Comparative analysis of in vitro processivity of HIV-1 reverse transcriptases containing mutations 65R, 74V, 184V and 65R+74V

Sharma, P. L.; Nettles, James H.; Feldman, Anya; Rapp, Kimberly L.; Schinazi, Raymond F.

doi:10.1016/j.antiviral.2009.06.002

Cited by 12 publications

(13 citation statements)

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“…Instead, we show that the decrease in DNA polymerase activity of SGS3 RT is likely due to decreased processivity (Fig.1B). A decrease in the in vitro processivity of L74V HIV-1 RT has been documented previously (Deval et al, 2004 and Sharma et al, 2009). …”

Section: 0 Discussionsupporting

confidence: 71%

Molecular mechanism of HIV-1 resistance to 3′-azido-2′,3′-dideoxyguanosine

Meteer¹,

Schinazi²,

Mellors³

et al. 2014

Antiviral Research

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We reported that 3’-azido-2’,3’-dideoxyguanosine (3’-azido-ddG) selected for the L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain of HIV-1 reverse transcriptase (RT). In this study, we have defined the molecular mechanisms of 3’-azido-ddG resistance by performing in-depth biochemical analyses of HIV-1 RT containing mutations L74V, F77L, V106I, L214F, R277K and K476N (SGS3). The SGS3 HIV-1 RT was from a single-genome-derived full-length RT sequence obtained from 3’-azido-ddG resistant HIV-1 selected in vitro. We also analyzed two additional constructs that either lacked the L74V mutation (SGS3-L74V) or the K476N mutation (SGS3-K476N). Pre-steady-state kinetic experiments revealed that the L74V mutation allows RT to effectively discriminate between the natural nucleotide (dGTP) and 3’-azido-ddG-triphosphate (3’-azido-ddGTP). 3’-azido-ddGTP discrimination was primarily driven by a decrease in 3’-azido-ddGTP binding affinity (Kd) and not by a decreased rate of incorporation (kpol). The L74V mutation was found to severely impair RT’s ability to excise the chain-terminating 3’-azido-ddG-monophosphate (3’-azido-ddGMP) moiety. However, the K476N mutation partially restored the enzyme’s ability to excise 3’-azido-ddGMP on an RNA/DNA, but not on a DNA/DNA, template/primer by selectively decreasing the frequency of secondary RNase H cleavage events. Collectively, these data provide strong additional evidence that the nucleoside base structure is major determinant of HIV-1 resistance to the 3’-azido-2’,3’-dideoxynucleosides.

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Section: 0 Discussionsupporting

confidence: 71%

Molecular mechanism of HIV-1 resistance to 3′-azido-2′,3′-dideoxyguanosine

Meteer¹,

Schinazi²,

Mellors³

et al. 2014

Antiviral Research

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“…RT processivity assays were performed as described elsewhere [13,24,25]. Briefly, stock viruses supernatants containing 1500 to 3000 ng equivalent of antigen p24 were centrifuged at 16,000 rpm for 2 h at 4°C.…”

Section: Methodsmentioning

confidence: 99%

A Leu to Ile but not Leu to Val change at HIV-1 reverse transcriptase codon 74 in the background of K65R mutation leads to an increased processivity of K65R+L74I enzyme and a replication competent virus

Chunduri

Rimland

Nurpeisov

et al. 2011

Virol J

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BackgroundThe major hurdle in the treatment of Human Immunodeficiency virus type 1 (HIV-1) includes the development of drug resistance-associated mutations in the target regions of the virus. Since reverse transcriptase (RT) is essential for HIV-1 replication, several nucleoside analogues have been developed to target RT of the virus. Clinical studies have shown that mutations at RT codon 65 and 74 which are located in β3-β4 linkage group of finger sub-domain of RT are selected during treatment with several RT inhibitors, including didanosine, deoxycytidine, abacavir and tenofovir. Interestingly, the co-selection of K65R and L74V is rare in clinical settings. We have previously shown that K65R and L74V are incompatible and a R→K reversion occurs at codon 65 during replication of the virus. Analysis of the HIV resistance database has revealed that similar to K65R+L74V, the double mutant K65R+L74I is also rare. We sought to compare the impact of L→V versus L→I change at codon 74 in the background of K65R mutation, on the replication of doubly mutant viruses.MethodsProviral clones containing K65R, L74V, L74I, K65R+L74V and K65R+L74I RT mutations were created in pNL4-3 backbone and viruses were produced in 293T cells. Replication efficiencies of all the viruses were compared in peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication capacity (RC) of mutant viruses in relation to wild type was calculated on the basis of antigen p24 production and RT activity, and paired analysis by student t-test was performed among RCs of doubly mutant viruses. Reversion at RT codons 65 and 74 was monitored during replication in PBM cells. In vitro processivity of mutant RTs was measured to analyze the impact of amino acid changes at RT codon 74.ResultsReplication kinetics plot showed that all of the mutant viruses were attenuated as compared to wild type (WT) virus. Although attenuated in comparison to WT virus and single point mutants K65R, L74V and L74I; the double mutant K65R+L74I replicated efficiently in comparison to K65R+L74V mutant. The increased replication capacity of K65R+L74I viruses in comparison to K65R+L74V viruses was significant at multiplicity of infection 0.01 (p = 0.0004). Direct sequencing and sequencing after population cloning showed a more pronounced reversion at codon 65 in viruses containing K65R+L74V mutations in comparison to viruses with K65R+L74I mutations. In vitro processivity assays showed increased processivity of RT containing K65R+L74I in comparison to K65R+L74V RT.ConclusionsThe improved replication kinetics of K65R+L74I virus in comparison to K65R+L74V viruses was due to an increase in the processivity of RT containing K65R+L74I mutations. These observations support the rationale behind structural functional analysis to understand the interactions among unique RT mutations that may emerge during the treatment with specific drug regimens.

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“…L74V in an NL4-3 or HXBc2 background results in impaired replication capacity compared with WT and a more common NRTI mutation, M184V (Deval et al, 2004;Sharma & Crumpacker, 1999;Sharma et al, 2009). One defect reported for L74V RT is a reduced processivity of primer extension, as discovered from analysis of the purified RT.…”

Section: Discussionmentioning

confidence: 99%

“…One defect reported for L74V RT is a reduced processivity of primer extension, as discovered from analysis of the purified RT. However, a purified RT having M184V showed an even greater decrease in processivity than the L74V mutant, despite the ability of M184V virus to replicate better than an L74V virus in cell culture assays (Sharma & Crumpacker, 1999;Sharma et al, 2009). Additionally, conflicting reports did not find a significant difference in processivity between WT and L74V RTs, suggesting that there may be other biochemical mechanisms that contribute to the L74V fitness defect (Boyer et al, 1998;Caliendo et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

L74V increases the reverse transcriptase content of HIV-1 virions with non-nucleoside reverse transcriptase drug-resistant mutations L100I+K103N and K101E+G190S, which results in increased fitness

Wang

Bambara

et al. 2013

Journal of General Virology

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The fitness of non-nucleoside reverse transcriptase inhibitor (NNRTI) drug-resistant reverse transcriptase (RT) mutants of HIV-1 correlates with the amount of RT in the virions and the RNase H activity of the RT. We wanted to understand the mechanism by which secondary NNRTI-resistance mutations, L100I and K101E, and the nucleoside resistance mutation, L74V, alter the fitness of K103N and G190S viruses. We measured the amount of RT in virions and the polymerization and RNase H activities of mutant RTs compared to wild-type, K103N and G190S. We found that L100I, K101E and L74V did not change the polymerization or RNase H activities of K103N or G190S RTs. However, L100I and K101E reduced the amount of RT in the virions and subsequent addition of L74V restored RT levels back to those of G190S or K103N alone. We conclude that fitness changes caused by L100I, K101E and L74V derive from their effects on RT content.

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Comparative analysis of in vitro processivity of HIV-1 reverse transcriptases containing mutations 65R, 74V, 184V and 65R+74V

Cited by 12 publications

References 44 publications

Molecular mechanism of HIV-1 resistance to 3′-azido-2′,3′-dideoxyguanosine

Molecular mechanism of HIV-1 resistance to 3′-azido-2′,3′-dideoxyguanosine

A Leu to Ile but not Leu to Val change at HIV-1 reverse transcriptase codon 74 in the background of K65R mutation leads to an increased processivity of K65R+L74I enzyme and a replication competent virus

L74V increases the reverse transcriptase content of HIV-1 virions with non-nucleoside reverse transcriptase drug-resistant mutations L100I+K103N and K101E+G190S, which results in increased fitness

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