone</name></author><author><name sortKey="Nal, B" uniqKey="Nal B">B. Nal</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Klumperman, J" uniqKey="Klumperman J">J. Klumperman</name></author><author><name sortKey="Locker, J X0a K" uniqKey="Locker J">J.
K. Locker</name></author><author><name sortKey="Meijer, A" uniqKey="Meijer A">A. Meijer</name></author><author><name sortKey="Horzinek, M X0a C" uniqKey="Horzinek M">M.
C. Horzinek</name></author><author><name sortKey="Geuze, H X0a J" uniqKey="Geuze H">H.
J. Geuze</name></author><author><name sortKey="Rottier, P X0a J" uniqKey="Rottier P">P.
J. Rottier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Salanueva, I X0a J" uniqKey="Salanueva I">I.
J. Salanueva</name></author><author><name sortKey="Carrascosa, J X0a L" uniqKey="Carrascosa J">J.
L. Carrascosa</name></author><author><name sortKey="Risco, C" uniqKey="Risco C">C. Risco</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Machamer, C X0a E" uniqKey="Machamer C">C.
E. Machamer</name></author><author><name sortKey="Mentone, S A" uniqKey="Mentone S">S. A. Mentone</name></author><author><name sortKey="Rose, J X0a K" uniqKey="Rose J">J.
K. Rose</name></author><author><name sortKey="Farquhar, M X0a G" uniqKey="Farquhar M">M.
G. Farquhar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Locker, J X0a K" uniqKey="Locker J">J.
K. Locker</name></author><author><name sortKey="Griffiths, G" uniqKey="Griffiths G">G. Griffiths</name></author><author><name sortKey="Horzinek, M X0a C" uniqKey="Horzinek M">M.
C. Horzinek</name></author><author><name sortKey="Rottier, P X0a J" uniqKey="Rottier P">P.
J. Rottier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Machamer, C X0a E" uniqKey="Machamer C">C.
E. Machamer</name></author><author><name sortKey="Grim, M G" uniqKey="Grim M">M. G. Grim</name></author><author><name sortKey="Esquela, A" uniqKey="Esquela A">A. Esquela</name></author><author><name sortKey="Chung, S X0a W" uniqKey="Chung S">S.
W. Chung</name></author><author><name sortKey="Rolls, M" uniqKey="Rolls M">M. Rolls</name></author><author><name sortKey="Ryan, K" uniqKey="Ryan K">K. Ryan</name></author><author><name sortKey="Swift, A X0a M" uniqKey="Swift A">A.
M. Swift</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Machamer, C X0a E" uniqKey="Machamer C">C.
E. Machamer</name></author><author><name sortKey="Rose, J X0a K" uniqKey="Rose J">J.
K. Rose</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Locker, J X0a K" uniqKey="Locker J">J.
K. Locker</name></author><author><name sortKey="Klumperman, J" uniqKey="Klumperman J">J. Klumperman</name></author><author><name sortKey="Oorschot, V" uniqKey="Oorschot V">V. Oorschot</name></author><author><name sortKey="Horzinek, M X0a C" uniqKey="Horzinek M">M.
C. Horzinek</name></author><author><name sortKey="Geuze, H X0a J" uniqKey="Geuze H">H.
J. Geuze</name></author><author><name sortKey="Rottier, P X0a J" uniqKey="Rottier P">P.
J. Rottier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Waguri, S" uniqKey="Waguri S">S. Waguri</name></author><author><name sortKey="Dewitte, F" uniqKey="Dewitte F">F. Dewitte</name></author><author><name sortKey="Le Borgne, R" uniqKey="Le Borgne R">R. Le Borgne</name></author><author><name sortKey="Rouille, Y" uniqKey="Rouille Y">Y. Rouillé</name></author><author><name sortKey="Uchiyama, Y" uniqKey="Uchiyama Y">Y. Uchiyama</name></author><author><name sortKey="Dubremetz, J F" uniqKey="Dubremetz J">J.-F. Dubremetz</name></author><author><name sortKey="Hoflack, B" uniqKey="Hoflack B">B. Hoflack</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dunn, K X0a W" uniqKey="Dunn K">K.
W. Dunn</name></author><author><name sortKey="Kamocka, M M" uniqKey="Kamocka M">M. M. Kamocka</name></author><author><name sortKey="Mcdonald, J X0a H" uniqKey="Mcdonald J">J.
H. McDonald</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Venditti, R" uniqKey="Venditti R">R. Venditti</name></author><author><name sortKey="Wilson, C" uniqKey="Wilson C">C. Wilson</name></author><author><name sortKey="De Matteis, M X0a A" uniqKey="De Matteis M">M.
A. De Matteis</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Geva, Y" uniqKey="Geva Y">Y. Geva</name></author><author><name sortKey="Schuldiner, M" uniqKey="Schuldiner M">M. Schuldiner</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Votsmeier, C" uniqKey="Votsmeier C">C. Votsmeier</name></author><author><name sortKey="Gallwitz, D" uniqKey="Gallwitz D">D. Gallwitz</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yang, Y" uniqKey="Yang Y">Y. Yang</name></author><author><name sortKey="Zhang, L" uniqKey="Zhang L">L. Zhang</name></author><author><name sortKey="Geng, H" uniqKey="Geng H">H. Geng</name></author><author><name sortKey="Deng, Y" uniqKey="Deng Y">Y. Deng</name></author><author><name sortKey="Huang, B" uniqKey="Huang B">B. Huang</name></author><author><name sortKey="Guo, Y" uniqKey="Guo Y">Y. Guo</name></author><author><name sortKey="Zhao, Z" uniqKey="Zhao Z">Z. Zhao</name></author><author><name sortKey="Tan, W" uniqKey="Tan W">W. Tan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bos, K" uniqKey="Bos K">K. Bos</name></author><author><name sortKey="Wraight, C" uniqKey="Wraight C">C. Wraight</name></author><author><name sortKey="Stanley, K X0a K" uniqKey="Stanley K">K.
K. Stanley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ponnambalam, S" uniqKey="Ponnambalam S">S. Ponnambalam</name></author><author><name sortKey="Rabouille, C" uniqKey="Rabouille C">C. Rabouille</name></author><author><name sortKey="Luzio, J X0a P" uniqKey="Luzio J">J.
P. Luzio</name></author><author><name sortKey="Nilsson, T" uniqKey="Nilsson T">T. Nilsson</name></author><author><name sortKey="Warren, G" uniqKey="Warren G">G. Warren</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Armstrong, J" uniqKey="Armstrong J">J. Armstrong</name></author><author><name sortKey="Patel, S" uniqKey="Patel S">S. Patel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nal, B" uniqKey="Nal B">B. Nal</name></author><author><name sortKey="Chan, C" uniqKey="Chan C">C. Chan</name></author><author><name sortKey="Kien, F" uniqKey="Kien F">F. Kien</name></author><author><name sortKey="Siu, L" uniqKey="Siu L">L. Siu</name></author><author><name sortKey="Tse, J" uniqKey="Tse J">J. Tse</name></author><author><name sortKey="Chu, K" uniqKey="Chu K">K. Chu</name></author><author><name sortKey="Kam, J" uniqKey="Kam J">J. Kam</name></author><author><name sortKey="Staropoli, I" uniqKey="Staropoli I">I. Staropoli</name></author><author><name sortKey="Crescenzo Chaigne, B" uniqKey="Crescenzo Chaigne B">B. Crescenzo-Chaigne</name></author><author><name sortKey="Escriou, N" uniqKey="Escriou N">N. Escriou</name></author><author><name sortKey="Van Der Werf, S" uniqKey="Van Der Werf S">S. van der Werf</name></author><author><name sortKey="Yuen, K X0a Y" uniqKey="Yuen K">K.
Y. Yuen</name></author><author><name sortKey="Altmeyer, R" uniqKey="Altmeyer R">R. Altmeyer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mcbride, C X0a E" uniqKey="Mcbride C">C.
E. McBride</name></author><author><name sortKey="Machamer, C X0a E" uniqKey="Machamer C">C.
E. Machamer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Locker, J X0a K" uniqKey="Locker J">J.
K. Locker</name></author><author><name sortKey="Opstelten, D X0a J" uniqKey="Opstelten D">D.
J. Opstelten</name></author><author><name sortKey="Ericsson, M" uniqKey="Ericsson M">M. Ericsson</name></author><author><name sortKey="Horzinek, M X0a C" uniqKey="Horzinek M">M.
C. Horzinek</name></author><author><name sortKey="Rottier, P X0a J" uniqKey="Rottier P">P.
J. Rottier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Heffernan, L X0a F" uniqKey="Heffernan L">L.
F. Heffernan</name></author><author><name sortKey="Simpson, J X0a C" uniqKey="Simpson J">J.
C. Simpson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Puthenveedu, M X0a A" uniqKey="Puthenveedu M">M.
A. Puthenveedu</name></author><author><name sortKey="Linstedt, A X0a D" uniqKey="Linstedt A">A.
D. Linstedt</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tu, L" uniqKey="Tu L">L. Tu</name></author><author><name sortKey="Banfield, D X0a K" uniqKey="Banfield D">D.
K. Banfield</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Biol Chem</journal-id><journal-id journal-id-type="iso-abbrev">J. Biol. Chem</journal-id><journal-id journal-id-type="hwp">jbc</journal-id><journal-id journal-id-type="pmc">jbc</journal-id><journal-id journal-id-type="publisher-id">JBC</journal-id><journal-title-group><journal-title>The Journal of Biological Chemistry</journal-title></journal-title-group><issn pub-type="ppub">0021-9258</issn><issn pub-type="epub">1083-351X</issn><publisher><publisher-name>American Society for Biochemistry and Molecular
Biology</publisher-name><publisher-loc>11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110,
U.S.A.</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31399512</article-id><article-id pub-id-type="pmc">6768645</article-id><article-id pub-id-type="publisher-id">RA119.008964</article-id><article-id pub-id-type="doi">10.1074/jbc.RA119.008964</article-id><article-categories><subj-group subj-group-type="heading"><subject>Cell Biology</subject></subj-group></article-categories><title-group><article-title>The C-terminal domain of the MERS coronavirus M protein contains a
<italic>trans</italic>-Golgi network localization signal</article-title><alt-title alt-title-type="short">MERS-CoV M protein TGN localization</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Perrier</surname><given-names>Anabelle</given-names></name><xref ref-type="aff" rid="aff1"></xref><xref ref-type="author-notes" rid="FN1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Bonnin</surname><given-names>Ariane</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Desmarets</surname><given-names>Lowiese</given-names></name><xref ref-type="aff" rid="aff1"></xref><xref ref-type="author-notes" rid="FN2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Danneels</surname><given-names>Adeline</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Goffard</surname><given-names>Anne</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Rouillé</surname><given-names>Yves</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Dubuisson</surname><given-names>Jean</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="false">https://orcid.org/0000-0002-9972-4054</contrib-id><name><surname>Belouzard</surname><given-names>Sandrine</given-names></name><xref ref-type="aff" rid="aff1"></xref><xref ref-type="corresp" rid="cor1"><sup>3</sup></xref></contrib><aff id="aff1">Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France</aff></contrib-group><author-notes><corresp id="cor1"><label>3</label> To whom correspondence should be addressed.
E-mail: <email>sandrine.belouzard@ibl.cnrs.fr</email>.</corresp><fn fn-type="supported-by" id="FN1"><label>1</label><p>Supported by a fellowship from the University of Lille and the Region
Hauts-de-France.</p></fn><fn fn-type="supported-by" id="FN2"><label>2</label><p>Supported by a fellowship from the Region Hauts-de-France.</p></fn><fn fn-type="edited-by"><p>Edited by Phyllis I. Hanson</p></fn></author-notes><pub-date pub-type="ppub"><day>27</day><month>9</month><year>2019</year></pub-date><pub-date pub-type="epub"><day>9</day><month>8</month><year>2019</year></pub-date><pub-date pub-type="pmc-release"><day>9</day><month>8</month><year>2019</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on the
<volume>294</volume><issue>39</issue><fpage>14406</fpage><lpage>14421</lpage><history><date date-type="received"><day>18</day><month>4</month><year>2019</year></date><date date-type="rev-recd"><day>6</day><month>8</month><year>2019</year></date></history><permissions><copyright-statement>© 2019 Perrier et al.</copyright-statement><copyright-year>2019</copyright-year><copyright-holder>Perrier et al.</copyright-holder><license><license-p>Published under exclusive license by The American Society for
Biochemistry and Molecular Biology, Inc.</license-p><license-p>This article is made available via the PMC Open Access Subset for
unrestricted re-use and analyses in any form or by any means with
acknowledgement of the original source. These permissions are granted for
the duration of the COVID-19 pandemic or until permissions are revoked in
writing. Upon expiration of these permissions, PMC is granted a perpetual
license to make this article available via PMC and Europe PMC, consistent
with existing copyright protections.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="zbc03919014406.pdf"></self-uri><abstract><p>Coronavirus M proteins represent the major protein component of the viral
envelope. They play an essential role during viral assembly by interacting with
all of the other structural proteins. Coronaviruses bud into the endoplasmic
reticulum (ER)–Golgi intermediate compartment (ERGIC), but the mechanisms
by which M proteins are transported from their site of synthesis, the ER, to the
budding site remain poorly understood. Here, we investigated the intracellular
trafficking of the Middle East respiratory syndrome coronavirus (MERS-CoV) M
protein. Subcellular localization analyses revealed that the MERS-CoV M protein
is retained intracellularly in the <italic>trans</italic>-Golgi network (TGN),
and we identified two motifs in the distal part of the C-terminal domain as
being important for this specific localization. We identified the first motif as
a functional diacidic DxE ER export signal, because substituting Asp-211 and
Glu-213 with alanine induced retention of the MERS-CoV M in the ER. The second
motif, <sup>199</sup>KxGxYR<sup>204</sup>, was responsible for retaining the M
protein in the TGN. Substitution of this motif resulted in MERS-CoV M leakage
toward the plasma membrane. We further confirmed the role of
<sup>199</sup>KxGxYR<sup>204</sup> as a TGN retention signal by using
chimeras between MERS-CoV M and the M protein of infectious bronchitis virus
(IBV). Our results indicated that the C-terminal domains of both proteins
determine their specific localization, namely TGN and
ERGIC/<italic>cis</italic>-Golgi for MERS-M and IBV-M, respectively. Our
findings indicate that MERS-CoV M protein localizes to the TGN because of the
combined presence of an ER export signal and a TGN retention motif.</p></abstract><kwd-group><kwd>viral protein</kwd><kwd>protein motif</kwd><kwd>protein sorting</kwd><kwd>intracellular trafficking</kwd><kwd>endoplasmic reticulum (ER)</kwd><kwd>coronavirus</kwd><kwd>MERS-CoV</kwd><kwd>Middle East respiratory syndrome</kwd><kwd>trans-Golgi network localization</kwd><kwd>plasma membrane</kwd></kwd-group><funding-group><award-group id="award1"><funding-source>Région Hauts de France</funding-source><award-id>Visionn-AIRR</award-id><principal-award-recipient><name><surname>Perrier</surname><given-names>Anabelle</given-names></name></principal-award-recipient><principal-award-recipient><name><surname>Belouzard</surname><given-names>Sandrine</given-names></name></principal-award-recipient></award-group><award-group id="award2"><funding-source>Région Hauts-de-France/ Lille university</funding-source><award-id>Graduate Student Fellowship</award-id><principal-award-recipient><name><surname>Perrier</surname><given-names>Anabelle</given-names></name></principal-award-recipient><principal-award-recipient><name><surname>Belouzard</surname><given-names>Sandrine</given-names></name></principal-award-recipient></award-group><award-group id="award3"><funding-source><institution-wrap><institution>Région Hauts-de-France </institution><institution-id institution-id-type="open-funder-registry">10.13039/501100010095</institution-id></institution-wrap></funding-source><award-id>Postdoctoral Fellowship</award-id><principal-award-recipient><name><surname>Desmarets</surname><given-names>Lowiese</given-names></name></principal-award-recipient></award-group></funding-group></article-meta></front><body><sec sec-type="intro"><title>Introduction</title><p>Coronaviruses (CoVs)<xref ref-type="fn" rid="FN3"><sup>4</sup></xref> are widespread
pathogens that can infect a wide variety of species among mammals and birds (<xref rid="B1" ref-type="bibr">1</xref>, <xref rid="B2" ref-type="bibr">2</xref>),
including humans, causing mostly respiratory and enteric symptoms. There are six
known coronaviruses infecting humans. The first human coronaviruses, HCoV-229E and
HCoV-OC43, were isolated in the 1960s from patients suffering from a common cold.
Administration of these viruses to volunteers rapidly confirmed their harmless
character. Therefore, research on coronaviruses had been mostly of veterinary
interest, but this changed recently with the emergence of two highly pathogenic
human coronaviruses causing severe pneumonia epidemics. First, the severe acute
respiratory syndrome coronavirus (SARS-CoV) appeared in 2002, and then the Middle
East respiratory syndrome coronavirus (MERS-CoV) appeared in 2012. Both viruses have
a zoonotic origin, showing that this virus family is a reservoir of emerging
pathogens, especially because of their high interspecies transmission (<xref rid="B3" ref-type="bibr">3</xref>).</p><p>Coronaviruses are enveloped positive single-stranded RNA viruses, with a very large
genome of 25–30 kb, belonging to the Coronaviridae family in the Nidovirales
order. The viral particle is composed of a lipid envelope in which at least three
structural proteins are anchored: the <italic>spike</italic> protein (S), the
<italic>envelope</italic> protein (E), and the <italic>membrane</italic> protein
(M). Inside the particle, the viral RNA is associated with the
<italic>nucleocapsid</italic> protein (N), forming a helical capsid. The S
protein triggers viral entry by binding to the cellular receptor and mediating
fusion of the viral envelope with the host cell membrane (<xref rid="B4" ref-type="bibr">4</xref>, <xref rid="B5" ref-type="bibr">5</xref>). The E protein is a
small protein with multiple roles during infection (<xref rid="B6" ref-type="bibr">6</xref>). The SARS-CoV E protein plays an important role in viral
pathogenesis, and this role can be linked to the ion channel activity of the protein
(<xref rid="B7" ref-type="bibr">7</xref>). The E protein is also involved in
viral assembly, trafficking, and egress of virions, by promoting membrane curvature
and viral fission and by inducing morphological changes of the compartments of the
secretory pathway (<xref rid="B8" ref-type="bibr">8</xref>, <xref rid="B9" ref-type="bibr">9</xref>).</p><p>The M protein is the most abundant protein of the envelope (<xref rid="B10" ref-type="bibr">10</xref>). Its length ranges from 217 to 230 amino acid residues in
most coronaviruses, but it can go up to 270 residues in some coronaviruses
(bottlenose dolphin coronavirus). It is a protein with three membrane-spanning
hydrophobic segments, a small N-terminal domain located outside the virion (or
inside the lumen of intracellular organelles), and a large C-terminal domain that
makes up half of the protein, inside the virion (or in the cytoplasm of infected
cells) (<xref rid="B10" ref-type="bibr">10</xref>). M proteins of some
alphacoronaviruses contain an additional hydrophobic segment that functions as a
signal peptide. The M protein is invariably glycosylated on its N-terminal domain.
However, there are differences in the type of glycosylation. The murine hepatitis
virus (MHV) and some other <italic>Betacoronavirus</italic> M proteins are
<italic>O</italic>-glycosylated, whereas <italic>Alpha</italic>- and
<italic>Deltacoronavirus</italic> M proteins are modified with
<italic>N</italic>-linked sugars. The glycosylation is dispensable for virus
assembly (<xref rid="B11" ref-type="bibr">11</xref>). The M protein is considered to
be the motor of the assembly of viral particles because it is able to interact with
all of the other structural proteins (<xref rid="B12" ref-type="bibr">12</xref><xref ref-type="bibr" rid="B13">–</xref><xref rid="B14" ref-type="bibr">14</xref>). Important M–M interactions have also been demonstrated
during assembly (<xref rid="B15" ref-type="bibr">15</xref>, <xref rid="B16" ref-type="bibr">16</xref>). For many coronaviruses, including the transmissible
gastroenteritis virus, MHV, and IBV, the co-expression of M and E proteins in cells
is sufficient to induce the production of virus-like particles (<xref rid="B17" ref-type="bibr">17</xref><xref ref-type="bibr" rid="B18">–</xref><xref rid="B19" ref-type="bibr">19</xref>), indicating that
these proteins are essential and sufficient to the assembly step. Only for SARS-CoV,
the nucleocapsid protein has been shown to be additionally required for the
production of virus-like particles (<xref rid="B20" ref-type="bibr">20</xref>). EM
observations have shown that coronaviruses bud inside the ER–Golgi
intermediate compartment (ERGIC) (<xref rid="B21" ref-type="bibr">21</xref>, <xref rid="B22" ref-type="bibr">22</xref>) and then travel through the Golgi. Although
the assembly step of coronaviruses occurs in the ERGIC compartment, it has been
shown for several coronaviruses that the M protein expressed alone in cells can go
beyond the assembly site in the secretory pathway (<xref rid="B21" ref-type="bibr">21</xref>). However, the subcellular localization of the M protein varies
between the different coronavirus species. Indeed, MHV M protein can reach the
<italic>trans</italic>-Golgi network (TGN), whereas IBV-M protein is retained in
the ERGIC and one or two cisternae of the <italic>cis</italic>-Golgi (<xref rid="B21" ref-type="bibr">21</xref>, <xref rid="B23" ref-type="bibr">23</xref>,
<xref rid="B24" ref-type="bibr">24</xref>). The intracellular retention of IBV-M
has been attributed to the first membrane-spanning segment and particularly to polar
residues located within this domain (<xref rid="B25" ref-type="bibr">25</xref>,
<xref rid="B26" ref-type="bibr">26</xref>). For MHV, both the TM1 and the last
22 amino acids seem to be important determinants for the intracellular localization
of the protein (<xref rid="B27" ref-type="bibr">27</xref>). The aim of this study
was to investigate the subcellular localization of the MERS-M protein and to
characterize the signals involved in its trafficking.</p></sec><sec sec-type="results"><title>Results</title><sec><title>MERS-CoV M localizes in the trans-Golgi network</title><p>The M protein is composed of a short N-terminal domain followed by three
membrane-spanning segments and a long C-terminal domain (<xref ref-type="fig" rid="F1">Fig. 1</xref><italic>A</italic>). To analyze the intracellular
trafficking of the MERS-CoV M protein, we transfected a vector expressing the
MERS-CoV M protein with a HA tag fused at its N terminus (HA-MERS-M) in HeLa
cells and compared the protein localization with different compartment markers
using immunofluorescence and confocal microscopy (<xref ref-type="fig" rid="F1">Fig. 1</xref><italic>B</italic>). As observed in <xref ref-type="fig" rid="F1">Fig. 1</xref><italic>B</italic>, MERS-CoV M shows a good
colocalization with TGN46 that localizes in the <italic>trans</italic>-Golgi
network. We confirmed this colocalization by co-transfection of the M protein
with the GFP fused to the transmembrane domain and cytosolic tail of the
cation-independent mannose 6-phosphate receptor (GFP-CI-MPR). This reporter has
been shown to be localized in the TGN in HeLa cells (<xref rid="B28" ref-type="bibr">28</xref>). The images were then analyzed using ImageJ to
calculate the Pearson correlation coefficient (PCC) for each co-staining (<xref ref-type="fig" rid="F1">Fig. 1</xref><italic>C</italic>). The PCC measures
the pixel-by-pixel covariance of the signal levels of two images (<xref rid="B29" ref-type="bibr">29</xref>). The PCC values range from −1 to
1. A PCC value of 1 is obtained for two images whose intensities of fluorescence
are linearly and perfectly related, whereas a value of −1 means that the
intensities are inversely related to one another. Values near 0 mean that the
intensities are uncorrelated. Our results confirm the localization of MERS-CoV M
in the TGN with a PCC of 0.878 (±0.014) and 0.852 (±0.012) for the
MERS-CoV M with TGN46 and GFP-CI-MPR markers, respectively. The PCCs for
MERS-CoV M and CD4 (a cell surface marker) or the calreticulin (an ER marker)
are below 0.4. However, the PCC between MERS-CoV M and the ERGIC marker,
ERGIC-53, is higher (0.524 ± 0.03).</p><fig id="F1" orientation="portrait" position="float"><label>Figure 1.</label><caption><p>A, <italic>schematic drawing</italic> of the MERS-M protein with the
sequence of residues 149–219 of the C-terminal domain.
<italic>B</italic> and <italic>C</italic>, subcellular localization
of the MERS-CoV M protein. Cells expressing HA-tagged M protein in
combination with the GFP-CI-MPR, ERGIC-53-GFP, or CD4 fused with GFP
were labeled with an anti-HA antibody. GFP-CI-MPR is a TGN marker,
ERGIC-53-GFP is an ER–Golgi intermediate compartment marker, and
CD4 is a protein expressed at the cell surface. To detect the ER
compartment or the TGN, cells were double-labeled for HA and CRT or
TGN46, as indicated. <italic>Bars</italic>, 20 μm. Pearson's
correlation coefficients were calculated for each combination of
co-staining. <italic>D</italic> and <italic>E</italic>, N-terminal or
C-terminal added tags have no effect on the TGN localization of the M
protein. Cells expressing untagged M protein (<italic>M</italic>), N- or
C-terminally V5-tagged M protein (<italic>V5-M</italic> and
<italic>M-V5</italic> respectively), N-terminally HA-tagged M
(<italic>HA-M</italic>), or C-terminally VSVG-tagged M
(<italic>M-VSVG</italic>) were double-labeled with anti-M antibody
together with an anti-TGN46 antibody. Pearson's correlation coefficients
were calculated. <italic>Error bars</italic>, S.E.</p></caption><graphic xlink:href="zbc0401911830001"></graphic></fig><p>To facilitate the detection of the M protein and particularly in co-staining
experiments, different M protein constructs were generated with different tags
either at the N terminus (HA or V5) or at the C terminus (V5 or VSVG). This
offers the advantage of using a panel of antibodies raised in different species.
To use these tools, we analyzed their intracellular localization to confirm that
adding a tag, regardless of their sequence or the position of the insertion, had
no effect on MERS-CoV M localization. We also generated a polyclonal antibody
raised against a C-terminal peptide of the M protein to detect the untagged
protein. This antibody was used to control the effect of the tags on the
subcellular localization of the M protein. Cells expressing M or the different
tagged versions of this protein were processed for double-label
immunofluorescent detection of the M protein and TGN46 (<xref ref-type="fig" rid="F1">Fig. 1</xref><italic>D</italic>). For each protein, the
colocalization level with the TGN46 marker was also quantified by calculating
the PCC (<xref ref-type="fig" rid="F1">Fig. 1</xref><italic>E</italic>). As
observed with HA-M, the untagged form of the protein presented a strong
colocalization with the TGN46 marker (<xref ref-type="fig" rid="F1">Fig.
1</xref>, <italic>D</italic> and <italic>E</italic>) with a PCC of 0.857
± 0.015. Similar results were obtained with the other tagged proteins.
Altogether, these results indicate that the MERS-CoV M protein expressed alone
is located in the TGN and that the tag added to our constructs does not alter
this localization.</p></sec><sec><title>The last 20 residues of the C-terminal domain of the MERS-CoV M protein are
important for the MERS-CoV M intracellular trafficking</title><p>To study the role of the C-terminal domain of MERS-CoV M in its intracellular
trafficking, we constructed serial deletion mutants of 20 residues. The deletion
of the last 20 residues of the MERS-CoV M protein (MΔ20) produced mutants
that were no longer localized in the TGN (<xref ref-type="fig" rid="F2">Fig.
2</xref><italic>A</italic>). Moreover, it induced a different intracellular
localization, depending on the tag used. Indeed, HA-MΔ20 co-localizes with
calreticulin, the ER marker, as shown by the double labeling in <xref ref-type="fig" rid="F2">Fig. 2</xref><italic>B</italic>, whereas
MΔ20-VSVG was located at the cell surface, as shown by the double labeling
of MΔ20-VSVG and CD4 (<xref ref-type="fig" rid="F2">Fig.
2</xref><italic>C</italic>). We measured the extent of colocalization for
HA-MΔ20 or MΔ20-VSVG with the ER, TGN, or cell surface marker by
measuring the PCC (<xref ref-type="fig" rid="F2">Fig.
2</xref><italic>D</italic>). The WT protein presented a PCC of 0.905 ±
0.007 for TGN46 and of 0.296 ± 0.014 and 0.239 ± 0.017 for CRT and
CD4, respectively. The HA-MΔ20 protein showed a decrease of the PCC for
TGN46 (0.369 ± 0.019) associated with an increase of the PCC with CRT
(0.855 ± 0.011). The HA-MΔ20 also showed a moderate increase in cell
surface localization with a PCC for CD4 of 0.539 ± 0.018. The PCC between
MΔ20-VSVG and TGN46 was also strongly decreased (0.331 ± 0.025);
however, the MΔ20-VSVG protein presented a strong increase of the PCC for
CD4 (0.79 ± 0.016) and a moderate increase of the PCC for CRT (0.475 ±
0.018). These results suggest that important intracellular trafficking motifs
are present in the distal part of the C-terminal domain of MERS-CoV M. Analysis
of the sequence of the last 20 residues showed that the deleted sequence
contains a potential diacidic ER export signal at positions 211–213
(<sup>211</sup>DIE<sup>213</sup>). This signal was first characterized for
the glycoprotein of VSV and is indeed present in the VSVG tag
(YT<underline>DIE</underline>MNRLGK) used in our experiments. It is likely
that a blockade of ER export arose from the deletion in HA-MΔ20 and the
loss of the DxE signal; however, the addition of the VSVG tag in the
MΔ20-VSVG protein restored the signal and rescued the ER export. Moreover,
the MΔ20-VSVG protein was mainly located at the cell surface instead of
being retained in TGN, suggesting that the distal part of the C-terminal domain
of the M protein also contains a determinant responsible for its localization in
the TGN.</p><fig id="F2" orientation="portrait" position="float"><label>Figure 2.</label><caption><p><bold>The distal part of MERS-M C-terminal domain contains motif(s)
involved in its subcellular localization.</bold> Cells expressing
the M protein (<italic>HA-M</italic> and <italic>M-VSVG</italic>) or the
M protein lacking its last 20 residues
(<italic>HA-M</italic>Δ<italic>20</italic> or
<italic>M</italic>Δ<italic>20-VSVG</italic>) or the M protein
with Asp-211 and Glu-213 mutated into Ala (<italic>HA-M-DxE</italic> or
<italic>M-DxE-VSVG</italic>) were processed for detection of M
protein using an anti-tag antibody. The TGN was detected by using an
anti-TGN46 (<italic>A</italic>), and the ER was detected by using an
anti-CRT antibody (<italic>B</italic>). The plasma membrane was labeled
by co-expression of CD4 fused to GFP together with the M protein
(<italic>C</italic>). <italic>Bars</italic>, 20 μm. Pearson's
correlation coefficients were calculated for each combination of
co-staining (<italic>D</italic>). <italic>Error bars</italic>, S.E.</p></caption><graphic xlink:href="zbc0401911830002"></graphic></fig></sec><sec><title>DxE is a functional ER export signal for MERS-CoV M</title><p>To confirm the role of the DxE signal in the ER export of the MERS-M protein, we
mutated the aspartic acid and glutamic acid into alanine (D211A/E213A) in the M
protein fused with an N-terminal HA tag (HA-M-DxE) or with a C-terminal VSVG tag
(M-DxE-VSVG) and analyzed the subcellular localization of the mutants in
confocal microscopy. Similarly to HA-MΔ20, HA-M-DxE was mainly localized
in the ER, whereas M-DxE-VSVG was localized in the TGN (<xref ref-type="fig" rid="F2">Fig. 2</xref>, <italic>A</italic> and <italic>B</italic>),
confirming that the VSVG tag is able to compensate the D211A/E213A mutation.
This result demonstrates that the DxE signal present in the C-terminal domain of
MERS-CoV M protein is a functional ER export signal involved in the trafficking
of the protein.</p></sec><sec><title>Four residues in the C-terminal domain mediate MERS-CoV M localization to the
TGN</title><p>The presence of the DxE signal explained the exit of the M protein from the ER
but not its retention in the <italic>trans</italic>-Golgi, because it is
commonly accepted that in nonpolarized cells, the constitutive secretory pathway
leads to the plasma membrane by default (<italic>i.e.</italic> in absence of
specific addressing/retention signals). Considering that and the fact that the
MΔ20-VSVG protein migrates to the cell surface, we looked for the presence
of another signal in the last 20 amino acids of the cytosolic tail, which could
be involved in the retention of MERS-CoV M in the TGN compartment. For this
purpose, we constructed three smaller C-terminal deletion mutants lacking 5, 10,
or 15 residues but keeping the VSVG tag to rescue the ER export. These mutants
were called MΔ5, MΔ10, and MΔ15. We then compared the
subcellular localization of these mutants with the WT and MΔ20 M proteins
(<xref ref-type="fig" rid="F3">Fig. 3</xref>, <italic>A–C</italic>).
Similar to what was observed for the WT M and in contrast to MΔ20 protein,
the MΔ5, MΔ10, and MΔ15 proteins co-localized with the TGN46
marker, indicating that the 5-amino acid sequence AGNYR, located between the
Δ20 and Δ15 deletions, is likely involved in the specific
localization of the protein in the TGN. To identify the residues involved in the
TGN localization of the protein, each amino acid (except the alanine) was
mutated individually into alanine in the MΔ15 protein. MΔ15-G201A,
MΔ15-N202A, MΔ15-Y203A, and MΔ15-R204A were expressed in HeLa
cells, and the subcellular localization of the proteins was analyzed by
fluorescence microscopy (<xref ref-type="fig" rid="F4">Fig. 4</xref>). The
proteins carrying the mutation G201A, Y203A, or R204A showed a reduced
colocalization with the TGN46 marker compared with the MΔ15 (<xref ref-type="fig" rid="F4">Fig. 4</xref>, <italic>A</italic> and
<italic>C</italic>), with partial export of the protein to the cell surface
(<xref ref-type="fig" rid="F4">Fig. 4</xref>, <italic>B</italic> and
<italic>C</italic>). This difference in intracellular trafficking is
illustrated by a decrease of PCC between the M mutants and TGN46 associated with
an increase of the PCC with CD4. These results indicate a role of these three
residues in the localization of the protein in the TGN. The mutation N202A had
no effect on the subcellular localization of the protein compared with the WT.
To confirm these results, the mutation G201A, Y203A, or R204A was also inserted
into the full-length protein. Analysis of the subcellular localization of these
mutants is shown in <xref ref-type="fig" rid="F5">Fig. 5</xref>. M-G201A,
M-Y203A, and M-R204A showed a reduced co-localization with TGN46 compared with
the WT M-VSVG and an increase of cell surface expression, resulting in an
increase of the PCC between the mutant and CD4. To ensure that we identified the
full motif involved in the TGN localization of the protein, we also mutated
three conserved residues (Tyr-195, Arg-197, or Lys-199) among the
betacoronaviruses located upstream of the Δ20 deletion. Interestingly,
mutation of the residue Lys-199 also resulted in an increase of the cell surface
expression of the protein (<xref ref-type="fig" rid="F5">Fig. 5</xref>).</p><fig id="F3" orientation="portrait" position="float"><label>Figure 3.</label><caption><p><bold>Subcellular localization of mutants with serial deletions of the
distal part of the MERS-CoV M protein.</bold> M protein with a
C-terminal VSVG tag (<italic>M</italic>) or M protein deleted of 5
(<italic>M</italic>Δ<italic>5</italic>), 10
(<italic>M</italic>Δ<italic>10</italic>), 15
(<italic>M</italic>Δ<italic>15</italic>), or 20 amino acid
residues (<italic>M</italic>Δ<italic>20</italic>) was expressed in
HeLa cells, and its localization either in the TGN or at the plasma
membrane was investigated by double labeling with TGN46
(<italic>A</italic>) or by co-expressing CD4 fused to GFP
(<italic>B</italic>). <italic>Bars</italic>, 20 μm. Pearson's
correlation coefficients were calculated for each combination of
co-staining (<italic>C</italic>). <italic>Error bars</italic>, S.E.</p></caption><graphic xlink:href="zbc0401911830003"></graphic></fig><fig id="F4" orientation="portrait" position="float"><label>Figure 4.</label><caption><p><bold>Identification of the MERS-CoV MΔ15 motif involved in its TGN
localization.</bold> Amino acid residues Gly-201, Asn-202, Tyr-203,
and Arg-204 located in the last 5 residues of MΔ15 (located in the
TGN) were mutated individually into alanine, and the subcellular
localization of the mutants was analyzed as described in the legend to
<xref ref-type="fig" rid="F3">Fig. 3</xref>. <italic>Bars</italic>,
20 μm. <italic>Error bars</italic>, S.E.</p></caption><graphic xlink:href="zbc0401911830004"></graphic></fig><fig id="F5" orientation="portrait" position="float"><label>Figure 5.</label><caption><p><bold>The KxGxYR motif is involved in MERS-M protein localization in
TGN.</bold> The mutations K199A, G201A, Y203A, and R204A were
introduced individually or together in the context of the full-length M
protein with a C-terminal VSVG tag. The subcellular localization of the
mutants was analyzed as described in the legend to <xref ref-type="fig" rid="F3">Fig. 3</xref>. <italic>Bars</italic>, 20 μm.
<italic>Error bars</italic>, S.E.</p></caption><graphic xlink:href="zbc0401911830005"></graphic></fig><p>We also constructed a quadruple mutant protein, M-K199A/G201A/Y203A/R204A
(M-KGYR). The extent of colocalization of the quadruple mutant (M-KGYR) with
TGN46 and CD4 was in the same range than those of the single mutants.</p><p>To confirm the cell surface expression of the M protein when the residues
Lys-199, Gly-201, Tyr-203, and Arg-204 are mutated, we performed a cell surface
biotinylation assay. The MERS-M protein contains an
<italic>N</italic>-glycosylation site in its N-terminal domain
(MS<underline>NMT</underline>QLTE); consequently, the migration profile of
the MERS-M protein in immunoblotting renders the quantification of the protein
amount difficult (see <xref ref-type="fig" rid="F6">Fig.
6</xref><italic>C</italic>), so we also mutated the
<italic>N</italic>-glycosylation site in the different mutants. First, we
verified that introducing the mutation N3Q had no effect on the intracellular
localization of the different proteins (M, MΔ20, M-DxE, M-K199A, M-G201A,
M-Y203A, M-R204A, and M-KGYR; data not shown). Plasma membrane proteins in cells
expressing the different mutants were labeled with nonpermeable biotin, and then
biotinylated proteins were precipitated with streptavidin-conjugated agarose
beads and analyzed by immunoblotting (<xref ref-type="fig" rid="F6">Fig.
6</xref>, <italic>A</italic> and <italic>B</italic>). The M protein is only
weakly expressed at the cell surface, with less than 1% of the total amount of
protein expression at the cell surface. Mutation of the residues Lys-199,
Gly-201, Tyr-203, and Arg-204 alone or in combination induced an increase in
cell surface detection of the M protein, with ∼13% of the total amount of
N3Q-M-KGYR located at the cell surface. Single mutations induced only moderate
increases of the cell surface expression compared with the quadruple mutant. The
N3Q-M-DxE was barely detected at the cell surface. As seen in an
immunofluorescent colocalization assay (<xref ref-type="fig" rid="F6">Fig.
6</xref><italic>B</italic>), the expression of the N3Q-MΔ20 mutant at
the cell surface was slightly increased compared with the WT protein. Together,
these results indicated that the residues Lys-199, Gly-201, Tyr-203, and Arg-204
of MERS-CoV M are involved in its specific localization in the TGN.</p><fig id="F6" orientation="portrait" position="float"><label>Figure 6.</label><caption><p><bold>Cell surface expression of M protein mutants.</bold> Plasma
membrane proteins of cells expressing the different M protein mutants
were labeled with nonpermeable biotin. Biotinylated proteins were
purified using streptavidin-conjugated agarose beads. Biotinylated M
proteins and total M proteins in cell lysates were detected by
immunoblotting (<italic>A</italic>) and quantified (<italic>B</italic>).
Results are expressed as the percentage of total M protein expressed at
the cell surface and are expressed as the mean of five independent
experiments. <italic>Error bars</italic>, S.E. Results were analyzed by
using an analysis of variance test (*, <italic>p</italic> < 0.1; **,
<italic>p</italic> < 0.01; ***, <italic>p</italic> < 0.001;
****, <italic>p</italic> < 0.0001). Glycosylation of M protein
mutants is shown. Lysates of cells expressing the M protein or the M
protein with the ER export signal mutated (<italic>M-DxE</italic>) or
the TGN localization motif mutated (<italic>M-KGYR</italic>) were
treated with EndoH or PNGase F. A lysate of cells expressing the M
protein with its <italic>N</italic>-glycosylation site mutated (N3Q) was
left untreated. N-terminal tagged proteins were detected by Western
blotting with an anti-V5 antibody (<italic>C</italic>). Endocytosis of M
protein and M-KGYR. Cells were transfected with vectors expressing the M
and M-K199A-G210A-Y203A-R204A (<italic>M-KGYR</italic>) proteins. Then
cell surface proteins were labeled with nonpermeable biotin at 4
°C. Endocytosis was allowed by incubating the cells for 30 min at
37 °C. Biotin of noninternalized proteins was cleaved with GSH.
Internalized M protein was detected after purification with
streptavidin-conjugated agarose beads by immunoblotting
(<italic>E</italic>). In each experiment, each condition was
performed in duplicate. For cell surface–associated protein, only
25% of the sample was loaded on the gel. For the controls of GSH
cleavage (without any internalization, 0 min) and for the samples
internalized (30 min), the totality of the samples were loaded on the
gel. Internalized M protein was quantified. The results are expressed as
the percentage of cell surface–associated M protein and are
expressed as the mean of three independent experiments. <italic>Error
bars</italic>, S.E. (<italic>E</italic> and <italic>F</italic>).</p></caption><graphic xlink:href="zbc0401911830006"></graphic></fig><p>We also investigated the <italic>N</italic>-glycosylation status of the DxE and
KxGxYR mutants. In the immunoblot, the WT protein migrated as three bands (<xref ref-type="fig" rid="F6">Fig. 6</xref><italic>C</italic>). A first band of
around 20–25 kDa corresponds to the unglycosylated M protein, confirmed
by the migration profile of the protein in which the
<italic>N</italic>-glycosylation site was abolished by mutation (N3Q-M) and by
treatment with PNGase F. A second band of ∼30 kDa and a third more
diffuse band migrating more slowly were also observed. As expected, the second
band is sensitive to endoglycosidase H (EndoH) treatment, showing that this band
corresponds to M proteins glycosylated in the ER that have not reached the Golgi
yet. The more diffuse band was not sensitive to EndoH treatment, suggesting that
this form is further modified in the Golgi. Only one band was observed with the
DxE mutant with a high intensity at 30 kDa sensitive to EndoH. The EndoH
sensitivity of the 30 kDa band likely reflects the accumulation of this protein
in the ER due to the lack of export signal. It is worth noticing that the
migration profile of the M-KGYR mutant in Western blotting showed an increased
<italic>N</italic>-glycosylation consistent with a better trafficking
through the Golgi (<xref ref-type="fig" rid="F6">Fig.
6</xref><italic>C</italic>). Altogether, these results confirm the presence of
two intracellular trafficking motifs in the C-terminal domain of the MERS-CoV M
protein: first the DxE motif is responsible for the ER export of the protein,
and then a second motif, KxGxYR, is involved in its retention in the TGN.</p></sec><sec><title>KxGxYR is a not an internalization signal and is not involved in M
oligomerization</title><p>Intracellular trafficking is a dynamic process with proteins that can undergo
cycles of cell surface expression and internalization. At steady state, the
localization of proteins at the plasma membrane results from an equilibrium
between anterograde intracellular trafficking and retrieval of protein by
endocytosis. Any inhibition of endocytosis would result in protein accumulation
at the cell surface. To test whether the KxGxYR motif is an endocytosis signal,
we analyzed the endocytosis of the M protein in a biotinylation assay. Proteins
expressed at the plasma membrane of cells expressing N3Q-M or N3Q-M-KGYR were
labeled at 4 °C with a nonpermeable cleavable biotin, and then endocytosis
was allowed by incubating the cells at 37 °C for 30 min. Noninternalized
biotin was then cleaved with GSH, and internalized proteins were detected by
immunoblotting. As shown in <xref ref-type="fig" rid="F6">Fig. 6</xref>(<italic>E</italic> and <italic>F</italic>), we did not detect any
difference in endocytosis levels between the WT protein and M-KGYR.</p><p>It has been proposed that oligomerization of MHV-M protein could be involved in
its TGN retention. Indeed, mutants that do not form oligomers were detected at
the cell surface. In cell lysates, the mutant N3Q-M-KGYR forms dimer in amounts
comparable with the WT protein (<xref ref-type="fig" rid="F6">Fig.
6</xref><italic>D</italic>). We also analyzed the formation of N3Q-M-KGYR
oligomers at the cell surface. To do so, the cell proteins at the cell surface
were biotinylated and cross-linked. The formation of multimers was detected by
immunoblotting (<xref ref-type="fig" rid="F6">Fig. 6</xref><italic>D</italic>).
As previously shown, expression of N3Q-M-KGYR at the cell surface was increased.
We detected the formation of dimers with a strong band migrating at 40 kDa and
also the formation of higher oligomers for both proteins, suggesting that the
increased cell surface expression conferred by the mutation of the motif KxGxYR
is not due to a defect in M–M interactions. These results indicate that
the KxGxYR signal is not involved in M oligomerization and is not an
internalization signal and that the localization of MERS-CoV M in the TGN is
likely due to a mechanism of retention, preventing the cell surface expression
of the protein.</p></sec><sec><title>IBV C-terminal domain is involved in its ERGIC localization</title><p>To confirm the role of the KxGxYR as a retention signal in the TGN, we tried to
transfer this signal on a protein expressed at the cell surface. We used CD4 as
a reporter; however, the chimeric proteins that were constructed presented
folding defects (data not shown). CD4 is a type-I transmembrane protein, whereas
the M protein has a very different architecture with three transmembrane
segments. Therefore, we constructed chimeras between MERS-CoV M and the M
protein of another coronavirus, IBV. This way, we were able to construct
chimeras that conserved the transmembrane domain structure of the protein, which
is likely important for the folding and localization of the protein. The IBV-M
protein expressed alone in cells is located in the ERGIC and
<italic>cis</italic>-Golgi (<xref rid="B26" ref-type="bibr">26</xref>), so
its localization can be distinguished from that of MERS-CoV M, using specific
compartment markers. In addition, it has been shown that the first transmembrane
segment of IBV-M is involved in the intracellular retention of the protein. The
amino acid sequences of the MERS- and IBV-M C-terminal extremity are not
conserved and are rather different (<xref ref-type="fig" rid="F7">Fig.
7</xref><italic>A</italic>).</p><fig id="F7" orientation="portrait" position="float"><label>Figure 7.</label><caption><p><bold>MERS-M and IBV-M sequence alignment (<italic>A</italic>) and
<italic>schematic drawings</italic> of the different IBV-M and
MERS-M chimeras that were constructed (<italic>B</italic>).</bold>First, the C-terminal domain of MERS-CoV M was replaced with that of
IBV-M (<italic>MERS-M/IBV-M</italic>), and the C-terminal domain of
IBV-M was replaced by that of MERS-CoV M with or without the mutation of
the KxGxYR motif (<italic>IBV-M/MERS-M-KGYR</italic> and
<italic>IBV-M/MERS-M</italic>, respectively). The first
membrane-spanning segment of MERS-CoV M was replaced with that of IBV-M
in the context of the MERS-M-KGYR
(<italic>TM1-IBV/MERS-M-KGYR</italic>), and the first membrane-spanning
segment of IBV-M was replaced by that of MERS-CoV M
(<italic>TM1-MERS/IBV-M</italic>). All of the chimeras were tagged
at their C-terminal extremity with a VSVG epitope.</p></caption><graphic xlink:href="zbc0401911830007"></graphic></fig><p>First, we constructed chimeras in which we switched the C-terminal domains of the
proteins: MERS-M/IBV-M and IBV-M/MERS-M or IBV-M/MERS-M-KGYR. We also replaced
the first transmembrane segment of the MERS-M-KGYR with that of IBV-M to test
whether the first transmembrane segment of IBV-M can retain MERS-M-KGYR
intracellularly (TM1-IBV/MERS-M-KGYR). Finally, we replaced the first
transmembrane segment of IBV-M with that of MERS-CoV M. Schematic drawings of
the different chimeras that were constructed are presented in <xref ref-type="fig" rid="F7">Fig. 7</xref><italic>B</italic>. The subcellular
localization of the chimeric proteins was then analyzed by fluorescence
microscopy. As shown in <xref ref-type="fig" rid="F8">Fig. 8</xref>, the
immunofluorescent staining of IBV-M and MERS-CoV M differed in their pattern,
with a compact perinuclear staining for MERS-CoV M and a punctuated staining for
IBV-M. Co-localization assessment of IBV-M with TGN46 and ERGIC-53 showed that
the protein mainly localizes within the ERGIC. The MERS-M/IBV-M protein
colocalized with the ERGIC-53 marker and not the TGN46 marker, and is thus
located in the ERGIC (<xref ref-type="fig" rid="F8">Fig.
8</xref><italic>C</italic>), whereas the IBV-M/MERS-M protein colocalized
with the TGN46 marker and not with the ERGIC-53 marker and is thus localized in
the TGN. In other words, the switch of the C-terminal domains of the IBV-M and
MERS-CoV M proteins caused a switch of their specific localizations, to the
ERGIC and the TGN, respectively. Interestingly, the IBV-M/MERS-KGYR protein
localized to the cell surface, confirming the role of the KxGxYR signal in the
specific localization of the MERS-M protein to the TGN. This result also
suggests that the first transmembrane segment of IBV-M is not able to retain
MERS-M-KGYR intracellularly. In accordance with this result, the chimera
TM1-IBV/MERS-M-KGYR was also located at the cell surface, and the chimera
TM1-MERS/IBV-M was located in the ERGIC compartment. These results are
unexpected based on previous reports on the role of the first transmembrane
segment of IBV-M in its intracellular retention. Moreover, these results
indicate that for both MERS-CoV M and IBV-M, the presence of the C-terminal
domain is critical to induce the specific localization of the protein.
Furthermore, we also confirmed the involvement of the KxGxYR signal in the
specific retention of MERS-CoV M to the TGN, even in the chimeric context.</p><fig id="F8" orientation="portrait" position="float"><label>Figure 8.</label><caption><p><bold>Subcellular localization of IBV-M and MERS-CoV M proteins
chimeras.</bold> The localization of the different chimeras in the
TGN or in the ERGIC compartments was investigated by immunofluorescent
double labeling by using an anti-VSVG antibody and an anti-TGN46
(<italic>A</italic>) or by expressing the ERGIC-53 marker conjugated
with GFP (<italic>B</italic>). <italic>Bars</italic>, 20 μm.
Pearson's correlation coefficients were calculated for each combination
of co-staining (<italic>C</italic>). <italic>Error bars</italic>,
S.E.</p></caption><graphic xlink:href="zbc0401911830008"></graphic></fig></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>Viruses divert the intracellular trafficking machinery, and studying the
intracellular trafficking of viral membrane proteins often helps to decipher the
mechanisms of protein sorting and leads to uncovering new sorting motifs. We
investigated the intracellular trafficking of the MERS-CoV M protein and identified
a well-known ER export signal. In addition, we identified a novel TGN retention
motif.</p><p>Specific targeting of viral structural proteins to the assembly site in the cell is
crucial for viral egress and spreading. The three envelope proteins of coronaviruses
E, M, and S are synthesized in the ER. Protein exit from the ER toward the Golgi
occurs at specific sites called ER exit sites and for most of the proteins relies on
the coat protein complex II (COPII). Assembly of the coat starts with the activation
of the GTPase Sar1 by Sec12, an integral ER guanine nucleotide exchange factor. This
allows the recruitment of the complex Sec23/24, forming the inner layer of the coat
followed by the recruitment of the outer layer of the coat formed by Sec13/31 (<xref rid="B30" ref-type="bibr">30</xref>). ER export signals, including LxxLE,
diacidic DxE, YNNSNP, or triple R, can interact directly with Sec24 or Sar1 and lead
to the recruitment of the COPII carriers (<xref rid="B31" ref-type="bibr">31</xref>). We identified a functional DxE motif in the C-terminal part of the
MERS-CoV M protein, and this signal is also present in the M protein of the porcine
epidemic diarrhea virus. Some <italic>Alpha</italic>- and
<italic>Gammacoronavirus</italic> M proteins, such as HCoV-229E, HCoV-NL63,
FIPV, or IBV, contain a diacidic ExE motif; however, it remains to be determined
whether these signals can also act as ER export signal because, in the yeast protein
Sys1p, ExE cannot compensate for the DxE signal (<xref rid="B32" ref-type="bibr">32</xref>).</p><p>After its exit from the ER, the MERS-CoV M protein reaches the Golgi, where the
protein undergoes further modification of its <italic>N</italic>-glycans, as shown
in our glycosidase resistance assays. As shown previously for MHV-M protein, we and
others (<xref rid="B33" ref-type="bibr">33</xref>) found that ectopically expressed
MERS-CoV M protein is mainly located in the TGN at steady state. In the cells, the
TGN is a sorting station where proteins are either sent to the cell surface or
diverted toward other endomembrane compartments. The localization of proteins in
specific biosynthetic compartments generally results from an equilibrium between
anterograde and retrograde movements of the proteins. For example, in
immunofluorescent labeling of the protein TGN38/46, the protein is located in the
TGN, but this localization is the consequence of a very dynamic process in which the
protein is transported to the plasma membrane and recycled back to the TGN after
internalization and sorting in endosomes. The SDYQRL motif in the C-terminal domain
of the protein is important for its retrieval from the cell surface (<xref rid="B34" ref-type="bibr">34</xref>). In addition, the transmembrane domain of
the protein also participates in the TGN localization of the protein by mediating
some retention in the TGN (<xref rid="B35" ref-type="bibr">35</xref>). An increase
of the transport of the protein to the cell surface can lead to the saturation of
the retrieval mechanism of the protein and its accumulation at the cell surface.</p><p>Here, we identified a KxGxYR motif in the MERS-CoV M protein that is involved in the
TGN localization of the protein. The mutation of any residue of this motif leads to
the accumulation of the protein at the plasma membrane. This signal is highly
conserved in the M proteins of <italic>Betacoronavirus</italic>. Interestingly,
Armstrong and Patel (<xref rid="B36" ref-type="bibr">36</xref>) previously reported
that deletion of the last 18 residues of the C-terminal domain of the MHV-M protein
induced a shift of the protein localization toward the cell surface. Interestingly,
this deletion is in the middle of the KxGxYR signal, leaving only the Lys and Gly
residues on the truncated protein. In another study, Locker <italic>et al.</italic>(<xref rid="B27" ref-type="bibr">27</xref>) also reported a deletion of 22
residues of the protein, leading to the accumulation of the protein at the cell
surface. Furthermore, in their study about the structural requirements of MHV-M
protein, De Haan <italic>et al.</italic> (<xref rid="B11" ref-type="bibr">11</xref>)
mentioned that one of their mutants containing a mutation of the KxGxYR motif
(Y211G) leaked to the cell surface. These data suggest that TGN localization of M
proteins may be a general feature of the betacoronaviruses and that the KxGxYR motif
is involved in this localization. It has been reported that the SARS-CoV M protein
is located in the Golgi; unfortunately, its precise localization within the Golgi
remains unclear (<xref rid="B37" ref-type="bibr">37</xref>, <xref rid="B38" ref-type="bibr">38</xref>).</p><p>Locker <italic>et al.</italic> (<xref rid="B39" ref-type="bibr">39</xref>) reported
that oligomerization of MHV-M protein is involved in the TGN retention of the
protein; however, our results show that it is unlikely that the KxGxYR motif is
involved in the oligomerization of the protein. As a small proportion of the
MERS-CoV M protein could be detected at the cell surface (less than 1% of the total
protein; see <xref ref-type="fig" rid="F6">Fig. 6</xref>, <italic>A</italic> and
<italic>B</italic>), we tested whether the KxGxYR motif could be an endocytosis
motif. The MERS-CoV M protein is retrieved from the cell surface by endocytosis;
however, we could not detect any defect of endocytosis of the M protein when the
KxGxYR motif was mutated (<xref ref-type="fig" rid="F6">Fig. 6</xref>,
<italic>E</italic> and <italic>F</italic>). This result argues against an
endocytic function of this motif and suggests a role as a retention signal. Indeed,
it was reported previously (<xref rid="B27" ref-type="bibr">27</xref>) that the
MHV-M protein does not cycle between the plasma membrane of the cell and the TGN but
rather acts as a TGN-resident protein. Another hypothesis is a cycle of the M
between the ER and the TGN with the KxGxYR motif acting as a retrieval signal from
the TGN to the ER. The mutation of this signal would inhibit the retrieval of the
protein, allowing its trafficking to the cell surface. The retrograde trafficking
ensures the constant recycling of proteins and lipids from the Golgi to the ER to
maintain their steady-state distribution and the composition and function of the
organelles themselves. Two distinct mechanisms are responsible for this retrograde
transport. The first one depends on the coat protein complex I (COPI), and the
second one is the COPI-independent pathway that is less characterized, involves the
Rab6 GTPase, and is composed of tubular rather than vesicular carriers (<xref rid="B40" ref-type="bibr">40</xref>). The formation of the COPI-coated vesicles
starts with the recruitment en bloc of the coatomer composed of seven subunits by
the Arf1 GTPase. Cargos carry specific signals in their cytosolic-exposed domain
mediating their recruitment by COPI. The best characterized motifs are the di-lysine
motifs KKxx or KxKxx that are recognized by the α-COP or β′-COP
subunit of the coatomer. Multimeric proteins, such as receptors or channels, contain
an arginine-based sorting signal (φRxR, where φ represents any
hydrophobic amino acid). A common feature of the motif involved in the protein
sorting toward the COPI-dependent pathway is the presence of basic residues. Because
of its content in basic residues, the KxGxYR motif might be recognized by the COPI
machinery to prevent its cell surface expression.</p><p>Nevertheless, the mechanism of action of the KxGxYR motif remains to be further
elucidated, particularly in the context of the viral infection. Indeed, the
retention of the protein may be important for the proper assembly of the viral
particle by promoting interaction with the other viral membrane components. How the
interaction with E or S may mask the KxGxYR retention signal or how these protein
complexes may further traffic through the biosynthetic pathway remains to be
clarified.</p><p>Furthermore, we cannot exclude the possibility that other domains of the protein may
also be implicated in the TGN localization of the protein. As mentioned above, the
transmembrane domain and an endocytic motif cooperate for the proper localization of
TGN38/46. The Golgi apparatus is a compartmentalized structure where glycosylation
occurs in an ordered process (<xref rid="B41" ref-type="bibr">41</xref>). The
successful completion of glycosylation relies in part on the proper distribution of
glycosyltransferases along the Golgi that will permit their action in a sequential
manner. Most of the glycosyltransferases are type II transmembrane proteins. They
consist of a short N-terminal domain exposed in the cytosol, a transmembrane domain,
a stem region, and an enzymatic domain. The nonuniform distribution of these enzymes
in the Golgi is likely maintained by a combination of retention and recycling
mechanisms (<xref rid="B42" ref-type="bibr">42</xref>). The mechanisms that ensure
the localization of glycosyltransferases in the Golgi are numerous and diverse.
These include the oligomerization status of the enzyme and complex formation, the
length of the transmembrane domain but also its composition, which may affect the
way it is interacting with the different lipid compositions that the protein
encounters in the different Golgi cisternae. The cytosolic domains also contain
motifs involved in the localization of the enzymes (<xref rid="B42" ref-type="bibr">42</xref>).</p><p>To confirm the role of the KxGxYR motif as a retention signal in the TGN, we
attempted to transfer the motif in CD4, a protein expressed at the plasma membrane.
Unfortunately, the different chimeras that we constructed were not folded properly.
This was likely due to the difference of protein structures, CD4 being a type I
transmembrane protein, whereas MERS-CoV M is a triple membrane-spanning protein.
Therefore, to further confirm the role of the KxGxYR motif, we constructed chimeras
between MERS-CoV M and IBV-M, as they show clear differences in subcellular
localization at steady state. Indeed, IBV-M is mainly expressed in the ERGIC and
<italic>cis</italic>-Golgi compartment. Previous studies reported the role of
the first transmembrane segment in the intracellular retention of the protein (<xref rid="B26" ref-type="bibr">26</xref>). This was shown by the replacement of the
transmembrane domain of the glycoprotein G of the vesicular stomatitis virus (VSVG)
with the first transmembrane segment of IBV-M, which resulted in the intracellular
retention of the protein, and four polar residues were shown to mediate this
retention (<xref rid="B25" ref-type="bibr">25</xref>). Surprisingly, when we
replaced the first membrane-spanning domain of MERS-CoV M with that of IBV-M in the
context where the KxGxYR motif is mutated into alanine residues
(TM1-IBV/MERS-M-KGYR), the protein was not located in the ERGIC but was still
transported to the cell surface. This difference may lie in the use of a full-length
coronavirus M protein instead of a reporter protein such as VSVG. Interestingly,
when we swapped the C-terminal domains of the proteins, we also switched their
specific localizations, suggesting that the IBV-M C-terminal domain contains a
signal(s) for the ERGIC localization of the protein and not the first membrane
segment as reported previously.</p><p>Altogether, our results suggest that the C-terminal domain of coronavirus M proteins
dictates their specific localization, the betacoronavirus M proteins being addressed
to the TGN, where they are retained by the action of the KxGxYR motif. The motif
mediating IBV-M localization in the ERGIC and <italic>cis</italic>-Golgi compartment
remains, however, to be determined. At this state of knowledge, we cannot exclude
the possibility that the membrane-spanning segments of the protein additionally
participate in the intracellular retention of these proteins and cooperate with the
C-terminal domain to prevent the expression of the protein at the cell surface.</p></sec><sec sec-type="methods"><title>Experimental procedures</title><sec><title>Plasmids</title><p>The coding sequence of the M protein was cloned in the pCDNA3.1(+) vector, with
or without a sequence coding for different tags, including HA, VSVG, and V5.
Total RNA from blood samples of an infected patient were extracted by using the
Nucleospin RNA kit (Macherey-Nagel) according to the manufacturer's
instructions. Then reverse transcription was performed using the high-capacity
cDNA reverse transcription kit (Applied Biosystems), and the M protein sequence
was amplified by two successive PCRs. First, the sequence was amplified by using
the two following primers: 5′-gacgagtgggtttaacgaact-3′ and
5′-ggggatgccataacaatgaaa-3′. Then, to insert the sequence in
expression vectors, the sequence was amplified with
5′-tcggatccaccatgtctaatatgacgcaactcactg-3′ (primer A) and
5′cagaattcctaagctcgaagcaatgcaa-3′ (primer B; untagged protein) or
by combination of primer A and
5′-tagaattcagctcgaagcaatgcaagttcaat-3′ (primer C; C-terminal
tagged protein) or with 5′-acggatccaatatgacgcaactcactgagg-3′
(primer D) with primer B (N-terminal tagged protein). PCR products were inserted
between the BamHI and EcoRI restriction sites of the different vectors.</p><p>M protein deletion mutants were generated by PCR by using either primer A or D in
combination with a reverse primer annealing at different positions of the M
sequence, with or without a stop codon with an EcoRI restriction site. M protein
point mutants were generated by site-directed mutagenesis using PCR. Overlapping
primers containing the mutation(s) of interest were designed and used for PCR on
a MERS-M WT template. PCRs were then gel-purified, digested, and cloned into a
pCDNA3.1-VSVG or pCDNA3.1-HA plasmid. The chimeric constructions were generated
by fusion PCR. For IBV-M/MERS-M-Ct, the sequence of IBV-M corresponding to
residues 1–101 were amplified with the forward primer
5′-ttaagctttccatgcccaacgagacaaattg-3′ and the reverse primer
5′-taaacagccgaatactctggatccaataac-3′, containing 10 bases
complementary to the MERS-M sequence at its 5′ extremity. The MERS-M
sequence between residues 100 and 219 was amplified by PCR using the forward
primer 5′-ccagagtATTcggctgtttatgagaactgg-3′ containing 10 bases
complementary to the IBV-M sequence and with the primer D. Then the two PCR
products were mixed and amplified using the forward primer that anneals the
IBV-M sequence and the primer D. Using the same strategy of overlapping
sequences for the internal primers, we constructed MERS-M/IBV-M-Ct composed of
the MERS-M<sub>1–102</sub> fused to IBV-M<sub>105–225</sub>. The
first transmembrane segment of the MERS-M-KGYR was replaced by the first
transmembrane segment of IBV-M by fusing IBV-M<sub>1–42</sub> to
MERS-M-KGYR<sub>41–219</sub> (TM1-IBV/MERS-M-KGYR), and the first
transmembrane segment of IBV-M was replaced by the first transmembrane segment
of MERS-M by fusing MERS-M<sub>1–40</sub> to
IBV-M<sub>43–225</sub> (TM1-MERS/IBV-M). The PCR products were
gel-purified and digested by BamHI and EcoRI and then inserted into the
pCDNA3.1-V5 expression vector. All of the constructs were verified by DNA
sequencing.</p><p>The plasmids coding the ERGIC-53 protein fused to the GFP and coding the
GFP-CI-MPR were kindly provided by Dr. Hauri (University of Basel) and Dr.
Hoflack (University of Dresden) respectively. The plasmid encoding the CD4-GFP
fusion protein was constructed by amplification of the GFP with the two primers
5′-AGACATGTAGCCCCATTGTGAGCAAGGGCGAGGAGCT-3′ and
5′-GGGTCGACTCACTTGTACAGCTCGTCCATGC-3′ and then inserted into the
PCI-CD4 between the AflIII and SalI restriction sites.</p></sec><sec><title>Cell culture and transfection</title><p>HeLa cells were maintained in minimal essential medium supplemented with 10%
fetal calf serum and 1% Glutamax. 24 h before transfection, HeLa cells were
plated in 24-well plates on coverslips or in 6-well plates. The next day,
plasmids encoding WT M protein or M mutant protein were transfected into HeLa
cells using TransIT®-LT1 transfection reagent (Mirus Bio).</p></sec><sec><title>Immunofluorescence and confocal microscopy</title><p>At 18 h post-transfection, cells were rinsed with PBS, fixed with 3% PFA, and
processed for immunofluorescence analysis. Cells were permeabilized with 0.1%
Triton X-100 in PBS for 5 min and then blocked with buffer containing 10% goat
or horse serum in PBS for 10 min. M protein was detected using anti-M pAbs
(rabbit, Proteogenix) or anti-tag antibodies: anti-HA mAbs (3F10,
Sigma-Aldrich), anti-VSVG mAbs (P5D4, produced in the laboratory of the
authors), or anti-V5 mAbs (Thermo Fisher Scientific). For co-localization
experiments, cells were double-labeled for M-proteins and cellular marker,
anti-CRT pAbs for endoplasmic reticulum (ER) and anti-TGN46 pAbs for TGN
(Bio-Rad). Primary antibodies were diluted in blocking buffer. In some cases,
intracellular compartments were stained by transfecting an expression vector for
a marker fused with GFP. For ERGIC and TGN compartments, cells were
co-transfected with M proteins and expression vectors for ERGIC53 and M6PR fused
to GFP, respectively. For cell surface staining, cells were transfected with a
vector expressing CD4 fused to GFP. After a 30-min incubation with primary
antibodies, cells were washed three times for 5 min with PBS. Then the cells
were incubated with fluorescent secondary antibodies
(cyanine-3–conjugated goat anti-mouse IgG; cyanine-3–conjugated
goat anti-rabbit IgG; Alexa 488–conjugated donkey anti-rat IgG;
cyanine-3–conjugated donkey anti-sheep IgG; Alexa 488–conjugated
donkey anti-mouse IgG; Alexa 555–conjugated goat anti-rat IgG) and 1
μg/ml 4′,6-diamidino-2-phenylindole.</p><p>Images were acquired using a laser-scanning confocal microscope LSM 880 (Zeiss)
using a ×63 oil immersion objective. Signals were sequentially collected
using single fluorescence excitation and acquisition settings to avoid
crossover.</p><p>The extent of colocalization was quantified by calculating the PCC using the
JACoP plugin of ImageJ. The PCC examines the relationship between the
intensities of the pixels of two channels in the same image. For each
calculation, at least 15 images were analyzed to obtain a PCC mean. A PCC of 1
indicates perfect correlation, 0 no correlation, and −1 perfect
anti-correlation.</p></sec><sec><title>Biotinylation and internalization assay</title><p>HeLa cells were seeded in 6-well plates and transfected the next day with
pCDNA3.1-V5-N3Q-M, pCDNA3.1-V5-N3Q-MΔ20, pCDNA3.1-V5-N3Q-M-D211A,E213A,
pCDNA3.1-V5-N3Q-M-K199A,G201A,Y203A,R204A, PCDNA3.1-V5-N3Q-M-K199A,
pCDNA3.1-V5-N3Q-M-G201A, pCDNA3.1-V5-N3Q-M-Y203A, or pCDNA3.1-V5-N3Q-M-R204A. At
24 h post-transfection, cells were rinsed on ice with ice-cold PBS and incubated
twice with 250 μg/ml EZ-Link<sup>TM</sup> Sulfo-NHS-SS-Biotin (Pierce)
diluted in PBS for 15 min to label cell surface proteins. Unfixed biotin was
then quenched by two sequential incubations of the cells for 10 min with 50
m<sc>m</sc> glycine/PBS.</p><p>For internalization assays, cells were biotinylated 48 h post-transfection and
then incubated at 37 °C for 30 min. The biotin of nonendocytosed proteins
was then cleaved upon three incubations of 20 min with GSH buffer (50
m<sc>m</sc> reduced GSH, 75 m<sc>m</sc> NaCl, 75 m<sc>m</sc> NaOH, 10% fetal
calf serum) followed by two incubations of 15 min with iodoacetamide buffer (50
m<sc>m</sc> iodoacetamide, 1% BSA, PBS).</p><p>Cells were then lysed with B1 buffer (50 m<sc>m</sc> Tris, pH 7.5, 100
m<sc>m</sc> NaCl, 2 m<sc>m</sc> EDTA, 1% Triton X-100, 0.1% SDS, protease
inhibitor mixture) on ice. Lysates were centrifuged at 14,000 rpm at 4 °C
for 5 min to remove cellular debris and were then incubated with 30 μl of
streptavidin-conjugated agarose beads (Sigma) for 2 h. Beads were then washed
serially with 1 ml of buffers B1, B2 (50 m<sc>m</sc> Tris, pH 7.5, 100
m<sc>m</sc> NaCl, 2 m<sc>m</sc> EDTA, 0.1% Triton X-100, 0.5% SDS, 0.5%
deoxycholate), B3 (50 m<sc>m</sc> Tris, pH 7.5, 500 m<sc>m</sc> NaCl, 2
m<sc>m</sc> EDTA, 0.1% Triton X-100), and B4 (50 m<sc>m</sc> Tris, pH 7.5,
100 m<sc>m</sc> NaCl, 2 m<sc>m</sc> EDTA). Proteins were resuspended in Laemmli
loading buffer and detected by immunoblotting. Samples were separated by
SDS-PAGE, and proteins were transferred on a nitrocellulose membrane (Amersham
Biosciences). Membrane-bound M proteins were then detected using a monoclonal
anti-V5 antibody and horseradish peroxidase–conjugated secondary
antibody. Detection was carried out by chemiluminescence (Pierce).</p></sec><sec><title>Glycosidase treatment</title><p>HeLa cells were transfected with vectors expressing V5-M, V5-M-DxE, V5-M-KGYR, or
V5-N3Q-M proteins. 24 h later, cells were lysed in B1 buffer. Then, 30 μl
of lysates were mock-treated or treated with PNGase F or endoglycosidase H
according to the manufacturer's instructions. Then proteins were separated on
SDS-PAGE and detected by immunoblotting.</p></sec><sec><title>M–M interaction assay</title><p>HeLa cells were seeded in 10-cm dishes and transfected with vectors expressing
V5-N3Q-M or V5-N3Q-M-KGYR. The next day, cell surface proteins were biotinylated
at 4 °C and cross-linked with 0.8% PFA in PBS for 10 min. Then PFA was
quenched by washing the cells with 50 m<sc>m</sc> NH<sub>4</sub>CL/PBS twice.
Cells were lysed with B1 buffer, and lysates were processed for streptavidin
precipitation as described previously. Proteins were resuspended in nonreducing
Laemmli loading buffer without heating and detected by immunoblotting.</p></sec></sec><sec><title>Author contributions</title><p>A. P. and S. B. formal analysis; A. P., A. B., L. D., A. D., and S. B. investigation;
A. P., J. D., and S. B. writing-original draft; A. P., A. B., L. D., A. G., Y. R.,
J. D., and S. B. writing-review and editing; L. D., A. G., Y. R., J. D., and S. B.
conceptualization; A. D. and Y. R. resources; J. D. and S. B. supervision; S. B.
funding acquisition.</p></sec></body><back><fn-group><fn fn-type="supported-by"><p>This work was supported by a Visionn-AIRR grant from Region Hauts-de-France.
<named-content content-type="COI-statement">The authors declare that they
have no conflicts of interest with the contents of this
article</named-content>.</p></fn></fn-group><fn-group content-type="abbreviations"><fn id="FN3"><label>4</label><p>The abbreviations used are: <def-list><def-item><term id="G1">CoV</term><def><p>coronavirus</p></def></def-item><def-item><term id="G2">HCoV</term><def><p>human CoV</p></def></def-item><def-item><term id="G3">SARS</term><def><p>severe acute respiratory syndrome</p></def></def-item><def-item><term id="G4">MERS</term><def><p>Middle East respiratory syndrome</p></def></def-item><def-item><term id="G5">S</term><def><p>E, M, and N protein</p></def></def-item><def-item><term id="G6">spike</term><def><p>envelope, membrane, and nucleocapsid protein, respectively</p></def></def-item><def-item><term id="G7">MHV</term><def><p>murine hepatitis virus</p></def></def-item><def-item><term id="G8">IBV</term><def><p>infectious bronchitis virus</p></def></def-item><def-item><term id="G9">ER</term><def><p>endoplasmic reticulum</p></def></def-item><def-item><term id="G10">ERGIC</term><def><p>ER–Golgi intermediate compartment</p></def></def-item><def-item><term id="G11">TGN</term><def><p><italic>trans</italic>-Golgi network</p></def></def-item><def-item><term id="G12">MPR</term><def><p>mannose 6-phosphate receptor</p></def></def-item><def-item><term id="G13">PCC</term><def><p>Pearson correlation coefficient</p></def></def-item><def-item><term id="G14">PNGase F</term><def><p>peptide:<italic>N</italic>-glycosidase F</p></def></def-item><def-item><term id="G15">EndoH</term><def><p>endoglycosidase H</p></def></def-item><def-item><term id="G16">COPI and COPII</term><def><p>coat protein complex I and II, respectively</p></def></def-item><def-item><term id="G17">HA</term><def><p>hemagglutinin</p></def></def-item><def-item><term id="G18">PFA</term><def><p>paraformaldehyde</p></def></def-item><def-item><term id="G19">pAb</term><def><p>polyclonal antibod</p></def></def-item><def-item><term id="G20">CRT</term><def><p>calreticulin.</p></def></def-item></def-list></p></fn></fn-group><ack><title>Acknowledgments</title><p>We thank Hans-Peter Hauri, Bernard Hoflack, and Gary Whittaker for providing
reagents. The immunofluorescence analyses were performed with the help of the
imaging core facility of the BioImaging Center Lille Nord-de-France.</p></ack><ref-list><title>References</title><ref id="B1"><label>1.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Masters</surname><given-names>P.
S.</given-names></name></person-group> (<year>2006</year>)
<article-title>The molecular biology of coronaviruses</article-title>.
<source>Adv. Virus Res</source>. <volume>66</volume>,
<fpage>193</fpage>–<lpage>292</lpage><pub-id pub-id-type="doi">10.1016/S0065-3527(06)66005-3</pub-id><pub-id pub-id-type="pmid">16877062</pub-id></mixed-citation></ref><ref id="B2"><label>2.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Perlman</surname><given-names>S.</given-names></name>, and
<name name-style="western"><surname>Netland</surname><given-names>J.</given-names></name></person-group>(<year>2009</year>) <article-title>Coronaviruses post-SARS: update on
replication and pathogenesis</article-title>. <source>Nat. Rev.
Microbiol</source>. <volume>7</volume>,
<fpage>439</fpage>–<lpage>450</lpage><pub-id pub-id-type="doi">10.1038/nrmicro2147</pub-id><pub-id pub-id-type="pmid">19430490</pub-id></mixed-citation></ref><ref id="B3"><label>3.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Corman</surname><given-names>V.
M.</given-names></name>, <name name-style="western"><surname>Muth</surname><given-names>D.</given-names></name>,
<name name-style="western"><surname>Niemeyer</surname><given-names>D.</given-names></name>, and
<name name-style="western"><surname>Drosten</surname><given-names>C.</given-names></name></person-group>(<year>2018</year>) <article-title>Hosts and sources of endemic human
coronaviruses</article-title>. <source>Adv. Virus Res</source>.
<volume>100</volume>, <fpage>163</fpage>–<lpage>188</lpage><pub-id pub-id-type="doi">10.1016/bs.aivir.2018.01.001</pub-id><pub-id pub-id-type="pmid">29551135</pub-id></mixed-citation></ref><ref id="B4"><label>4.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Belouzard</surname><given-names>S.</given-names></name>,
<name name-style="western"><surname>Millet</surname><given-names>J.
K.</given-names></name>,
<name name-style="western"><surname>Licitra</surname><given-names>B.
N.</given-names></name>, and
<name name-style="western"><surname>Whittaker</surname><given-names>G.
R.</given-names></name></person-group> (<year>2012</year>)
<article-title>Mechanisms of coronavirus cell entry mediated by the viral
spike protein</article-title>. <source>Viruses</source><volume>4</volume>, <fpage>1011</fpage>–<lpage>1033</lpage><pub-id pub-id-type="doi">10.3390/v4061011</pub-id><pub-id pub-id-type="pmid">22816037</pub-id></mixed-citation></ref><ref id="B5"><label>5.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Heald-Sargent</surname><given-names>T.</given-names></name>, and
<name name-style="western"><surname>Gallagher</surname><given-names>T.</given-names></name></person-group>(<year>2012</year>) <article-title>Ready, set, fuse! The coronavirus spike
protein and acquisition of fusion competence</article-title>.
<source>Viruses</source><volume>4</volume>, <fpage>557</fpage>–<lpage>580</lpage><pub-id pub-id-type="doi">10.3390/v4040557</pub-id><pub-id pub-id-type="pmid">22590686</pub-id></mixed-citation></ref><ref id="B6"><label>6.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Ruch</surname><given-names>T.
R.</given-names></name>, and
<name name-style="western"><surname>Machamer</surname><given-names>C.
E.</given-names></name></person-group> (<year>2012</year>)
<article-title>The coronavirus E protein: assembly and
beyond</article-title>. <source>Viruses</source><volume>4</volume>, <fpage>363</fpage>–<lpage>382</lpage><pub-id pub-id-type="doi">10.3390/v4030363</pub-id><pub-id pub-id-type="pmid">22590676</pub-id></mixed-citation></ref><ref id="B7"><label>7.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Nieto-Torres</surname><given-names>J.
L.</given-names></name>,
<name name-style="western"><surname>Verdiá-Báguena</surname><given-names>C.</given-names></name>,
<name name-style="western"><surname>Castaño-Rodriguez</surname><given-names>C.</given-names></name>,
<name name-style="western"><surname>Aguilella</surname><given-names>V.
M.</given-names></name>, and
<name name-style="western"><surname>Enjuanes</surname><given-names>L.</given-names></name></person-group>(<year>2015</year>) <article-title>Relevance of viroporin ion channel
activity on viral replication and pathogenesis</article-title>.
<source>Viruses</source><volume>7</volume>, <fpage>3552</fpage>–<lpage>3573</lpage><pub-id pub-id-type="doi">10.3390/v7072786</pub-id><pub-id pub-id-type="pmid">26151305</pub-id></mixed-citation></ref><ref id="B8"><label>8.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Westerbeck</surname><given-names>J.
W.</given-names></name>, and
<name name-style="western"><surname>Machamer</surname><given-names>C.
E.</given-names></name></person-group> (<year>2019</year>)
<article-title>The infectious bronchitis coronavirus envelope protein alters
Golgi pH to protect spike protein and promote release of infectious
virus</article-title>. <source>J. Virol</source>. <volume>93</volume>,
<fpage>e00015</fpage>–<lpage>19</lpage><pub-id pub-id-type="doi">10.1128/JVI.00015-19</pub-id><pub-id pub-id-type="pmid">30867314</pub-id></mixed-citation></ref><ref id="B9"><label>9.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Westerbeck</surname><given-names>J.
W.</given-names></name>, and
<name name-style="western"><surname>Machamer</surname><given-names>C.
E.</given-names></name></person-group> (<year>2015</year>)
<article-title>A coronavirus E protein is present in two distinct pools with
different effects on assembly and the secretory pathway</article-title>.
<source>J. Virol</source>. <volume>89</volume>,
<fpage>9313</fpage>–<lpage>9323</lpage><pub-id pub-id-type="doi">10.1128/JVI.01237-15</pub-id><pub-id pub-id-type="pmid">26136577</pub-id></mixed-citation></ref><ref id="B10"><label>10.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>de Haan</surname><given-names>C. A.
M.</given-names></name>, and
<name name-style="western"><surname>Rottier</surname><given-names>P. J.
M.</given-names></name></person-group> (<year>2005</year>)
<article-title>Molecular interactions in the assembly of
coronaviruses</article-title>. <source>Adv. Virus Res</source>.
<volume>64</volume>, <fpage>165</fpage>–<lpage>230</lpage><pub-id pub-id-type="doi">10.1016/S0065-3527(05)64006-7</pub-id><pub-id pub-id-type="pmid">16139595</pub-id></mixed-citation></ref><ref id="B11"><label>11.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>de Haan</surname><given-names>C.
A.</given-names></name>, <name name-style="western"><surname>Kuo</surname><given-names>L.</given-names></name>,
<name name-style="western"><surname>Masters</surname><given-names>P.
S.</given-names></name>,
<name name-style="western"><surname>Vennema</surname><given-names>H.</given-names></name>, and
<name name-style="western"><surname>Rottier</surname><given-names>P.
J.</given-names></name></person-group> (<year>1998</year>)
<article-title>Coronavirus particle assembly: primary structure requirements
of the membrane protein</article-title>. <source>J. Virol</source>.
<volume>72</volume>, <fpage>6838</fpage>–<lpage>6850</lpage><pub-id pub-id-type="pmid">9658133</pub-id></mixed-citation></ref><ref id="B12"><label>12.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Opstelten</surname><given-names>D.
J.</given-names></name>,
<name name-style="western"><surname>Raamsman</surname><given-names>M.
J.</given-names></name>,
<name name-style="western"><surname>Wolfs</surname><given-names>K.</given-names></name>,
<name name-style="western"><surname>Horzinek</surname><given-names>M.
C.</given-names></name>, and
<name name-style="western"><surname>Rottier</surname><given-names>P.
J.</given-names></name></person-group> (<year>1995</year>)
<article-title>Envelope glycoprotein interactions in coronavirus
assembly</article-title>. <source>J. Cell Biol</source>.
<volume>131</volume>, <fpage>339</fpage>–<lpage>349</lpage><pub-id pub-id-type="doi">10.1083/jcb.131.2.339</pub-id><pub-id pub-id-type="pmid">7593163</pub-id></mixed-citation></ref><ref id="B13"><label>13.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>de Haan</surname><given-names>C.
A.</given-names></name>, <name name-style="western"><surname>Smeets</surname><given-names>M.</given-names></name>,
<name name-style="western"><surname>Vernooij</surname><given-names>F.</given-names></name>,
<name name-style="western"><surname>Vennema</surname><given-names>H.</given-names></name>, and
<name name-style="western"><surname>Rottier</surname><given-names>P.
J.</given-names></name></person-group> (<year>1999</year>)
<article-title>Mapping of the coronavirus membrane protein domains involved
in interaction with the spike protein</article-title>. <source>J.
Virol</source>. <volume>73</volume>,
<fpage>7441</fpage>–<lpage>7452</lpage><pub-id pub-id-type="pmid">10438834</pub-id></mixed-citation></ref><ref id="B14"><label>14.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Arndt</surname><given-names>A.
L.</given-names></name>, <name name-style="western"><surname>Larson</surname><given-names>B. J.</given-names></name>, and
<name name-style="western"><surname>Hogue</surname><given-names>B.
G.</given-names></name></person-group> (<year>2010</year>)
<article-title>A conserved domain in the coronavirus membrane protein tail
is important for virus assembly</article-title>. <source>J. Virol</source>.
<volume>84</volume>, <fpage>11418</fpage>–<lpage>11428</lpage><pub-id pub-id-type="doi">10.1128/JVI.01131-10</pub-id><pub-id pub-id-type="pmid">20719948</pub-id></mixed-citation></ref><ref id="B15"><label>15.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Tseng</surname><given-names>Y.-T.</given-names></name>,
<name name-style="western"><surname>Wang</surname><given-names>S.-M.</given-names></name>,
<name name-style="western"><surname>Huang</surname><given-names>K.-J.</given-names></name>,
<name name-style="western"><surname>Lee</surname><given-names>A.
I.-R.</given-names></name>,
<name name-style="western"><surname>Chiang</surname><given-names>C.-C.</given-names></name>, and
<name name-style="western"><surname>Wang</surname><given-names>C.-T.</given-names></name></person-group>(<year>2010</year>) <article-title>Self-assembly of severe acute respiratory
syndrome coronavirus membrane protein</article-title>. <source>J. Biol.
Chem</source>. <volume>285</volume>,
<fpage>12862</fpage>–<lpage>12872</lpage><pub-id pub-id-type="doi">10.1074/jbc.M109.030270</pub-id><pub-id pub-id-type="pmid">20154085</pub-id></mixed-citation></ref><ref id="B16"><label>16.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>de Haan</surname><given-names>C. A.
M.</given-names></name>, <name name-style="western"><surname>Vennema</surname><given-names>H.</given-names></name>, and
<name name-style="western"><surname>Rottier</surname><given-names>P. J.
M.</given-names></name></person-group> (<year>2000</year>)
<article-title>Assembly of the coronavirus envelope: homotypic interactions
between the M proteins</article-title>. <source>J. Virol</source>.
<volume>74</volume>, <fpage>4967</fpage>–<lpage>4978</lpage><pub-id pub-id-type="doi">10.1128/JVI.74.11.4967-4978.2000</pub-id><pub-id pub-id-type="pmid">10799570</pub-id></mixed-citation></ref><ref id="B17"><label>17.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Baudoux</surname><given-names>P.</given-names></name>,
<name name-style="western"><surname>Carrat</surname><given-names>C.</given-names></name>,
<name name-style="western"><surname>Besnardeau</surname><given-names>L.</given-names></name>,
<name name-style="western"><surname>Charley</surname><given-names>B.</given-names></name>, and
<name name-style="western"><surname>Laude</surname><given-names>H.</given-names></name></person-group>(<year>1998</year>) <article-title>Coronavirus pseudoparticles formed with
recombinant M and E proteins induce α interferon synthesis by
leukocytes</article-title>. <source>J. Virol</source>. <volume>72</volume>,
<fpage>8636</fpage>–<lpage>8643</lpage><pub-id pub-id-type="pmid">9765403</pub-id></mixed-citation></ref><ref id="B18"><label>18.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Vennema</surname><given-names>H.</given-names></name>,
<name name-style="western"><surname>Godeke</surname><given-names>G.
J.</given-names></name>,
<name name-style="western"><surname>Rossen</surname><given-names>J.
W.</given-names></name>,
<name name-style="western"><surname>Voorhout</surname><given-names>W.
F.</given-names></name>,
<name name-style="western"><surname>Horzinek</surname><given-names>M.
C.</given-names></name>,
<name name-style="western"><surname>Opstelten</surname><given-names>D.
J.</given-names></name>, and
<name name-style="western"><surname>Rottier</surname><given-names>P.
J.</given-names></name></person-group> (<year>1996</year>)
<article-title>Nucleocapsid-independent assembly of coronavirus-like
particles by co-expression of viral envelope protein genes</article-title>.
<source>EMBO J</source>. <volume>15</volume>,
<fpage>2020</fpage>–<lpage>2028</lpage><pub-id pub-id-type="doi">10.1002/j.1460-2075.1996.tb00553.x</pub-id><pub-id pub-id-type="pmid">8617249</pub-id></mixed-citation></ref><ref id="B19"><label>19.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Lim</surname><given-names>K.
P.</given-names></name>, and <name name-style="western"><surname>Liu</surname><given-names>D. X.</given-names></name></person-group>(<year>2001</year>) <article-title>The missing link in coronavirus assembly:
retention of the avian coronavirus infectious brochitis virus envelope
protein in the pre-Golgi compartments and physical interaction between the
envelope and membrane proteins</article-title>. <source>J. Biol.
Chem</source>. <volume>276</volume>,
<fpage>17515</fpage>–<lpage>17523</lpage><pub-id pub-id-type="doi">10.1074/jbc.M009731200</pub-id><pub-id pub-id-type="pmid">11278557</pub-id></mixed-citation></ref><ref id="B20"><label>20.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Siu</surname><given-names>Y.
L.</given-names></name>, <name name-style="western"><surname>Teoh</surname><given-names>K. T.</given-names></name>,
<name name-style="western"><surname>Lo</surname><given-names>J.</given-names></name>,
<name name-style="western"><surname>Chan</surname><given-names>C.
M.</given-names></name>,
<name name-style="western"><surname>Kien</surname><given-names>F.</given-names></name>,
<name name-style="western"><surname>Escriou</surname><given-names>N.</given-names></name>,
<name name-style="western"><surname>Tsao</surname><given-names>S.
W.</given-names></name>,
<name name-style="western"><surname>Nicholls</surname><given-names>J.
M.</given-names></name>,
<name name-style="western"><surname>Altmeyer</surname><given-names>R.</given-names></name>,
<name name-style="western"><surname>Peiris</surname><given-names>J. S.
M.</given-names></name>,
<name name-style="western"><surname>Bruzzone</surname><given-names>R.</given-names></name>, and
<name name-style="western"><surname>Nal</surname><given-names>B.</given-names></name></person-group>(<year>2008</year>) <article-title>The M, E, and N structural proteins of
the severe acute respiratory syndrome coronavirus are required for efficient
assembly, trafficking, and release of virus-like particles</article-title>.
<source>J. Virol</source>. <volume>82</volume>,
<fpage>11318</fpage>–<lpage>11330</lpage><pub-id pub-id-type="doi">10.1128/JVI.01052-08</pub-id><pub-id pub-id-type="pmid">18753196</pub-id></mixed-citation></ref><ref id="B21"><label>21.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Klumperman</surname><given-names>J.</given-names></name>,
<name name-style="western"><surname>Locker</surname><given-names>J.
K.</given-names></name>,
<name name-style="western"><surname>Meijer</surname><given-names>A.</given-names></name>,
<name name-style="western"><surname>Horzinek</surname><given-names>M.
C.</given-names></name>,
<name name-style="western"><surname>Geuze</surname><given-names>H.
J.</given-names></name>, and
<name name-style="western"><surname>Rottier</surname><given-names>P.
J.</given-names></name></person-group> (<year>1994</year>)
<article-title>Coronavirus M proteins accumulate in the Golgi complex beyond
the site of virion budding</article-title>. <source>J. Virol</source>.
<volume>68</volume>, <fpage>6523</fpage>–<lpage>6534</lpage><pub-id pub-id-type="pmid">8083990</pub-id></mixed-citation></ref><ref id="B22"><label>22.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Salanueva</surname><given-names>I.
J.</given-names></name>,
<name name-style="western"><surname>Carrascosa</surname><given-names>J.
L.</given-names></name>, and
<name name-style="western"><surname>Risco</surname><given-names>C.</given-names></name></person-group>(<year>1999</year>) <article-title>Structural maturation of the
transmissible gastroenteritis coronavirus</article-title>. <source>J.
Virol</source>. <volume>73</volume>,
<fpage>7952</fpage>–<lpage>7964</lpage><pub-id pub-id-type="pmid">10482542</pub-id></mixed-citation></ref><ref id="B23"><label>23.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Machamer</surname><given-names>C.
E.</given-names></name>, <name name-style="western"><surname>Mentone</surname><given-names>S. A.</given-names></name>,
<name name-style="western"><surname>Rose</surname><given-names>J.
K.</given-names></name>, and
<name name-style="western"><surname>Farquhar</surname><given-names>M.
G.</given-names></name></person-group> (<year>1990</year>)
<article-title>The E1 glycoprotein of an avian coronavirus is targeted to
the cis Golgi complex</article-title>. <source>Proc. Natl. Acad. Sci.
U.S.A</source>. <volume>87</volume>,
<fpage>6944</fpage>–<lpage>6948</lpage><pub-id pub-id-type="doi">10.1073/pnas.87.18.6944</pub-id><pub-id pub-id-type="pmid">2169615</pub-id></mixed-citation></ref><ref id="B24"><label>24.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Locker</surname><given-names>J.
K.</given-names></name>,
<name name-style="western"><surname>Griffiths</surname><given-names>G.</given-names></name>,
<name name-style="western"><surname>Horzinek</surname><given-names>M.
C.</given-names></name>, and
<name name-style="western"><surname>Rottier</surname><given-names>P.
J.</given-names></name></person-group> (<year>1992</year>)
<article-title><italic>O</italic>-glycosylation of the coronavirus M
protein: differential localization of sialyltransferases in
<italic>N</italic>- and <italic>O</italic>-linked
glycosylation</article-title>. <source>J. Biol. Chem</source>.
<volume>267</volume>, <fpage>14094</fpage>–<lpage>14101</lpage><pub-id pub-id-type="pmid">1629209</pub-id></mixed-citation></ref><ref id="B25"><label>25.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Machamer</surname><given-names>C.
E.</given-names></name>, <name name-style="western"><surname>Grim</surname><given-names>M. G.</given-names></name>,
<name name-style="western"><surname>Esquela</surname><given-names>A.</given-names></name>,
<name name-style="western"><surname>Chung</surname><given-names>S.
W.</given-names></name>,
<name name-style="western"><surname>Rolls</surname><given-names>M.</given-names></name>,
<name name-style="western"><surname>Ryan</surname><given-names>K.</given-names></name>, and
<name name-style="western"><surname>Swift</surname><given-names>A.
M.</given-names></name></person-group> (<year>1993</year>)
<article-title>Retention of a cis Golgi protein requires polar residues on
one face of a predicted α-helix in the transmembrane
domain</article-title>. <source>Mol. Biol. Cell</source><volume>4</volume>, <fpage>695</fpage>–<lpage>704</lpage><pub-id pub-id-type="doi">10.1091/mbc.4.7.695</pub-id><pub-id pub-id-type="pmid">8400455</pub-id></mixed-citation></ref><ref id="B26"><label>26.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Machamer</surname><given-names>C.
E.</given-names></name>, and
<name name-style="western"><surname>Rose</surname><given-names>J.
K.</given-names></name></person-group> (<year>1987</year>)
<article-title>A specific transmembrane domain of a coronavirus E1
glycoprotein is required for its retention in the Golgi
region</article-title>. <source>J. Cell Biol</source>. <volume>105</volume>,
<fpage>1205</fpage>–<lpage>1214</lpage><pub-id pub-id-type="doi">10.1083/jcb.105.3.1205</pub-id><pub-id pub-id-type="pmid">2821010</pub-id></mixed-citation></ref><ref id="B27"><label>27.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Locker</surname><given-names>J.
K.</given-names></name>,
<name name-style="western"><surname>Klumperman</surname><given-names>J.</given-names></name>,
<name name-style="western"><surname>Oorschot</surname><given-names>V.</given-names></name>,
<name name-style="western"><surname>Horzinek</surname><given-names>M.
C.</given-names></name>,
<name name-style="western"><surname>Geuze</surname><given-names>H.
J.</given-names></name>, and
<name name-style="western"><surname>Rottier</surname><given-names>P.
J.</given-names></name></person-group> (<year>1994</year>)
<article-title>The cytoplasmic tail of mouse hepatitis virus M protein is
essential but not sufficient for its retention in the Golgi
complex</article-title>. <source>J. Biol. Chem</source>.
<volume>269</volume>, <fpage>28263</fpage>–<lpage>28269</lpage><pub-id pub-id-type="pmid">7961764</pub-id></mixed-citation></ref><ref id="B28"><label>28.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Waguri</surname><given-names>S.</given-names></name>,
<name name-style="western"><surname>Dewitte</surname><given-names>F.</given-names></name>,
<name name-style="western"><surname>Le Borgne</surname><given-names>R.</given-names></name>,
<name name-style="western"><surname>Rouillé</surname><given-names>Y.</given-names></name>,
<name name-style="western"><surname>Uchiyama</surname><given-names>Y.</given-names></name>,
<name name-style="western"><surname>Dubremetz</surname><given-names>J.-F.</given-names></name>, and
<name name-style="western"><surname>Hoflack</surname><given-names>B.</given-names></name></person-group>(<year>2003</year>) <article-title>Visualization of TGN to endosome
trafficking through fluorescently labeled MPR and AP-1 in living
cells</article-title>. <source>Mol. Biol. Cell</source>.
<volume>14</volume>, <fpage>142</fpage>–<lpage>155</lpage><pub-id pub-id-type="doi">10.1091/mbc.e02-06-0338</pub-id><pub-id pub-id-type="pmid">12529433</pub-id></mixed-citation></ref><ref id="B29"><label>29.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Dunn</surname><given-names>K.
W.</given-names></name>, <name name-style="western"><surname>Kamocka</surname><given-names>M. M.</given-names></name>, and
<name name-style="western"><surname>McDonald</surname><given-names>J.
H.</given-names></name></person-group> (<year>2011</year>)
<article-title>A practical guide to evaluating colocalization in biological
microscopy</article-title>. <source>Am. J. Physiol. Cell Physiol</source>.
<volume>300</volume>, <fpage>C723</fpage>–<lpage>C742</lpage><pub-id pub-id-type="doi">10.1152/ajpcell.00462.2010</pub-id><pub-id pub-id-type="pmid">21209361</pub-id></mixed-citation></ref><ref id="B30"><label>30.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Venditti</surname><given-names>R.</given-names></name>,
<name name-style="western"><surname>Wilson</surname><given-names>C.</given-names></name>, and
<name name-style="western"><surname>De Matteis</surname><given-names>M.
A.</given-names></name></person-group> (<year>2014</year>)
<article-title>Exiting the ER: what we know and what we
don't</article-title>. <source>Trends Cell Biol</source>.
<volume>24</volume>, <fpage>9</fpage>–<lpage>18</lpage><pub-id pub-id-type="doi">10.1016/j.tcb.2013.08.005</pub-id><pub-id pub-id-type="pmid">24076263</pub-id></mixed-citation></ref><ref id="B31"><label>31.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Geva</surname><given-names>Y.</given-names></name>, and
<name name-style="western"><surname>Schuldiner</surname><given-names>M.</given-names></name></person-group>(<year>2014</year>) <article-title>The back and forth of cargo exit from the
endoplasmic reticulum</article-title>. <source>Curr. Biol</source>.
<volume>24</volume>, <fpage>R130</fpage>–<lpage>R136</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2013.12.008</pub-id><pub-id pub-id-type="pmid">24502791</pub-id></mixed-citation></ref><ref id="B32"><label>32.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Votsmeier</surname><given-names>C.</given-names></name>, and
<name name-style="western"><surname>Gallwitz</surname><given-names>D.</given-names></name></person-group>(<year>2001</year>) <article-title>An acidic sequence of a putative yeast
Golgi membrane protein binds COPII and facilitates ER
export</article-title>. <source>EMBO J</source>. <volume>20</volume>,
<fpage>6742</fpage>–<lpage>6750</lpage><pub-id pub-id-type="doi">10.1093/emboj/20.23.6742</pub-id><pub-id pub-id-type="pmid">11726510</pub-id></mixed-citation></ref><ref id="B33"><label>33.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Yang</surname><given-names>Y.</given-names></name>,
<name name-style="western"><surname>Zhang</surname><given-names>L.</given-names></name>,
<name name-style="western"><surname>Geng</surname><given-names>H.</given-names></name>,
<name name-style="western"><surname>Deng</surname><given-names>Y.</given-names></name>,
<name name-style="western"><surname>Huang</surname><given-names>B.</given-names></name>,
<name name-style="western"><surname>Guo</surname><given-names>Y.</given-names></name>,
<name name-style="western"><surname>Zhao</surname><given-names>Z.</given-names></name>, and
<name name-style="western"><surname>Tan</surname><given-names>W.</given-names></name></person-group>(<year>2013</year>) <article-title>The structural and accessory proteins M,
ORF 4a, ORF 4b, and ORF 5 of Middle East respiratory syndrome coronavirus
(MERS-CoV) are potent interferon antagonists</article-title>.
<source>Protein Cell</source><volume>4</volume>, <fpage>951</fpage>–<lpage>961</lpage><pub-id pub-id-type="doi">10.1007/s13238-013-3096-8</pub-id><pub-id pub-id-type="pmid">24318862</pub-id></mixed-citation></ref><ref id="B34"><label>34.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Bos</surname><given-names>K.</given-names></name>,
<name name-style="western"><surname>Wraight</surname><given-names>C.</given-names></name>, and
<name name-style="western"><surname>Stanley</surname><given-names>K.
K.</given-names></name></person-group> (<year>1993</year>)
<article-title>TGN38 is maintained in the <italic>trans</italic>-Golgi
network by a tyrosine-containing motif in the cytoplasmic
domain</article-title>. <source>EMBO J</source>. <volume>12</volume>,
<fpage>2219</fpage>–<lpage>2228</lpage><pub-id pub-id-type="doi">10.1002/j.1460-2075.1993.tb05870.x</pub-id><pub-id pub-id-type="pmid">8491209</pub-id></mixed-citation></ref><ref id="B35"><label>35.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Ponnambalam</surname><given-names>S.</given-names></name>,
<name name-style="western"><surname>Rabouille</surname><given-names>C.</given-names></name>,
<name name-style="western"><surname>Luzio</surname><given-names>J.
P.</given-names></name>,
<name name-style="western"><surname>Nilsson</surname><given-names>T.</given-names></name>, and
<name name-style="western"><surname>Warren</surname><given-names>G.</given-names></name></person-group>(<year>1994</year>) <article-title>The TGN38 glycoprotein contains two
non-overlapping signals that mediate localization to the
<italic>trans</italic>-Golgi network</article-title>. <source>J. Cell
Biol</source>. <volume>125</volume>,
<fpage>253</fpage>–<lpage>268</lpage><pub-id pub-id-type="doi">10.1083/jcb.125.2.253</pub-id><pub-id pub-id-type="pmid">8163544</pub-id></mixed-citation></ref><ref id="B36"><label>36.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Armstrong</surname><given-names>J.</given-names></name>, and
<name name-style="western"><surname>Patel</surname><given-names>S.</given-names></name></person-group>(<year>1991</year>) <article-title>The Golgi sorting domain of coronavirus
E1 protein</article-title>. <source>J. Cell Sci</source>.
<volume>98</volume>, <fpage>567</fpage>–<lpage>575</lpage><pub-id pub-id-type="pmid">1860903</pub-id></mixed-citation></ref><ref id="B37"><label>37.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Nal</surname><given-names>B.</given-names></name>,
<name name-style="western"><surname>Chan</surname><given-names>C.</given-names></name>,
<name name-style="western"><surname>Kien</surname><given-names>F.</given-names></name>,
<name name-style="western"><surname>Siu</surname><given-names>L.</given-names></name>,
<name name-style="western"><surname>Tse</surname><given-names>J.</given-names></name>,
<name name-style="western"><surname>Chu</surname><given-names>K.</given-names></name>,
<name name-style="western"><surname>Kam</surname><given-names>J.</given-names></name>,
<name name-style="western"><surname>Staropoli</surname><given-names>I.</given-names></name>,
<name name-style="western"><surname>Crescenzo-Chaigne</surname><given-names>B.</given-names></name>,
<name name-style="western"><surname>Escriou</surname><given-names>N.</given-names></name>,
<name name-style="western"><surname>van der Werf</surname><given-names>S.</given-names></name>,
<name name-style="western"><surname>Yuen</surname><given-names>K.
Y.</given-names></name>, and
<name name-style="western"><surname>Altmeyer</surname><given-names>R.</given-names></name></person-group>(<year>2005</year>) <article-title>Differential maturation and subcellular
localization of severe acute respiratory syndrome coronavirus surface
proteins S, M and E</article-title>. <source>J. Gen. Virol</source>.
<volume>86</volume>, <fpage>1423</fpage>–<lpage>1434</lpage><pub-id pub-id-type="doi">10.1099/vir.0.80671-0</pub-id><pub-id pub-id-type="pmid">15831954</pub-id></mixed-citation></ref><ref id="B38"><label>38.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>McBride</surname><given-names>C.
E.</given-names></name>, and
<name name-style="western"><surname>Machamer</surname><given-names>C.
E.</given-names></name></person-group> (<year>2010</year>)
<article-title>A single tyrosine in the severe acute respiratory syndrome
coronavirus membrane protein cytoplasmic tail is important for efficient
interaction with spike protein</article-title>. <source>J. Virol</source>.
<volume>84</volume>, <fpage>1891</fpage>–<lpage>1901</lpage><pub-id pub-id-type="doi">10.1128/JVI.02458-09</pub-id><pub-id pub-id-type="pmid">20007283</pub-id></mixed-citation></ref><ref id="B39"><label>39.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Locker</surname><given-names>J.
K.</given-names></name>,
<name name-style="western"><surname>Opstelten</surname><given-names>D.
J.</given-names></name>,
<name name-style="western"><surname>Ericsson</surname><given-names>M.</given-names></name>,
<name name-style="western"><surname>Horzinek</surname><given-names>M.
C.</given-names></name>, and
<name name-style="western"><surname>Rottier</surname><given-names>P.
J.</given-names></name></person-group> (<year>1995</year>)
<article-title>Oligomerization of a
<italic>trans</italic>-Golgi/<italic>trans</italic>-Golgi network
retained protein occurs in the Golgi complex and may be part of its
retention</article-title>. <source>J. Biol. Chem</source>.
<volume>270</volume>, <fpage>8815</fpage>–<lpage>8821</lpage><pub-id pub-id-type="doi">10.1074/jbc.270.15.8815</pub-id><pub-id pub-id-type="pmid">7721788</pub-id></mixed-citation></ref><ref id="B40"><label>40.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Heffernan</surname><given-names>L.
F.</given-names></name>, and
<name name-style="western"><surname>Simpson</surname><given-names>J.
C.</given-names></name></person-group> (<year>2014</year>)
<article-title>The trials and tubule-ations of Rab6 involvement in
Golgi-to-ER retrograde transport</article-title>. <source>Biochem. Soc.
Trans</source>. <volume>42</volume>,
<fpage>1453</fpage>–<lpage>1459</lpage><pub-id pub-id-type="doi">10.1042/BST20140178</pub-id><pub-id pub-id-type="pmid">25233431</pub-id></mixed-citation></ref><ref id="B41"><label>41.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Puthenveedu</surname><given-names>M.
A.</given-names></name>, and
<name name-style="western"><surname>Linstedt</surname><given-names>A.
D.</given-names></name></person-group> (<year>2005</year>)
<article-title>Subcompartmentalizing the Golgi apparatus</article-title>.
<source>Curr. Opin. Cell Biol</source>. <volume>17</volume>,
<fpage>369</fpage>–<lpage>375</lpage><pub-id pub-id-type="doi">10.1016/j.ceb.2005.06.006</pub-id><pub-id pub-id-type="pmid">15975779</pub-id></mixed-citation></ref><ref id="B42"><label>42.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Tu</surname><given-names>L.</given-names></name>, and
<name name-style="western"><surname>Banfield</surname><given-names>D.
K.</given-names></name></person-group> (<year>2010</year>)
<article-title>Localization of Golgi-resident
glycosyltransferases</article-title>. <source>Cell. Mol. Life Sci</source>.
<volume>67</volume>, <fpage>29</fpage>–<lpage>41</lpage><pub-id pub-id-type="doi">10.1007/s00018-009-0126-z</pub-id><pub-id pub-id-type="pmid">19727557</pub-id></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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