Structure of a Covalently Trapped Catalytic Complex of HIV-1 Reverse Transcriptase: Implications for Drug Resistance

Huang, Huifang; Chopra, Rajiv; Verdine, Gregory L.; Harrison, Stephen C.

doi:10.1126/science.282.5394.1669

Cited by 1,393 publications

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References 45 publications

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“…The numbers at the beginning of the sequences indicate the number of residues from the N-terminus of the RT-RH omitted from the Figure. minal nucleotide of the primer (the cleaved labelled RNA fragment has a length of 18 nt). In comparison, HIV-1 enzyme cleaved the RNA between positions 18 and 19, in keeping with biochemical and crystallographic data, indicating that, in HIV-1 RT, the distance between the positions of RH cleavage and DNA polymerase active sites is 18-20 bp [20][21][22][23][24][25][26][27]. The effect of primer extension on RH cleavage was examined ( Figure 5B, ' jdNTP ').…”

Section: Figure 6 Sequence Alignment Of Ty1 and Hiv-1 Rtsmentioning

confidence: 56%

Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase–RNase H domain exhibits polymerase and RNase H activities

Wilhelm

Boutabout

Wilhelm³

2000

Biochemical Journal

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Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl2, NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.

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Section: Figure 6 Sequence Alignment Of Ty1 and Hiv-1 Rtsmentioning

confidence: 56%

Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase–RNase H domain exhibits polymerase and RNase H activities

Wilhelm

Boutabout

Wilhelm³

2000

Biochemical Journal

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“…Residues K65 and K70 both reside in the ␤3-␤4 loop in the "fingers" subdomain of the 66-kDa subunit of HIV-1 RT. In the crystal structure of the ternary HIV-1 RT-T/P-TTP and RT-T/P-TNV-DP complexes, the -amino group of K65 interacts with the ␥-phosphate of the bound nucleotide substrate (13,31). Modeling studies suggest that the K65R mutation in HIV-1 RT prevents optimal positioning of the NRTI-TP in the active site, which results in decreased catalytic efficiency of incorporation (27,29).…”

Section: Discussionmentioning

confidence: 99%

Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

Sluis‐Cremer

Sheen

Zelina

et al. 2007

Antimicrob Agents Chemother

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The K70E mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has become more prevalent in clinical samples, particularly in isolates derived from patients for whom triple-nucleoside regimens that include tenofovir (TNV), abacavir, and lamivudine (3TC) failed. To elucidate the molecular mechanism by which this mutation confers resistance to these nucleoside RT inhibitors (NRTI), we conducted detailed biochemical analyses comparing wild-type (WT), K70E, and K65R HIV-1 RT. Pre-steady-state kinetic experiments demonstrate that the K70E mutation in HIV-1 RT allows the enzyme to discriminate between the natural deoxynucleoside triphosphate substrate and the NRTI triphosphate (NRTI-TP). Compared to the WT enzyme, K70E RT showed 2.1-, 2.3-, and 3.5-foldhigher levels of resistance toward TNV-diphosphate, carbovir-TP, and 3TC-TP, respectively. By comparison, K65R RT demonstrated 12.4-, 12.0-, and 13.1-fold-higher levels of resistance, respectively, toward the same analogs. NRTI-TP discrimination by the K70E (and K65R) mutation was primarily due to decreased rates of NRTI-TP incorporation and not to changes in analog binding affinity. The K65R and K70E mutations also profoundly impaired the ability of RT to excise 3 -azido-2 ,3 -dideoxythymidine monophosphate (AZT-MP) and other NRTI-MP from the 3 end of a chain-terminated primer. When introduced into an enzyme with the thymidine analog mutations (TAMs) M41L, L210W, and T215Y, the K70E mutation inhibited ATP-mediated excision of AZT-MP. Taken together, these findings indicate that the K70E mutation, like the K65R mutation, reduces susceptibility to NRTI by selectively decreasing NRTI-TP incorporation and is antagonistic to TAM-mediated nucleotide excision.

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“…The recently described structure of the catalytic complex of RT with a deoxynucleoside triphosphate template and primer sheds new insight on the possible way that nucleoside analog mutations confer resistance (8). Residue 215 is located near the incoming nucleotide-binding site.…”

Section: Discussionmentioning

confidence: 99%

A Novel Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutational Pattern Confers Phenotypic Lamivudine Resistance in the Absence of Mutation 184V

Hertogs¹,

Bloor²,

Vroey³

et al. 2000

Antimicrob Agents Chemother

108

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We describe a new human immunodeficiency virus type 1 (HIV-1) mutational pattern associated with phenotypic resistance to lamivudine (3TC) in the absence of the characteristic replacement of methionine by valine at position 184 (M184V) of reverse transcriptase. Combined genotypic and phenotypic analyses of clinical isolates revealed the presence of moderate levels of phenotypic resistance (between 4-and 50-fold) to 3TC in a subset of isolates that did not harbor the M184V mutation. Mutational cluster analysis and comparison with the phenotypic data revealed a significant correlation between moderate phenotypic 3TC resistance and an increased incidence of replacement of glutamic acid by aspartic acid or alanine and of valine by isoleucine at residues 44 and 118 of reverse transcriptase, respectively. This occurred predominantly in those isolates harboring zidovudine resistance-associated mutations (41L, 215Y). The requirement of the combination of mutations 41L and 215Y with mutations 44D and 44A and/or 118I for phenotypic 3TC resistance was confirmed by site-directed mutagenesis experiments. These data support the assumption that HIV-1 may have access to several different genetic pathways to escape drug pressure or that the increase in the frequency of particular mutations may affect susceptibility to drugs that have never been part of a particular regimen.The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) variants is almost always observed during the course of treatment of patients with antiretroviral drugs (3, 10, 14-16, 18, 21, 27 Strategies, abstr. 19, p. 15, 1998). The mutational profile of the resistant viruses generally is characteristic for the particular drug(s) taken. For example, mutations at codons 41, 67, 70, 210, 215, and 219 of reverse transcriptase (RT) typically confer resistance to zidovudine (ZDV) (6,12,13,27). Similarly, mutation M184V in RT has been shown to be specifically associated with high-level (Ն50-fold) phenotypic resistance to lamivudine (3TC) (1,22,28). No "specific" mutation(s) associated with moderate levels of phenotypic resistance (4-to Ͻ50-fold) to 3TC has been described before. Those mutations that confer moderate (4-to Ͻ50-fold) levels of phenotypic resistance to 3TC reported previously always appeared in the context of a constellation of mutations that confer resistance to multiple nucleoside analogues or as a cross-resistance phenomenon that appears with the emergence of resistance to another nucleoside analogue. This has been the case for the nucleoside multidrug resistance complex of mutations Q151M, F77L, F116Y, A62V, and V75I, although the increase in the level of phenotypic resistance to 3TC in viruses that harbor those mutations is slight (9,20,24,25). In the case of the insertion mutations near position 69 of RT, a notable increase in the frequency of 3TC resistance has been reported together with an increased frequency of phenotypic resistance to other nucleosides (2, 17, 29). The K65R mutation appears infrequently during the course of tr...

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Structure of a Covalently Trapped Catalytic Complex of HIV-1 Reverse Transcriptase: Implications for Drug Resistance

Cited by 1,393 publications

References 45 publications

Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase–RNase H domain exhibits polymerase and RNase H activities

Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase–RNase H domain exhibits polymerase and RNase H activities

Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

A Novel Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutational Pattern Confers Phenotypic Lamivudine Resistance in the Absence of Mutation 184V

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