The role of steric hindrance in 3TC resistance of human immunodeficiency virus type-1 reverse transcriptase
Identifieur interne : 001805 ( Istex/Corpus ); précédent : 001804; suivant : 001806The role of steric hindrance in 3TC resistance of human immunodeficiency virus type-1 reverse transcriptase
Auteurs : Hong-Qiang Gao ; Paul L. Boyer ; Stefan G. Sarafianos ; Edward Arnold ; Stephen H. HughesSource :
- Journal of Molecular Biology [ 0022-2836 ] ; 2000.
English descriptors
- KwdEn :
- 3TC, BSA, HIV-1, PR, RNase H, RT, drug resistance, dsDNA, reverse transcriptase.
- Teeft :
- 3tctp, 3tctp binding, Active site, Amino, Analog, Assay, Binary, Biol, Cleavage, Cognate, Cognate dntp, Dctp, Dideoxy, Dideoxy nucleoside, Dntp, Dntps, England biolabs, Feng anderson, Ftctp, Grice, Higher concentration, Higher concentrations, Higher salt conditions, Hindrance, Huang, Human virus, Human virus type, Loading buffer, Mgcl2, Multiple cleavages, Mutant, Mutant enzymes, Mutation, Natl acad, Normal dntp, Normal dntps, Nucleic, Nucleic acid, Nucleoside, Nucleoside analogs, Oligonucleotide, Oligonucleotides, Oxathiolane ring, Polymerase, Polymerase assays, Primary effect, Primer, Primer strand, Resistance figure, Ribose ring, Rnase, Room temperature, Salt concentrations, Shift assay, Shift assays, Steric, Steric hindrance, Template strand, Ternary, Transcriptase, Triphosphate, Various concentrations.
Abstract
Abstract: Treating HIV infections with drugs that block viral replication selects for drug-resistant strains of the virus. Particular inhibitors select characteristic resistance mutations. In the case of the nucleoside analogs 3TC and FTC, resistant viruses are selected with mutations at amino acid residue 184 of reverse transcriptase (RT). The initial change is usually to M184I; this virus is rapidly replaced by a variant carrying the mutation M184V. 3TC and FTC are taken up by cells and converted into 3TCTP and FTCTP. The triphosphate forms of these nucleoside analogs are incorporated into DNA by HIV-1 RT and act as chain terminators. Both of the mutations, M184I and M184V, provide very high levels of resistance in vivo; purified HIV-1 RT carrying M184V and M184I also shows resistance to 3TCTP and FTCTP in in vitro polymerase assays. Amino acid M184 is part of the dNTP binding site of HIV-1 RT. Structural studies suggest that the mechanism of resistance of HIV-1 RTs carrying the M184V or M184I mutation involves steric hindrance, which could either completely block the binding of 3TCTP and FTCTP or allow binding of these nucleoside triphosphate molecules but only in a configuration that would prevent incorporation. The available kinetic data are ambiguous: one group has reported that the primary effect of the mutations is at the level of 3TCTP binding; another, at the level of incorporation. We have approached this problem using assays that monitor the ability of HIV-1 RT to undergo a conformational change upon binding a dNTP. These studies show that both wild-type RT and the drug-resistant variants can bind 3TCTP at the polymerase active site; however, the binding to M184V and M184I is somewhat weaker and is sensitive to salt. We propose that the drug-resistant variants bind 3TCTP in a strained configuration that is salt-sensitive and is not catalytically competent.
Url:
DOI: 10.1006/jmbi.2000.3823
Links to Exploration step
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<front><div type="abstract" xml:lang="en">Abstract: Treating HIV infections with drugs that block viral replication selects for drug-resistant strains of the virus. Particular inhibitors select characteristic resistance mutations. In the case of the nucleoside analogs 3TC and FTC, resistant viruses are selected with mutations at amino acid residue 184 of reverse transcriptase (RT). The initial change is usually to M184I; this virus is rapidly replaced by a variant carrying the mutation M184V. 3TC and FTC are taken up by cells and converted into 3TCTP and FTCTP. The triphosphate forms of these nucleoside analogs are incorporated into DNA by HIV-1 RT and act as chain terminators. Both of the mutations, M184I and M184V, provide very high levels of resistance in vivo; purified HIV-1 RT carrying M184V and M184I also shows resistance to 3TCTP and FTCTP in in vitro polymerase assays. Amino acid M184 is part of the dNTP binding site of HIV-1 RT. Structural studies suggest that the mechanism of resistance of HIV-1 RTs carrying the M184V or M184I mutation involves steric hindrance, which could either completely block the binding of 3TCTP and FTCTP or allow binding of these nucleoside triphosphate molecules but only in a configuration that would prevent incorporation. The available kinetic data are ambiguous: one group has reported that the primary effect of the mutations is at the level of 3TCTP binding; another, at the level of incorporation. We have approached this problem using assays that monitor the ability of HIV-1 RT to undergo a conformational change upon binding a dNTP. These studies show that both wild-type RT and the drug-resistant variants can bind 3TCTP at the polymerase active site; however, the binding to M184V and M184I is somewhat weaker and is sensitive to salt. We propose that the drug-resistant variants bind 3TCTP in a strained configuration that is salt-sensitive and is not catalytically competent.</div>
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<abstract>Abstract: Treating HIV infections with drugs that block viral replication selects for drug-resistant strains of the virus. Particular inhibitors select characteristic resistance mutations. In the case of the nucleoside analogs 3TC and FTC, resistant viruses are selected with mutations at amino acid residue 184 of reverse transcriptase (RT). The initial change is usually to M184I; this virus is rapidly replaced by a variant carrying the mutation M184V. 3TC and FTC are taken up by cells and converted into 3TCTP and FTCTP. The triphosphate forms of these nucleoside analogs are incorporated into DNA by HIV-1 RT and act as chain terminators. Both of the mutations, M184I and M184V, provide very high levels of resistance in vivo; purified HIV-1 RT carrying M184V and M184I also shows resistance to 3TCTP and FTCTP in in vitro polymerase assays. Amino acid M184 is part of the dNTP binding site of HIV-1 RT. Structural studies suggest that the mechanism of resistance of HIV-1 RTs carrying the M184V or M184I mutation involves steric hindrance, which could either completely block the binding of 3TCTP and FTCTP or allow binding of these nucleoside triphosphate molecules but only in a configuration that would prevent incorporation. The available kinetic data are ambiguous: one group has reported that the primary effect of the mutations is at the level of 3TCTP binding; another, at the level of incorporation. We have approached this problem using assays that monitor the ability of HIV-1 RT to undergo a conformational change upon binding a dNTP. These studies show that both wild-type RT and the drug-resistant variants can bind 3TCTP at the polymerase active site; however, the binding to M184V and M184I is somewhat weaker and is sensitive to salt. We propose that the drug-resistant variants bind 3TCTP in a strained configuration that is salt-sensitive and is not catalytically competent.</abstract>
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<note>Edited by A. R. Fersht</note>
<note type="content">Section title: Regular article</note>
<note type="content">Figure 1: Effects of binding the cognate dNTP to the polymerase active site of HIV-1 RT on RNase H activity. RNase H assays were performed with a radioactively labeled 81-base RNA derived from the genome of HIV-1 (Gao et al., 1998). This RNA was hybridized with one of the four related DNA oligonucleotides that either have a normal 3′ nucleotide or a dideoxy 3′ nucleotide. The oligonucleotides are either 19 or 20 bases long and bind to positions (on the RNA) 34 to 52 (3452 and dd3452) or 33 to 52 (3352 and dd3352). Incubation times were varied between 0.25 minute and 16 minutes for each sample. The digests were fractionated on a 15 % polyacrylamide sequencing gel; the RNA fragments were detected by autoradiography. The sequences of the RNA template and the complementary DNA oligonucleotide were chosen so that, after RNase H digestion, all of label remains in the 5′ fragment simplifying the interpretation of the data (Gao et al., 1998). The migration of DNA markers was used to gauge the sizes of the products of RNA cleavage; although the sizes of the DNA markers and the RNA cleavage products do not correspond directly, we have calibrated their relative migration (Gao et al., 1998). When HIV-1 binds to nucleic acid, the polymerase active site is at the 3′-end of the primer, the RNase H active site is aligned with the template strand approximately 17 bases 3′ (on the template strand). This corresponds to the initial sites of RNase H cleavage, approximately 17 bases from the 3′-end of the primer. These produce RNA fragments that migrate on the gel between the 50-base and 60-base DNA oligonucleotide markers. In the absence of a cognate dNTP (final concentration 300 μM), longer incubation produced cleavages approximately −8 from the 3′-end of the primer. These are the RNA bands that migrate at positions between the 40-base and 50-base DNA markers. Note that the addition of the cognate base altered the position of the primary cleavage (compare dd3452 and dd3452+dCTP in the last eight lanes on the right).</note>
<note type="content">Figure 2: Effects of other nucleosides on RNase H cleavage. RNase H assays and sample analysis were done as described in the legend to Figure 1 and in Materials and Methods. (a) Effects of adding the cognate nucleoside (mono-, di- and triphosphate). RNase H digests were done with the substrate created with the dideoxy terminated 20-base DNA oligonucleotide (dd3352) in the presence of dCTP, dCDP, dCMP, and ddCTP (see Materials and Methods). (b) Effects of adding non-cognate nucleoside triphosphates.</note>
<note type="content">Figure 3: Effects of various concentrations of dCTP and 3TCTP on RNase H cleavage. The RNase H substrate was prepared with the dideoxy terminated 20-base oligonucleotide (dd3352) and the RNase H reactions were performed in the presence of various concentrations (0.3-300 μM) of dCTP or 3TCTP. The reactions were done and the cleavage products were analyzed as described in Materials and Methods and in the legend to Figure 1. (a)-(c) RNase H reactions were performed with wild-type RT or M184V or M184I in the presence of dCTP. (d)-(f) RNase H reactions were performed with wild-type HIV-1 RT or M184V or M184I in the presence of 3TCTP.</note>
<note type="content">Figure 4: Gel shift assay to measure the ability of dCTP and 3TCTP to form the closed complex. HIV-1 RT (either wild-type or the M184V mutant) was allowed to form a complex with a radioactively labeled DNA substrate in which the 3′-end of the primer strand was dideoxy terminated and various concentrations of nucleoside triphosphate, either dCTP or 3TCTP (see Materials and Methods). After a five minute incubation at room temperature, the salt level was increased to 100 mM KCl and an unlabeled chase substrate, poly(rC)oligo(dG), was added. The reactions were then incubated for five minutes at 37°C and fractioned on a 6 % Novex polyacrylamide DNA retardation gel. Autoradiographs of the gels run with reactions that contained wild-type HIV-1 RT (a) or the M184V mutant (b). (c) Graph of the data shown in (a) and (b). The radioactivity in the upper band on the gels shown in (a) and (b) was determined using a phosphorimager.</note>
<note type="content">Figure 5: Effects of higher salt concentrations on RNase H cleavage in the presence of dCTP or 3TCTP. The assays were similar to those shown in Figure 3, except that the buffer was adjusted to be similar to the buffer used in the gel shift assays shown in Figure 4 (see Materials and Methods). (a)-(c) RNase H assays were done either with wild-type HIV-1 RT or the M184V or M1841 mutants, in the presence of dCTP. (d)-(f) RNase H reactions were performed with wild-type HIV-1 RT or the M184V or M184I mutants in the presence of 3TCTP.</note>
<note type="content">Figure 6: Model for effects of steric hindrance on 3TCTP binding by the M184V variant. The model shows one of the active-site aspartate residues (Asp185) and the adjacent residue at position 184 (valine in the Figure). The β-branched side-chain of valine causes the oxathiolane ring of 3TCTP to move from the position it would occupy when bound to the wild-type enzyme (blue) to a new position (magenta). This increases the distance to between the 3′OH of the last base of the primer and the α-phosphate group of 3TCTP. The position of the fingers (the α-carbon backbone is shown as a tube, blue for wild-type RT, magenta for the mutant) is also affected.</note>
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<note type="biography">Present address: H.-Q. Gao, P. L. Boyer, and S. H. Hughes, HIV Drug Resistance Program, National Cancer Institute-FCRDC, P.O. Box B, Building 539, Room 130A, Frederick, MD 21702-1201, USA.</note>
<affiliation>Present address: H.-Q. Gao, P. L. Boyer, and S. H. Hughes, HIV Drug Resistance Program, National Cancer Institute-FCRDC, P.O. Box B, Building 539, Room 130A, Frederick, MD 21702-1201, USA.</affiliation>
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<note type="biography">Present address: H.-Q. Gao, P. L. Boyer, and S. H. Hughes, HIV Drug Resistance Program, National Cancer Institute-FCRDC, P.O. Box B, Building 539, Room 130A, Frederick, MD 21702-1201, USA.</note>
<affiliation>Present address: H.-Q. Gao, P. L. Boyer, and S. H. Hughes, HIV Drug Resistance Program, National Cancer Institute-FCRDC, P.O. Box B, Building 539, Room 130A, Frederick, MD 21702-1201, USA.</affiliation>
<affiliation>ABL-Basic Research Program NCI-Frederick Cancer Research and Development Center P.O. Box B, Frederick MD 21702-1201, USA</affiliation>
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<author xml:id="author-0002"><persName><forename type="first">Stefan G</forename>
<surname>Sarafianos</surname>
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<affiliation>Center for Advanced Biotechnology and Medicine (CABM) and Rutgers University Chemistry Department, 679 Hoes Lane Piscataway, NJ 08854-5638, USA</affiliation>
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<author xml:id="author-0003"><persName><forename type="first">Edward</forename>
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<note type="biography">Present address: H.-Q. Gao, P. L. Boyer, and S. H. Hughes, HIV Drug Resistance Program, National Cancer Institute-FCRDC, P.O. Box B, Building 539, Room 130A, Frederick, MD 21702-1201, USA.</note>
<note type="correspondence"><p>Corresponding author</p>
</note>
<affiliation>Present address: H.-Q. Gao, P. L. Boyer, and S. H. Hughes, HIV Drug Resistance Program, National Cancer Institute-FCRDC, P.O. Box B, Building 539, Room 130A, Frederick, MD 21702-1201, USA.</affiliation>
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<abstract xml:lang="en"><p>Abstract: Treating HIV infections with drugs that block viral replication selects for drug-resistant strains of the virus. Particular inhibitors select characteristic resistance mutations. In the case of the nucleoside analogs 3TC and FTC, resistant viruses are selected with mutations at amino acid residue 184 of reverse transcriptase (RT). The initial change is usually to M184I; this virus is rapidly replaced by a variant carrying the mutation M184V. 3TC and FTC are taken up by cells and converted into 3TCTP and FTCTP. The triphosphate forms of these nucleoside analogs are incorporated into DNA by HIV-1 RT and act as chain terminators. Both of the mutations, M184I and M184V, provide very high levels of resistance in vivo; purified HIV-1 RT carrying M184V and M184I also shows resistance to 3TCTP and FTCTP in in vitro polymerase assays. Amino acid M184 is part of the dNTP binding site of HIV-1 RT. Structural studies suggest that the mechanism of resistance of HIV-1 RTs carrying the M184V or M184I mutation involves steric hindrance, which could either completely block the binding of 3TCTP and FTCTP or allow binding of these nucleoside triphosphate molecules but only in a configuration that would prevent incorporation. The available kinetic data are ambiguous: one group has reported that the primary effect of the mutations is at the level of 3TCTP binding; another, at the level of incorporation. We have approached this problem using assays that monitor the ability of HIV-1 RT to undergo a conformational change upon binding a dNTP. These studies show that both wild-type RT and the drug-resistant variants can bind 3TCTP at the polymerase active site; however, the binding to M184V and M184I is somewhat weaker and is sensitive to salt. We propose that the drug-resistant variants bind 3TCTP in a strained configuration that is salt-sensitive and is not catalytically competent.</p>
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<item><term>HIV-1</term>
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<item><term>reverse transcriptase</term>
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<item><term>3TC</term>
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<item><term>drug resistance</term>
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<item><term>RNase H</term>
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<item><term>RT</term>
<term>reverse transcriptase</term>
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<item><term>PR</term>
<term>protease</term>
</item>
<item><term>HIV-1</term>
<term>human immunodeficiency virus type 1</term>
</item>
<item><term>dsDNA</term>
<term>double-stranded DNA</term>
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<item><term>BSA</term>
<term>bovine serum albumin</term>
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<ce:title>The role of steric hindrance in 3TC resistance of human immunodeficiency virus type-1 reverse transcriptase<ce:cross-ref refid="FN1"><ce:sup>1</ce:sup>
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<ce:footnote id="FN1"><ce:label>1</ce:label>
<ce:note-para><ce:bold><ce:italic>Edited by A. R. Fersht</ce:italic>
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<ce:author-group><ce:author><ce:given-name>Hong-Qiang</ce:given-name>
<ce:surname>Gao</ce:surname>
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<ce:author><ce:given-name>Paul L</ce:given-name>
<ce:surname>Boyer</ce:surname>
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<ce:cross-ref refid="FN2"><ce:sup>2</ce:sup>
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<ce:author><ce:given-name>Stefan G</ce:given-name>
<ce:surname>Sarafianos</ce:surname>
<ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup>
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</ce:author>
<ce:author><ce:given-name>Edward</ce:given-name>
<ce:surname>Arnold</ce:surname>
<ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup>
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</ce:author>
<ce:author><ce:given-name>Stephen H</ce:given-name>
<ce:surname>Hughes</ce:surname>
<ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup>
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<ce:cross-ref refid="FN2"><ce:sup>2</ce:sup>
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<ce:cross-ref refid="COR1">*</ce:cross-ref>
<ce:e-address>hughes@NCIFCRF.GOV</ce:e-address>
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<ce:affiliation id="AFF1"><ce:label>1</ce:label>
<ce:textfn>ABL-Basic Research Program NCI-Frederick Cancer Research and Development Center P.O. Box B, Frederick MD 21702-1201, USA</ce:textfn>
</ce:affiliation>
<ce:affiliation id="AFF2"><ce:label>2</ce:label>
<ce:textfn>Center for Advanced Biotechnology and Medicine (CABM) and Rutgers University Chemistry Department, 679 Hoes Lane Piscataway, NJ 08854-5638, USA</ce:textfn>
</ce:affiliation>
<ce:correspondence id="COR1"><ce:label>*</ce:label>
<ce:text>Corresponding author</ce:text>
</ce:correspondence>
<ce:footnote id="FN2"><ce:label>2</ce:label>
<ce:note-para>Present address: H.-Q. Gao, P. L. Boyer, and S. H. Hughes, HIV Drug Resistance Program, National Cancer Institute-FCRDC, P.O. Box B, Building 539, Room 130A, Frederick, MD 21702-1201, USA.</ce:note-para>
</ce:footnote>
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<ce:abstract><ce:section-title>Abstract</ce:section-title>
<ce:abstract-sec><ce:simple-para>Treating HIV infections with drugs that block viral replication selects for drug-resistant strains of the virus. Particular inhibitors select characteristic resistance mutations. In the case of the nucleoside analogs 3TC and FTC, resistant viruses are selected with mutations at amino acid residue 184 of reverse transcriptase (RT). The initial change is usually to M184I; this virus is rapidly replaced by a variant carrying the mutation M184V. 3TC and FTC are taken up by cells and converted into 3TCTP and FTCTP. The triphosphate forms of these nucleoside analogs are incorporated into DNA by HIV-1 RT and act as chain terminators. Both of the mutations, M184I and M184V, provide very high levels of resistance <ce:italic>in vivo</ce:italic>
; purified HIV-1 RT carrying M184V and M184I also shows resistance to 3TCTP and FTCTP in <ce:italic>in vitro</ce:italic>
polymerase assays. Amino acid M184 is part of the dNTP binding site of HIV-1 RT. Structural studies suggest that the mechanism of resistance of HIV-1 RTs carrying the M184V or M184I mutation involves steric hindrance, which could either completely block the binding of 3TCTP and FTCTP or allow binding of these nucleoside triphosphate molecules but only in a configuration that would prevent incorporation. The available kinetic data are ambiguous: one group has reported that the primary effect of the mutations is at the level of 3TCTP binding; another, at the level of incorporation. We have approached this problem using assays that monitor the ability of HIV-1 RT to undergo a conformational change upon binding a dNTP. These studies show that both wild-type RT and the drug-resistant variants can bind 3TCTP at the polymerase active site; however, the binding to M184V and M184I is somewhat weaker and is sensitive to salt. We propose that the drug-resistant variants bind 3TCTP in a strained configuration that is salt-sensitive and is not catalytically competent.</ce:simple-para>
</ce:abstract-sec>
</ce:abstract>
<ce:keywords><ce:section-title>Keywords</ce:section-title>
<ce:keyword><ce:text>HIV-1</ce:text>
</ce:keyword>
<ce:keyword><ce:text>reverse transcriptase</ce:text>
</ce:keyword>
<ce:keyword><ce:text>3TC</ce:text>
</ce:keyword>
<ce:keyword><ce:text>drug resistance</ce:text>
</ce:keyword>
<ce:keyword><ce:text>RNase H</ce:text>
</ce:keyword>
</ce:keywords>
<ce:keywords class="abr"><ce:section-title>Abbreviations</ce:section-title>
<ce:keyword><ce:text>RT</ce:text>
<ce:keyword><ce:text>reverse transcriptase</ce:text>
</ce:keyword>
</ce:keyword>
<ce:keyword><ce:text>PR</ce:text>
<ce:keyword><ce:text>protease</ce:text>
</ce:keyword>
</ce:keyword>
<ce:keyword><ce:text>HIV-1</ce:text>
<ce:keyword><ce:text>human immunodeficiency virus type 1</ce:text>
</ce:keyword>
</ce:keyword>
<ce:keyword><ce:text>dsDNA</ce:text>
<ce:keyword><ce:text>double-stranded DNA</ce:text>
</ce:keyword>
</ce:keyword>
<ce:keyword><ce:text>BSA</ce:text>
<ce:keyword><ce:text>bovine serum albumin</ce:text>
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<name type="personal"><namePart type="given">Stephen H</namePart>
<namePart type="family">Hughes</namePart>
<affiliation>E-mail: hughes@NCIFCRF.GOV</affiliation>
<affiliation>ABL-Basic Research Program NCI-Frederick Cancer Research and Development Center P.O. Box B, Frederick MD 21702-1201, USA</affiliation>
<description>Corresponding author</description>
<description>Present address: H.-Q. Gao, P. L. Boyer, and S. H. Hughes, HIV Drug Resistance Program, National Cancer Institute-FCRDC, P.O. Box B, Building 539, Room 130A, Frederick, MD 21702-1201, USA.</description>
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<dateIssued encoding="w3cdtf">2000</dateIssued>
<dateModified encoding="w3cdtf">2000-04-25</dateModified>
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<abstract lang="en">Abstract: Treating HIV infections with drugs that block viral replication selects for drug-resistant strains of the virus. Particular inhibitors select characteristic resistance mutations. In the case of the nucleoside analogs 3TC and FTC, resistant viruses are selected with mutations at amino acid residue 184 of reverse transcriptase (RT). The initial change is usually to M184I; this virus is rapidly replaced by a variant carrying the mutation M184V. 3TC and FTC are taken up by cells and converted into 3TCTP and FTCTP. The triphosphate forms of these nucleoside analogs are incorporated into DNA by HIV-1 RT and act as chain terminators. Both of the mutations, M184I and M184V, provide very high levels of resistance in vivo; purified HIV-1 RT carrying M184V and M184I also shows resistance to 3TCTP and FTCTP in in vitro polymerase assays. Amino acid M184 is part of the dNTP binding site of HIV-1 RT. Structural studies suggest that the mechanism of resistance of HIV-1 RTs carrying the M184V or M184I mutation involves steric hindrance, which could either completely block the binding of 3TCTP and FTCTP or allow binding of these nucleoside triphosphate molecules but only in a configuration that would prevent incorporation. The available kinetic data are ambiguous: one group has reported that the primary effect of the mutations is at the level of 3TCTP binding; another, at the level of incorporation. We have approached this problem using assays that monitor the ability of HIV-1 RT to undergo a conformational change upon binding a dNTP. These studies show that both wild-type RT and the drug-resistant variants can bind 3TCTP at the polymerase active site; however, the binding to M184V and M184I is somewhat weaker and is sensitive to salt. We propose that the drug-resistant variants bind 3TCTP in a strained configuration that is salt-sensitive and is not catalytically competent.</abstract>
<note type="footnote">Edited by A. R. Fersht</note>
<note type="content">Section title: Regular article</note>
<note type="content">Figure 1: Effects of binding the cognate dNTP to the polymerase active site of HIV-1 RT on RNase H activity. RNase H assays were performed with a radioactively labeled 81-base RNA derived from the genome of HIV-1 (Gao et al., 1998). This RNA was hybridized with one of the four related DNA oligonucleotides that either have a normal 3′ nucleotide or a dideoxy 3′ nucleotide. The oligonucleotides are either 19 or 20 bases long and bind to positions (on the RNA) 34 to 52 (3452 and dd3452) or 33 to 52 (3352 and dd3352). Incubation times were varied between 0.25 minute and 16 minutes for each sample. The digests were fractionated on a 15 % polyacrylamide sequencing gel; the RNA fragments were detected by autoradiography. The sequences of the RNA template and the complementary DNA oligonucleotide were chosen so that, after RNase H digestion, all of label remains in the 5′ fragment simplifying the interpretation of the data (Gao et al., 1998). The migration of DNA markers was used to gauge the sizes of the products of RNA cleavage; although the sizes of the DNA markers and the RNA cleavage products do not correspond directly, we have calibrated their relative migration (Gao et al., 1998). When HIV-1 binds to nucleic acid, the polymerase active site is at the 3′-end of the primer, the RNase H active site is aligned with the template strand approximately 17 bases 3′ (on the template strand). This corresponds to the initial sites of RNase H cleavage, approximately 17 bases from the 3′-end of the primer. These produce RNA fragments that migrate on the gel between the 50-base and 60-base DNA oligonucleotide markers. In the absence of a cognate dNTP (final concentration 300 μM), longer incubation produced cleavages approximately −8 from the 3′-end of the primer. These are the RNA bands that migrate at positions between the 40-base and 50-base DNA markers. Note that the addition of the cognate base altered the position of the primary cleavage (compare dd3452 and dd3452+dCTP in the last eight lanes on the right).</note>
<note type="content">Figure 2: Effects of other nucleosides on RNase H cleavage. RNase H assays and sample analysis were done as described in the legend to Figure 1 and in Materials and Methods. (a) Effects of adding the cognate nucleoside (mono-, di- and triphosphate). RNase H digests were done with the substrate created with the dideoxy terminated 20-base DNA oligonucleotide (dd3352) in the presence of dCTP, dCDP, dCMP, and ddCTP (see Materials and Methods). (b) Effects of adding non-cognate nucleoside triphosphates.</note>
<note type="content">Figure 3: Effects of various concentrations of dCTP and 3TCTP on RNase H cleavage. The RNase H substrate was prepared with the dideoxy terminated 20-base oligonucleotide (dd3352) and the RNase H reactions were performed in the presence of various concentrations (0.3-300 μM) of dCTP or 3TCTP. The reactions were done and the cleavage products were analyzed as described in Materials and Methods and in the legend to Figure 1. (a)-(c) RNase H reactions were performed with wild-type RT or M184V or M184I in the presence of dCTP. (d)-(f) RNase H reactions were performed with wild-type HIV-1 RT or M184V or M184I in the presence of 3TCTP.</note>
<note type="content">Figure 4: Gel shift assay to measure the ability of dCTP and 3TCTP to form the closed complex. HIV-1 RT (either wild-type or the M184V mutant) was allowed to form a complex with a radioactively labeled DNA substrate in which the 3′-end of the primer strand was dideoxy terminated and various concentrations of nucleoside triphosphate, either dCTP or 3TCTP (see Materials and Methods). After a five minute incubation at room temperature, the salt level was increased to 100 mM KCl and an unlabeled chase substrate, poly(rC)oligo(dG), was added. The reactions were then incubated for five minutes at 37°C and fractioned on a 6 % Novex polyacrylamide DNA retardation gel. Autoradiographs of the gels run with reactions that contained wild-type HIV-1 RT (a) or the M184V mutant (b). (c) Graph of the data shown in (a) and (b). The radioactivity in the upper band on the gels shown in (a) and (b) was determined using a phosphorimager.</note>
<note type="content">Figure 5: Effects of higher salt concentrations on RNase H cleavage in the presence of dCTP or 3TCTP. The assays were similar to those shown in Figure 3, except that the buffer was adjusted to be similar to the buffer used in the gel shift assays shown in Figure 4 (see Materials and Methods). (a)-(c) RNase H assays were done either with wild-type HIV-1 RT or the M184V or M1841 mutants, in the presence of dCTP. (d)-(f) RNase H reactions were performed with wild-type HIV-1 RT or the M184V or M184I mutants in the presence of 3TCTP.</note>
<note type="content">Figure 6: Model for effects of steric hindrance on 3TCTP binding by the M184V variant. The model shows one of the active-site aspartate residues (Asp185) and the adjacent residue at position 184 (valine in the Figure). The β-branched side-chain of valine causes the oxathiolane ring of 3TCTP to move from the position it would occupy when bound to the wild-type enzyme (blue) to a new position (magenta). This increases the distance to between the 3′OH of the last base of the primer and the α-phosphate group of 3TCTP. The position of the fingers (the α-carbon backbone is shown as a tube, blue for wild-type RT, magenta for the mutant) is also affected.</note>
<subject><genre>article-category</genre>
<topic>Regular article</topic>
</subject>
<subject lang="en"><genre>Keywords</genre>
<topic>HIV-1</topic>
<topic>reverse transcriptase</topic>
<topic>3TC</topic>
<topic>drug resistance</topic>
<topic>RNase H</topic>
</subject>
<subject lang="en"><genre>Abbreviations</genre>
<topic>RT : reverse transcriptase</topic>
<topic>PR : protease</topic>
<topic>HIV-1 : human immunodeficiency virus type 1</topic>
<topic>dsDNA : double-stranded DNA</topic>
<topic>BSA : bovine serum albumin</topic>
</subject>
<relatedItem type="host"><titleInfo><title>Journal of Molecular Biology</title>
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<dateIssued encoding="w3cdtf">2000</dateIssued>
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<identifier type="ISSN">0022-2836</identifier>
<identifier type="PII">S0022-2836(00)X0245-8</identifier>
<part><date>2000</date>
<detail type="volume"><number>300</number>
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<detail type="issue"><number>2</number>
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<extent unit="issue-pages"><start>235</start>
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<identifier type="DOI">10.1006/jmbi.2000.3823</identifier>
<identifier type="PII">S0022-2836(00)93823-3</identifier>
<accessCondition type="use and reproduction" contentType="copyright">©2000 Academic Press</accessCondition>
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