Mutational studies of human immunodeficiency virus type 1 reverse transcriptase : the involvement of residues 183 and 184 in the fidelity of DNA synthesis

Bakhanashvili, Mary; Avidan, Orna; Hizi, Amnon

doi:10.1016/0014-5793(96)00747-8

Cited by 47 publications

(53 citation statements)

References 38 publications

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“…All our previous studies with a variety of RTs have indicated that the fidelity parameters (i.e. misinsertion and mispair extension) depend primarily on the sequences of nucleic acids copied, rather than on whether DNA or RNA were copied [15,17,20,23]. Therefore, in the present study we have analyzed only DNA substrates as representing both DNA and RNA.…”

Section: Resultsmentioning

confidence: 99%

“…We have also found that the relatively low fidelity of DNA synthesis exhibited by HIV RTs is shared by the RT of EIAV, that belongs to the lentivirus subfamily of retroviruses. Using parameters of both the capacity to incorporate a wrong nucleotide of the 3′ end of the nascent DNA strand and the ability to extend the preformed 3′ mispairs, it was suggested that the overall rates of error proneness in RTs is as follows : lentiviral RTs Ͼ AMV RT Ͼ MLV RT [14,15,17,20,30].…”

Section: Discussionmentioning

confidence: 99%

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DNA synthesis exhibited by the reverse transcriptase of mouse mammary tumor virus : Processivity and fidelity of misinsertion and mispair extension

Taube

Avidan

Bakhanashvili

et al. 1998

European Journal of Biochemistry

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We have recently expressed in bacteria an enzymatically active reverse transcriptase (RT) of mouse mammary tumor virus (MMTV), a mammalian retrovirus with a typical B-type morphology [Taube, R., Loya, S., Avidan, O., Perach, M. & Hizi, A. (1998) Biochemical J. 329, 579Ϫ587]. The purified recombinant protein was shown to possess the catalytic activities characteristic of retroviral reverse transcriptases. In the present study, we have analyzed two basic parameters characteristic of the DNA polymerase activity of the novel MMTV RT, namely the processivity and the fidelity of DNA synthesis. Two features related to fidelity were studied, the capacity to misinsert wrong nucleotides at the 3′ end of the nascent DNA strand and the ability to extend 3′ mispairs. The studied properties of MMTV RT were compared with those of the RT purified from virions of avian myeloblastosis virus (AMV), since AMV RT shows a relatively high sequence similarity to MMTV RT. MMTV RT shows a relative processivity of DNA synthesis which is as high as the reference AMV RT. Regarding fidelity of DNA synthesis, MMTV RT shows a fidelity of misinsertion lower than that of AMV RT, whereas its capacity to elongate mispaired DNA is lower than that of AMV RT indicating a somewhat higher fidelity. These fidelity properties are discussed also in the context of the RTs of lentiviruses, especially those of HIV, which were reported to exhibit an exceptionally low fidelity of DNA synthesis. It is clear that MMTV RT has a fidelity higher than that of lentiviral RTs.Keywords : mouse mammary tumor virus; avian myeloblastosis virus; reverse transcriptase ; DNA synthesis; processivity.The replication of retroviruses is initiated by the reverse transcription of the viral single-stranded RNA genome into a double-stranded linear DNA, which is subsequently integrated into the host cell chromosomal DNA. This critical step in the early life cycle of the viruses, is catalyzed by the viral reverse transcriptase (RT). The plus-strand viral RNA is copied by the RNA-dependent DNA polymerase activity of RT, producing RNA · DNA hybrids. Subsequently, the ribonuclease H activity of the RT specifically degrades the viral RNA in the RNA · DNA heteroduplex. Finally, a double-stranded linear DNA is obtained by the synthesis of the second DNA strand, catalyzed by DNA-dependent DNA polymerase activity of the same enzyme [1Ϫ4].Mouse mammary tumor virus (MMTV) is unique among retroviruses in its ability to replicate and function as a carcinogenic agent in mammary epithelial cells, resulting from transcriptional regulation by steroid hormones. Moreover, MMTV is the only infectious retrovirus with a distinct type-B morphology that was isolated both as an endogenous and an exogenous virus [5Ϫ8]. Therefore, it was of interest to study the reverse transcriptase of this retrovirus. We have recently expressed in bacteria the enzymatically active recombinant RT derived from the BR6 strain of milk-transmitted MMTV [9] by constructing a plasmid that induces in bacteria the synthesis of this e...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

DNA synthesis exhibited by the reverse transcriptase of mouse mammary tumor virus : Processivity and fidelity of misinsertion and mispair extension

Taube

Avidan

Bakhanashvili

et al. 1998

European Journal of Biochemistry

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“…In HIV-1, mutations M184V, K65R, and Q151M were shown to reduce the polymerase activity of the RT enzyme and increase the fidelity of reverse transcription (41,(47)(48)(49)(50)(51). A previous study showed that Q151M in the HIV-2 RT enzyme also results in reduced polymerase activity (16).…”

Section: Figmentioning

confidence: 99%

Mutation V111I in HIV-2 Reverse Transcriptase Increases the Fitness of the Nucleoside Analogue-Resistant K65R and Q151M Viruses

Deuzing

Charpentier

Wright

et al. 2015

J Virol

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Infection with HIV-2 can ultimately lead to AIDS, although disease progression is much slower than with HIV-1. HIV-2 patients are mostly treated with a combination of nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and protease inhibitors designed for HIV-1. Many studies have described the development of HIV-1 resistance to NRTIs and identified mutations in the polymerase domain of RT. Recent studies have shown that mutations in the connection and RNase H domains of HIV-1 RT may also contribute to resistance. However, only limited information exists regarding the resistance of HIV-2 to NRTIs. In this study, therefore, we analyzed the polymerase, connection, and RNase H domains of RT in HIV-2 patients failing NRTI-containing therapies. Besides the key resistance mutations K65R, Q151M, and M184V, we identified a novel mutation, V111I, in the polymerase domain. This mutation was significantly associated with mutations K65R and Q151M. Sequencing of the connection and RNase H domains of the HIV-2 patients did not reveal any of the mutations that were reported to contribute to NRTI resistance in HIV-1. We show that V111I does not strongly affect drug susceptibility but increases the replication capacity of the K65R and Q151M viruses. Biochemical assays demonstrate that V111I restores the polymerization defects of the K65R and Q151M viruses but negatively affects the fidelity of the HIV-2 RT enzyme. Molecular dynamics simulations were performed to analyze the structural changes mediated by V111I. This showed that V111I changed the flexibility of the 110-to-115 loop region, which may affect deoxynucleoside triphosphate (dNTP) binding and polymerase activity. IMPORTANCEMutation V111I in the HIV-2 reverse transcriptase enzyme was identified in patients failing therapies containing nucleoside analogues. We show that the V111I change does not strongly affect the sensitivity of HIV-2 to nucleoside analogues but increases the fitness of viruses with drug resistance mutations K65R and Q151M.

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“…For example, Rous sarcoma virus RT contains the YMDD motif, the RTs of retroelements such as 297 (Gypsy-like group) and Int 32 (Line-like group) contain both the YLDD and YADD sequences, and the poliovirus RNA polymerase contains the YGDD motif (41). Mutations in the YXDD motif can abolish enzymatic activity and alter the processivity and fidelity of RT (3,6,21,25,37). The prevalent mutation in HIV-1 RT associated with 3TC resistance is the M184V substitution (16,46,53), which confers a level of resistance 1,000 times greater than that displayed by the wildtype enzyme (16,53).…”

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confidence: 99%

Wild-Type and YMDD Mutant Murine Leukemia Virus Reverse Transcriptases Are Resistant to 2′,3′-Dideoxy-3′-Thiacytidine

Halvas

Svarovskaia

Freed

et al. 2000

J Virol

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The antiretroviral nucleoside analog 2,3-dideoxy-3-thiacytidine (3TC) is a potent inhibitor of wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). A methionine-to-valine or methionine-to-isoleucine substitution at residue 184 in the HIV-1 YMDD motif, which is located at the RT active site, leads to a high level of resistance to 3TC. We sought to determine whether 3TC can inhibit the replication of wild-type murine leukemia virus (MLV), which contains V223 at the YVDD active site motif of the MLV RT, and of the V223M, V223I, V223A, and V223S mutant RTs. Surprisingly, the wild type and all four of the V223 mutants of MLV RT were highly resistant to 3TC. These results indicate that determinants outside the YVDD motif of MLV RT confer a high level of resistance to 3TC. Therefore, structural differences among similar RTs might result in widely divergent sensitivities to antiretroviral nucleoside analogs.

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Mutational studies of human immunodeficiency virus type 1 reverse transcriptase : the involvement of residues 183 and 184 in the fidelity of DNA synthesis

Cited by 47 publications

References 38 publications

DNA synthesis exhibited by the reverse transcriptase of mouse mammary tumor virus : Processivity and fidelity of misinsertion and mispair extension

DNA synthesis exhibited by the reverse transcriptase of mouse mammary tumor virus : Processivity and fidelity of misinsertion and mispair extension

Mutation V111I in HIV-2 Reverse Transcriptase Increases the Fitness of the Nucleoside Analogue-Resistant K65R and Q151M Viruses

Wild-Type and YMDD Mutant Murine Leukemia Virus Reverse Transcriptases Are Resistant to 2′,3′-Dideoxy-3′-Thiacytidine

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