Phenotypic Mechanism of HIV-1 Resistance to 3‘-Azido-3‘-deoxythymidine (AZT):  Increased Polymerization Processivity and Enhanced Sensitivity to Pyrophosphate of the Mutant Viral Reverse Transcriptase

Arion, Dominique; Kaushik, Neerja; McCormick, Susan; Borkow, Gadi; Parniak, Michael A.

doi:10.1021/bi981200e

Cited by 319 publications

(366 citation statements)

References 41 publications

Supporting

Mentioning

353

Contrasting

Unclassified

Order By: Relevance

“…This resistance mechanism has been termed NRTI discrimination. The second mechanism involves the selective removal of the chain-terminating NRTI-MP from the prematurely terminated DNA chain (1,21). This mechanism has been termed NRTI excision.…”

Section: Resultsmentioning

confidence: 99%

“…We hypothesize that this putative interaction between K65 and E70 may alter the location of K65, thereby affecting NRTI-TP incorporation. HIV-1 RT has the innate capacity to unblock NRTI-MPterminated primers in the presence of physiological concentrations of ATP or PPi (1,22). Biochemical studies show that TAMs confer resistance to AZT and other NRTI by accelerating the rate at which the terminal AZT-MP (or other NRTI-MP) is removed through phosphorolytic cleavage (3,21).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

Sluis‐Cremer

Sheen

Zelina

et al. 2007

Antimicrob Agents Chemother

View full text Add to dashboard Cite

The K70E mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has become more prevalent in clinical samples, particularly in isolates derived from patients for whom triple-nucleoside regimens that include tenofovir (TNV), abacavir, and lamivudine (3TC) failed. To elucidate the molecular mechanism by which this mutation confers resistance to these nucleoside RT inhibitors (NRTI), we conducted detailed biochemical analyses comparing wild-type (WT), K70E, and K65R HIV-1 RT. Pre-steady-state kinetic experiments demonstrate that the K70E mutation in HIV-1 RT allows the enzyme to discriminate between the natural deoxynucleoside triphosphate substrate and the NRTI triphosphate (NRTI-TP). Compared to the WT enzyme, K70E RT showed 2.1-, 2.3-, and 3.5-foldhigher levels of resistance toward TNV-diphosphate, carbovir-TP, and 3TC-TP, respectively. By comparison, K65R RT demonstrated 12.4-, 12.0-, and 13.1-fold-higher levels of resistance, respectively, toward the same analogs. NRTI-TP discrimination by the K70E (and K65R) mutation was primarily due to decreased rates of NRTI-TP incorporation and not to changes in analog binding affinity. The K65R and K70E mutations also profoundly impaired the ability of RT to excise 3 -azido-2 ,3 -dideoxythymidine monophosphate (AZT-MP) and other NRTI-MP from the 3 end of a chain-terminated primer. When introduced into an enzyme with the thymidine analog mutations (TAMs) M41L, L210W, and T215Y, the K70E mutation inhibited ATP-mediated excision of AZT-MP. Taken together, these findings indicate that the K70E mutation, like the K65R mutation, reduces susceptibility to NRTI by selectively decreasing NRTI-TP incorporation and is antagonistic to TAM-mediated nucleotide excision.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

Sluis‐Cremer

Sheen

Zelina

et al. 2007

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…The other class of resistance mechanism involves repair (or unblocking) of the analog-terminated DNA chain, using PP i or a nucleoside 5Ј-NTP as a cosubstrate for the pyrophosphorolysis reaction (4,7). Mutations in RT such as M41L, D67N, K70R, L210W, T215Y/F, or K219Q/E/N (referred as thymidine analog-associated mutations) increase the unblocking activity in the case of resistance to AZT and d4T (32,33).…”

Section: Discussionmentioning

confidence: 99%

Mechanistic Insights into the Suppression of Drug Resistance by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Using α-Boranophosphate Nucleoside Analogs

Deval

Alvarez

Selmi

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

A class of amino acid substitutions in drug-resistant HIV-1 reverse transcriptase (RT) is responsible for the selectively impaired incorporation of the nucleotide analog inhibitor into DNA. We have shown previously that ␣-boranophosphate nucleoside analogs suppress RT-mediated resistance when the catalytic rate is responsible for drug resistance such as in the case of K65R and dideoxy (dd)NTPs, and Q151M toward AZTTP and ddNTPs. Here, we extend this property to BH 3 -d4TTP and BH 3 -3TCTP toward their clinically relevant mutants Q151M and M184V, respectively. Pre-steady-state kinetics on mutants of the Q151M RT family reveal a 3-5-fold resistance to d4TTP. This resistance is suppressed using BH 3 -d4TTP. Likewise, resistance to 3TCTP by M184V RT (30-fold) and K65R/M184V RT (180-fold) is suppressed using BH 3 -3TCTP because of a 160-fold acceleration of the catalytic constant k pol . Mechanistic insights into the rate enhancement were obtained using various ␣-boranophosphate nucleotides. The presence of the BH 3 group renders k pol independent of amino acid substitutions present in RT. Indeed, the ϳ100-fold decrease in polymerase activity caused by the R72A substitution is restored to wild-type levels using BH 3 -dTTP. Metal ion titration studies show that ␣-boranophosphate nucleoside analogs enhance 3-8-fold the binding of Mg 2؉ ions to the active site of the RT⅐DNA⅐dNTP complex and alleviate the requirement of critical amino acids involved in phosphodiester bond formation. To our knowledge, this is the first example of rescue of polymerase activity by means of a nucleotide analog.

show abstract

“…Nucleoside analogue inhibitors of the retroviral reverse transcriptase (NRTIs) 1 represent important components in currently used drug regimens to treat infection with the human immunodeficiency virus, type 1 (HIV-1). 3Ј-Azido-3Ј-deoxythymidine (zidovudine or AZT), 2Ј,3Ј-dideoxyinosine (didanosine), and (Ϫ)-␤-L-2Ј,3Ј-dideoxy-3Ј-thyacytidine (lamivudine) are prominent members of this class of compounds.…”

mentioning

confidence: 99%

“…NRTIs are intracellularly phosphorylated and act as chain terminators. 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).…”

mentioning

confidence: 99%

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

Marchand

Götte

2003

Journal of Biological Chemistry

146

View full text Add to dashboard Cite

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.

show abstract

Phenotypic Mechanism of HIV-1 Resistance to 3‘-Azido-3‘-deoxythymidine (AZT): Increased Polymerization Processivity and Enhanced Sensitivity to Pyrophosphate of the Mutant Viral Reverse Transcriptase

Cited by 319 publications

References 41 publications

Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

Mechanistic Insights into the Suppression of Drug Resistance by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Using α-Boranophosphate Nucleoside Analogs

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

Contact Info

Product

Resources

About