Fidelity analysis of HIV‐1 reverse transcriptase mutants with an altered amino‐acid sequence at residues Leu74, Glu89, Tyr115, Tyr183 and Met184

Jonckheere, Heidi; Clercq, Erik De; Anné, Jozef

doi:10.1046/j.1432-1327.2000.01272.x

Cited by 39 publications

(32 citation statements)

References 34 publications

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“…Part of this reorientation of the active site may be reflected in a change in the interaction between the amino acid at position 74 and the flipped out template base. This hypothesis is supported by a fidelity study that showed that RT L74V has an increased tendency to cause frameshift mutations (65). In a recent report, where similar resistance was observed to D4TTP by the V75T mutation, it was suggested that residues in the "template grip" region may interact with Gln 151 in a manner that can increase the selectivity of RT to nucleotide analogs (32).…”

Section: Role For the Hydrophobic Nature Of Cbvtp's Ribose Ring Inmentioning

confidence: 85%

Mechanistic Studies to Understand the Progressive Development of Resistance in Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Abacavir

Ray

Basavapathruni

Anderson

2002

Journal of Biological Chemistry

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Abacavir has been shown to select for multiple resistant mutations in the human immunodeficiency type 1 (HIV-1) pol gene. In an attempt to understand the molecular mechanism of resistance in response to abacavir, and nucleoside analogs in general, a set of reverse transcriptase mutants were studied to evaluate their kinetics of nucleotide incorporation and removal. It was found that, similar to the multidrug-resistant mutant reverse transcriptase (RT) Q151M, the mutations L74V, M184V, and a triple mutant containing L74V/Y115F/M184V all caused increased selectivity for dGTP over the active metabolite of abacavir (carbovir triphosphate). However, the magnitude of resistance observed in cell culture to abacavir in previous studies was less than that observed to other compounds. Our mechanistic studies suggest that this may be due to carbovir triphosphate decreasing the overall effect on its efficiency of incorporation by forming strong hydrophobic interactions in the RT active site. Unlike RT AZTR , no increase in the rate of ATP-or PP imediated chain terminator removal relative to RT WT could be detected for any of the mutants. However, marked decreases in the steady-state rate may serve as a mechanism for increased removal of a chain-terminating carbovir monophosphate by increasing the time spent at the primer terminus for some of the mutants studied. The triple mutant showed no advantage in selectivity over RT M184V and was severely impaired in its ability to remove a chain terminator, giving no kinetic basis for its increased resistance in a cellular system. Biochemical properties including percentage of active sites, fidelity, and processivity may suggest that the triple mutant's increased resistance to abacavir in cell culture is perhaps due to a fitness advantage, although further cellular studies are needed to verify this hypothesis. These data serve to further the understanding of how mutations in RT confer resistance to nucleoside analogs.

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Section: Role For the Hydrophobic Nature Of Cbvtp's Ribose Ring Inmentioning

confidence: 85%

Mechanistic Studies to Understand the Progressive Development of Resistance in Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Abacavir

Ray

Basavapathruni

Anderson

2002

Journal of Biological Chemistry

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“…The average viral titer obtained with wild-type HIV-1 RT was 1.7 ϫ 10 4 CFU/50 ng of p24 CA. The n-fold decrease in mutation frequency represents an average change in mutation frequency for mutants obtained in in vitro or in vivo forward mutation assays using the lacZ alphapeptide reporter gene (8,16,17,31,39,40,59,67,77; reviewed in reference 72). The following symbols for RT genotypes were used: ϩ, wild type; F, Q151N; E, Q151M; s, M184I; ᮀ, M184V; OE, F116Y; ‚, F116W; , Y115F; ƒ, L74V; ࡗ, K65R; छ, E89G.…”

Section: Discussionmentioning

confidence: 99%

Antiretroviral Drug Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Increase Template-Switching Frequency

Nikolenko

Svarovskaia

Delviks

et al. 2004

J Virol

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Template-switching events during reverse transcription are necessary for completion of retroviral replication and recombination. Structural determinants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that influence its template-switching frequency are not known. To identify determinants of HIV-1 RT that affect the frequency of template switching, we developed an in vivo assay in which RT template-switching events during viral replication resulted in functional reconstitution of the green fluorescent protein gene. A survey of single amino acid substitutions near the polymerase active site or deoxynucleoside triphosphate-binding site of HIV-1 RT indicated that several substitutions increased the rate of RT template switching. Several mutations associated with resistance to antiviral nucleoside analogs (K65R, L74V, E89G, Q151N, and M184I) dramatically increased RT template-switching frequencies by two-to sixfold in a single replication cycle. In contrast, substitutions in the RNase H domain (H539N, D549N) decreased the frequency of RT template switching by twofold. Depletion of intracellular nucleotide pools by hydroxyurea treatment of cells used as targets for infection resulted in a 1.8-fold increase in the frequency of RT template switching. These results indicate that the dynamic steady state between polymerase and RNase H activities is an important determinant of HIV-1 RT template switching and establish that HIV-1 recombination occurs by the previously described dynamic copy choice mechanism. These results also indicate that mutations conferring resistance to antiviral drugs can increase the frequency of RT template switching and may influence the rate of retroviral recombination and viral evolution.One of the most important mechanisms contributing to genetic variation and evolution of retroviral populations is recombination during viral replication (9, 74). As a result of recombination and selection pressure, a significant proportion of circulating human immunodeficiency virus type 1 (HIV-1) strains are recombinants (25,42,53,61,63,68). It was shown that recombination leads to the rapid emergence of viruses that are resistant to multiple antiviral drugs, ensuring their continued propagation (21,35,45,81). The HIV-1 recombination rate has been shown to be significantly higher than that of gammaretroviruses (30,60,84). In a single replication cycle, the HIV-1 recombination rate was reported to be 10-fold higher than that of spleen necrosis virus and murine leukemia virus (MLV) (4, 26, 30). The reasons for this difference in the rates of recombination are not well defined. Recombination occurs during reverse transcription as a result of reverse transcriptase (RT) switching templates between copackaged RNA molecules during viral DNA synthesis (26). Therefore, the frequency with which viral RNAs from different proviruses are copackaged as well as the template-switching properties of RT could influence the rate of recombination.It is not known whether viral RNAs derived from two differen...

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“…These differences could be due, in part, to the different mutation targets used. In this study, the entire lacZ gene was used as a mutation target, whereas in many of the cell-free fidelity studies, the lacZ␣ gene was used as a target (2,5,11,27,29,30,58,65). In cell-free systems, synthesis of one DNA strand is used to determine these rates.…”

Section: Discussionmentioning

confidence: 99%

“…The Q151N RT variant significantly decreased virus mutant frequencies while the K154A RT variant was not found to have a significant influence on virus mutant frequencies (Table 1). Intriguingly, another substitution in the dNTP binding site, Y115A, has been previously reported to decrease fidelity by a factor of 4 using the lacZ␣ gene (27). However, the Y115F and Y115V RT variants were found in lacZ␣ cell-free fidelity assays to have slightly lower error rates than that of wt RT (5).…”

Section: Analysis Of Hiv-1 Rt Single-amino-acid Variants On Virus Mutmentioning

confidence: 98%

Influence of Reverse Transcriptase Variants, Drugs, and Vpr on Human Immunodeficiency Virus Type 1 Mutant Frequencies

Mansky

Rouzic

Bénichou

et al. 2003

J Virol

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The evolution of drug resistance is a major complication of human immunodeficiency virus type 1 (HIV-1) chemotherapy. HIV-1 reverse transcriptase (RT) is a major target of antiretroviral therapy and ultimately the target of drug resistance mutations. Previous studies have indicated that drug-resistant HIV-1 RTs can alter HIV-1 mutant frequencies. In this study, we have tested a panel of HIV-1 RT variants for their ability to influence virus mutant frequencies. The RT variants tested included drug-resistant RT variants as well as other variants analyzed in enzyme fidelity studies with the lacZ␣ gene as a mutation target and/or implicated as being important for enzyme fidelity by structural studies. Combinations of mutations that alone had a statistically significant influence on virus mutant frequencies resulted in different mutant frequency phenotypes. Furthermore, when virus replication occurred in the presence of drugs [e.g., 3-azido-3-deoxythymidine, (؊)2/,3-dideoxy-3-thiacytidine, hydroxyurea, thymidine, or thioguanine] with selected RT variants, virus mutant frequencies increased. Similarly, Vpr variants deficient for binding to the uracil DNA glycosylase repair enzyme were observed to influence HIV-1 virus mutant frequencies when tested alone or in combination with RT variants. In summary, these observations indicate that HIV-1 mutant frequencies can significantly change by single amino acid substitutions in RT and that these effects can be altered by additional mutations in RT, by drugs, and/or by expression of Vpr variants. Such altered virus mutant frequencies could impact HIV-1 dynamics and evolution in small population sizes.

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Fidelity analysis of HIV‐1 reverse transcriptase mutants with an altered amino‐acid sequence at residues Leu74, Glu89, Tyr115, Tyr183 and Met184

Cited by 39 publications

References 34 publications

Mechanistic Studies to Understand the Progressive Development of Resistance in Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Abacavir

Mechanistic Studies to Understand the Progressive Development of Resistance in Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Abacavir

Antiretroviral Drug Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Increase Template-Switching Frequency

Influence of Reverse Transcriptase Variants, Drugs, and Vpr on Human Immunodeficiency Virus Type 1 Mutant Frequencies

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