The influence of 3TC resistance mutation M184I on the fidelity and error specificity of human immunodeficiency virus type 1 reverse transcriptase

Rezende, Lisa F.; Drosopoulos, William C.; Prasad, Vinayaka R.

doi:10.1093/nar/26.12.3066

Cited by 75 publications

(70 citation statements)

References 34 publications

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“…The M184V HIV-1 RT, which showed an ϳ2-fold increase fidelity in M13 lacZ␣ forward mutation assay (62), showed a maximum 2.4ϫ higher fidelity than wild-type RT in the kinetic analysis. Interestingly, the K d increase contributes to the high fidelity nature of M184V.…”

Section: Active Site Concentrations Of Mulv Rt On Matched T/p-mentioning

confidence: 95%

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Mechanistic Differences in RNA-dependent DNA Polymerization and Fidelity between Murine Leukemia Virus and HIV-1 Reverse Transcriptases

Skasko

Weiss

Reynolds

et al. 2005

Journal of Biological Chemistry

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We compared the mechanistic and kinetic properties of murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV-1) reverse transcriptases (RTs) during RNA-dependent DNA polymerization and mutation synthesis using pre-steady-state kinetic analysis. First, MuLV RT showed 6.5-121.6-fold lower binding affinity (K d ) to deoxynucleotide triphosphate (dNTP) substrates than HIV-1 RT, although the two RTs have similar incorporation rates (k pol ). Second, compared with HIV-1 RT, MuLV RT showed dramatic reduction during multiple dNTP incorporations at low dNTP concentrations. Presumably, due to its low dNTP binding affinity, the dNTP binding step becomes rate-limiting in the multiple rounds of the dNTP incorporation by MuLV RT, especially at low dNTP concentrations. Third, similar fold differences between MuLV and HIV-1 RTs in the K d and k pol values to correct and incorrect dNTPs were observed. This indicates that these two RT proteins have similar misinsertion fidelities. Fourth, these two RT proteins have different mechanistic capabilities regarding mismatch extension. MuLV RT has a 3.1-fold lower mismatch extension fidelity, compared with HIV-1 RT. Finally, MuLV RT has a 3.8-fold lower binding affinity to mismatched template/primer (T/P) substrate compared with HIV-1 RT. Our data suggest that the active site of MuLV RT has an intrinsically low dNTP binding affinity, compared with HIV-1 RT. In addition, instead of the misinsertion step, the mismatch extension step, which varies between MuLV and HIV-1 RTs, contributes to their fidelity differences. The implications of these kinetic differences between MuLV and HIV-1 RTs on viral cell type specificity and mutagenesis are discussed.Retroviruses encode a versatile DNA polymerase called reverse transcriptase (RT).1 The function of RT is to synthesize linear double-stranded proviral DNAs from single-stranded positive sense viral RNA genomes during viral replication. In order to catalyze this process, RTs perform several distinct enzymatic reactions including RNA-dependent DNA polymerization, DNA-dependent DNA polymerization, strand transfer, and RNase H cleavage (1). The DNA polymerase activity of RTs has been targeted, using various types of RT inhibitors such as nucleoside substrate-like compounds (i.e. azidothymidine (AZT) and didanosine (ddI)) (2), as a means to reduce viral replication in infected individuals. Lentiviruses such as human immunodeficiency virus type 1 (HIV-1) uniquely infect terminally differentiated/nondividing cells (i.e. macrophages) as well as dividing cells (i.e. activated CD4ϩ T cells), whereas oncoretroviruses such as murine leukemia virus (MuLV) productively replicate mainly in dividing cells (3,4). Numerous studies have reported that actively dividing cells have higher cellular deoxynucleotide triphosphate (dNTP) concentrations than nondividing cells (5). Recently, it was reported that the cellular dNTP concentration of human macrophages (ϳ40 nM) is ϳ100 times lower than that of dividing CD4 ϩ T cells (ϳ5 M) (6). Therefore, RTs ...

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Section: Active Site Concentrations Of Mulv Rt On Matched T/p-mentioning

confidence: 95%

“…In addition, M184V HIV-1 RT also showed reduced mismatch primer extension capability (63). M184I HIV-1 RT showed slightly higher fidelity than M184V (62). However, the pre-steady-state kinetic analysis has not been reported.…”

Section: Active Site Concentrations Of Mulv Rt On Matched T/p-mentioning

confidence: 98%

Mechanistic Differences in RNA-dependent DNA Polymerization and Fidelity between Murine Leukemia Virus and HIV-1 Reverse Transcriptases

Skasko

Weiss

Reynolds

et al. 2005

Journal of Biological Chemistry

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“…Interestingly, pre-steady-state kinetic studies of the M184V mutant demonstrated that the M184V mutation only slightly (1-to 2-fold) affects K d (dNTP binding affinity), but not k pol (conformational change/chemical catalysis) steps of HIV-1 RT with normal dNTP substrates, implying that the Val mutation does not significantly affect the structural architecture of the HIV-1 RT dNTP binding pocket (10 -12). In addition, the M184I RT showed a greater fold increase of enzyme fidelity than the M184V RT, even though the effects of the mutations on fidelity are relatively small (13)(14)(15). It was also reported that the Met-184 mutations alter the mutation spectrum of HIV-1 RT (13).…”

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confidence: 96%

“…In addition, the M184I RT showed a greater fold increase of enzyme fidelity than the M184V RT, even though the effects of the mutations on fidelity are relatively small (13)(14)(15). It was also reported that the Met-184 mutations alter the mutation spectrum of HIV-1 RT (13).…”

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confidence: 96%

Reduced dNTP Binding Affinity of 3TC-resistant M184I HIV-1 Reverse Transcriptase Variants Responsible for Viral Infection Failure in Macrophage

Jamburuthugoda

Santos-Velazquez

Skasko

et al. 2008

Journal of Biological Chemistry

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We characterized HIV-1 reverse transcriptase (RT) variants either with or without the (؊)-2,3-deoxy-3-thiacytidine-resistant M184I mutation isolated from a single HIV-1 infected patient. First, unlike variants with wild-type M184, M184I RT variants displayed significantly reduced DNA polymerase activity at low dNTP concentrations, which is indicative of reduced dNTP binding affinity. Second, the M184I variant displayed a ϳ10-to 13-fold reduction in dNTP binding affinity, compared with the Met-184 variant. However, the k pol values of these two RTs were similar. Third, unlike HIV-1 vectors with wild-type RT, the HIV-1 vector harboring M184I RT failed to transduce cell types containing low dNTP concentrations, such as human macrophage, likely due to the reduced DNA polymerization activity of the M184I RT under low cellular dNTP concentration conditions. Finally, we compared the binary complex structures of wild-type and M184I RTs. The Ile mutation at position 184 with a longer and more rigid ␤-branched side chain, which was previously known to alter the RT-template interaction, also appears to deform the shape of the dNTP binding pocket. This can restrict ground state dNTP binding and lead to inefficient DNA synthesis particularly at low dNTP concentrations, ultimately contributing to viral replication failure in macrophage and instability in vivo of the M184I mutation.(Ϫ)-2Ј,3Ј-Deoxy-3Ј-thiacytidine (3TC), 3 a deoxycytidine analog reverse transcriptase (RT) inhibitor, has been routinely included in the anti-HIV-1 drug regime (1, 2). During 3TC therapy, two amino acid substitutions at position 184 of RT are sequentially selected, conferring viral resistance to this drug. The M184I mutation is detected earlier, and eventually replaced with the M184V mutation. One initial explanation for the delayed selection of the M184V mutation is the requirement of two nucleotide mutations to develop the final M184V mutation from the wild-type methionine codon (ATG). In contrast, the M184I mutation requires only a single nucleotide mutation from the Met codon, which may result in its early selection during 3TC therapy (3-7).Structural analysis later provided a functional explanation for the instability in vivo of the M184I mutation. The M184I mutation alters the RT interaction with the template nucleotide more significantly than the M184V mutation, leading to more severely decreased processivity, compared with the M184V RT (8). Indeed, the distributive DNA synthesis catalyzed by the M184I RT has been a major explanation for the instability in vivo of this mutation and its rapid transition to the M184V mutation. The long ␤-branched side chains of these two mutations efficiently block the entry of 3TCTP to the active site (8, 9). Interestingly, pre-steady-state kinetic studies of the M184V mutant demonstrated that the M184V mutation only slightly (1-to 2-fold) affects K d (dNTP binding affinity), but not k pol (conformational change/chemical catalysis) steps of HIV-1 RT with normal dNTP substrates, implying that the Val mutation d...

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“…The polymerase of one isolate of 3TC-resistant virus contains a mutation, M184V, which is thought to result in reduced incorporation of chain-terminating nucleotide analogs (7). The M184V reverse transcriptase may display altered fidelity of DNA synthesis, though there is some controversy as to whether fidelity is increased or decreased (7)(8)(9)(10)(11). Could variants of RNA viruses with increased replicative fidelity display resistance to ribavirin?…”

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confidence: 99%

A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity

Pfeiffer

Kirkegaard

2003

Proc. Natl. Acad. Sci. U.S.A.

384

442

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Ribavirin is a nucleotide analog that can be incorporated by viral polymerases, causing mutations by allowing base mismatches. It is currently used therapeutically as an antiviral drug during hepatitis C virus infections. During the amplification of poliovirus genomic RNA or hepatitis C replicons, error frequency is known to increase upon ribavirin treatment. This observation has led to the hypothesis that ribavirin's antiviral activity results from error catastrophe caused by increased mutagenesis of viral genomes. Here, we describe the generation of ribavirin-resistant poliovirus by serial viral passage in the presence of increasing concentrations of the drug. Ribavirin resistance can be caused by a single amino acid change, G64S, in the viral polymerase in an unresolved portion of the fingers domain. Compared with wild-type virus, ribavirinresistant poliovirus displays increased fidelity of RNA synthesis in the absence of ribavirin and increased survival both in the presence of ribavirin and another mutagen, 5-azacytidine. Ribavirin-resistant poliovirus represents an unusual class of viral drug resistance: resistance to a mutagen through increased fidelity.

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The influence of 3TC resistance mutation M184I on the fidelity and error specificity of human immunodeficiency virus type 1 reverse transcriptase

Cited by 75 publications

References 34 publications

Mechanistic Differences in RNA-dependent DNA Polymerization and Fidelity between Murine Leukemia Virus and HIV-1 Reverse Transcriptases

Mechanistic Differences in RNA-dependent DNA Polymerization and Fidelity between Murine Leukemia Virus and HIV-1 Reverse Transcriptases

Reduced dNTP Binding Affinity of 3TC-resistant M184I HIV-1 Reverse Transcriptase Variants Responsible for Viral Infection Failure in Macrophage

A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity

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