Correlation between Viral Resistance to Zidovudine and Resistance at the Reverse Transcriptase Level for a Panel of Human Immunodeficiency Virus Type 1 Mutants

Lennerstrand, Johan; Hertogs, Kurt; Stammers, D.K.; Larder, Brendan A.

doi:10.1128/jvi.75.15.7202-7205.2001

Cited by 53 publications

(61 citation statements)

References 26 publications

(22 reference statements)

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“…This makes sense, considering the intrinsic relationship between the two processes and would suggest that a mutant that mediates resistance by removal would not show dramatic reductions in the incorporation of an analog. In support of this hypothesis, studies on RT AZTR , which appears to confer resistance by removing chain-terminating AZTMP (34,36,37,40) and D4TMP (39,68,69), have shown very little or no reduction in the incorporation of AZTMP (25,35). Consistent with previous reports suggesting that AZT resistance mutations cause resistance by increasing the rate of dNMP removal a role in resistance to abacavir.…”

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

confidence: 73%

“…Accordingly, this may increase the overall amount of removal by prolonging the time RT spends at the 3Ј terminus no matter what the removing agent (PP i or ATP) and in the absence of any marked change in the kinetic rate of removal. These results taken together suggest that both modes of removal may be active in vivo (40), although further studies are needed to clarify inconsistencies in the literature.…”

Section: Compounds Currently Approved By the Food And Drug Administramentioning

confidence: 80%

“…Interestingly, RT Q151M also showed an elevated rate of ATP-mediated removal of dGMP; however, it was slightly slower than RT WT at removing CBVMP. This suggests that removal may play a role in Q151M-mediated resistance to some compounds, although studies have suggested removal to be unimportant in its resistance to AZT (40) or D4T (68).…”

mentioning

confidence: 96%

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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 Insupporting

confidence: 73%

Section: Compounds Currently Approved By the Food And Drug Administramentioning

confidence: 80%

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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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“…Naeger et al employed an assay that measured excision at a single position; this may explain why they did not see an effect on excision (see Results and Discussion). Lennerstrand et al (15) reported that M184V reduces AZTMP excision when combined with the AZT resistance mutations M41L, D67N, K70R, and T215Y but not when combined with mutations M41L and T215Y.…”

mentioning

confidence: 99%

The M184V Mutation Reduces the Selective Excision of Zidovudine 5′-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1

Boyer

Sarafianos

Arnold

et al. 2002

J Virol

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The M184V mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) causes resistance to lamivudine, but it also increases the sensitivity of the virus to zidovudine (3-azido-3-deoxythymidine; AZT). This sensitization to AZT is seen both in the presence and the absence of the mutations that confer resistance to AZT. AZT resistance is due to enhanced excision of AZT 5-monophosphate (AZTMP) from the end of the primer by the RT of the resistant virus. Published data suggest that the excision reaction involves pyrophosphorolysis but that the likely in vivo pyrophosphate donor is not pyrophosphate but ATP. The mutations that lead to AZT resistance enhance ATP binding and, in so doing, enhance pyrophosphorolysis. The excision reaction is specific for AZT because HIV-1 RT, which can form a closed complex with a dideoxyterminated primer and an incoming deoxynucleoside triphosphate (dNTP), does not form the closed complex with an AZTMP-terminated primer and an incoming dNTP. This means that an AZTMP-terminated primer has better access to the site where it can be excised. The M184V mutation alters the polymerase active site in a fashion that specifically interferes with ATP-mediated excision of AZTMP from the end of the primer strand. The M184V mutation does not affect the incorporation of AZT 5-triphosphate (AZTTP), either in the presence or the absence of mutations that enhance AZTMP excision. However, in the presence of ATP, the M184V mutation does decrease the ability of HIV-1 RT to carry out AZTMP excision. Based on these results, and on the results of other excision experiments, we present a model to explain how the M184V mutation affects AZTMP excision.Although combination therapy has been useful in the treatment of human immunodeficiency virus type 1 (HIV-1) infections, the development of drug resistance is a major problem. The drugs that have been approved for HIV-1 therapy target two viral enzymes, reverse transcriptase (RT) and protease (PR). There are two types of drugs that block reverse transcription, nucleoside analogs and nonnucleoside RT inhibitors. Because there are just three classes of approved anti-HIV-1 drugs, there is considerable cross-resistance within each of the three drug classes. This exacerbates the problem of drug resistance and places a premium on therapies that involve combinations of drugs that do not induce significant cross-resistance.Nucleoside analogs lack the 3Ј OH that is present in normal deoxynucleoside triphosphates (dNTPs). If a nucleoside analog is stably incorporated into viral DNA by HIV-1 RT, it acts as a chain terminator and blocks viral DNA synthesis. Viruses that are resistant to nucleoside analogs have specific mutations in RT. Because these drug-resistant RTs must be able to incorporate normal dNTPs reasonably efficiently, resistance must involve an increase in the discrimination between normal dNTPs and the analogs. This increased discrimination can arise either as a specific block to the incorporation of the nucleoside analog or by selective e...

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“…Previous biochemical studies have shown that the incorporated chain terminator can be removed from the primer terminus through phosphorolytic cleavage in the presence of either pyrophosphate (PP i ) (1) or in the presence of ribonucleotide triphosphates (NTPs) (2), which were shown to act as pyrophosphate donors. Increasing evidence suggests that the latter reaction pathway provides an important mechanism for HIV resistance to AZT and, to a certain degree, also to other NRTIs (3)(4)(5)(6)(7)(8)(9)(10)(11).…”

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confidence: 99%

Site-specific Footprinting Reveals Differences in the Translocation Status of HIV-1 Reverse Transcriptase

Marchand

Götte

2003

Journal of Biological Chemistry

146

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Resistance to nucleoside analogue inhibitors of the reverse transcriptase of the HIV-1 often involves phosphorolytic excision of the incorporated chain terminator. Previous crystallographic and modeling studies suggested that this reaction could only occur when the enzyme resides in a pre-translocational stage. Here we studied mechanisms of polymerase translocation using novel site-specific footprinting techniques. Classical footprinting approaches, based on the detection of protected nucleic acid residues, are not sensitive enough to visualize subtle structural differences at single nucleotide resolution. Thus, we developed chemical footprinting techniques that give rise to hyperreactive cleavage on the template strand mediated through specific contacts with the enzyme. Two specific cuts served as markers that defined the position of the polymerase and RNase H domain, respectively. We show that the presence of the next correct dNTP, following the incorporated chain terminator, caused a shift in the position of the two cuts a single nucleotide further downstream. The footprints point to monotonic sliding motions and provide compelling evidence for the existence of an equilibrium between pre-and post-translocational stages. Our data show that enzyme translocation is reversible and uncoupled from nucleotide incorporation and the release of pyrophosphate. This translocational equilibrium ensures access to the pre-translocational stage after incorporation of the chain terminator. The efficiency of excision correlates with an increase in the population of complexes that exist in the pre-translocational stage, and we show that the latter configuration is preferred with an enzyme that contains mutations associated with resistance to nucleoside analogue inhibitors.

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Correlation between Viral Resistance to Zidovudine and Resistance at the Reverse Transcriptase Level for a Panel of Human Immunodeficiency Virus Type 1 Mutants

Cited by 53 publications

References 26 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

The M184V Mutation Reduces the Selective Excision of Zidovudine 5′-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1

Site-specific Footprinting Reveals Differences in the Translocation Status of HIV-1 Reverse Transcriptase

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