Site-directed Mutagenesis of Arginine 72 of HIV-1 Reverse Transcriptase

Sarafianos, Stefan G.; Pandey, Virendra N.; Kaushik, Neerja; Modak, Mukund J.

doi:10.1074/jbc.270.34.19729

Cited by 71 publications

(82 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In some of our assays, k pol is increased more than 150-fold, for example when BH 3 -3TCTP is used instead of the unsubstituted 3TCTP. An efficient incorporation of BH 3 -TTP is even achieved using the R72A substitution, which decreases the polymerase activity ϳ100-fold (15,31,34,35). Hence, these enzymatic results highlight the general potential given by BH 3 derivatives to circumvent resistance brought by almost any discrimination-associated mutation.…”

Section: Discussionmentioning

confidence: 78%

“…It was thus of interest to know whether other substitutions or other catalytically critical amino acid residues would also be turned into catalytically irrelevant amino acids by the ␣-boranophosphate group. Arg 72 is such an amino acid, as its mutation to alanine nearly abrogates polymerase activity (31). Lys 65 is in the vicinity of the ␤-and ␥-phosphates of the incoming nucleotide, and K65R-mediated drug resistance is suppressed by the BH 3 substitution.…”

Section: The Role Of the Bh 3 Group In The Suppression Of Drugmentioning

confidence: 99%

See 1 more Smart Citation

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

Section: Discussionmentioning

confidence: 78%

Section: The Role Of the Bh 3 Group In The Suppression Of Drugmentioning

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

show abstract

“…Residue Ala 114 also interacts with the dNTP ␤-phosphate in the RT⅐T/P⅐dNTP ternary complex (20). Some studies have suggested that mutagenesis of RT residues interacting with the dNTP phosphates may directly affect RT pyrophosphorolytic activity (21,22). 2 In addition to the A114S mutation, a number of other resistance mutations appear to resensitize AZT-resistant HIV-1 to AZT.…”

Section: Discussionmentioning

confidence: 99%

Mechanism by Which Phosphonoformic Acid Resistance Mutations Restore 3′-Azido-3′-deoxythymidine (AZT) Sensitivity to AZT-resistant HIV-1 Reverse Transcriptase

Arion

Sluis‐Cremer²,

Parniak

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

The development of phosphonoformic acid (PFA) resistance against a background of 3-azido-3-deoxythymidine (AZT) resistance in human immunodeficiency virus type 1 (HIV-1) restores viral sensitivity to AZT. High level AZT resistance requires multiple mutations (D67N/K70R/T215F/K219Q). In order to characterize the mechanism of PFA resistance-mediated resensitization to AZT, the A114S mutation associated with PFA resistance was introduced into the reverse transcriptase (RT) of both wild type and drug-resistant virus. We previously showed that pyrophosphorolytic removal of chainterminating AZT is the primary mechanism of the AZT resistance phenotype (Arion, D., Kaushik, N., McCormick, S., Borkow, G., and Parniak, M. A. (1998) Biochemistry 37, 15908 -15917). Introduction of A114S into the AZT resistance background significantly diminishes both the enhanced pyrophosphorolytic activity and the DNA synthesis processivity associated with the AZTresistant RT. The A114S mutation also alters the nucleotide-dependent phosphorolysis activity associated with AZT resistance. The presence of the A114S mutation therefore severely impairs the mutant enzyme's ability to excise chain-terminating AZT. The decrease in phosphorolytic activity of RT conferred by the PFA resistance A114S mutation resensitizes AZT-resistant HIV-1 to AZT by allowing the latter to again function as a chain terminator of viral DNA synthesis. These data further underscore the importance of phosphorolytic removal of chain-terminating AZT as the primary mechanism of HIV-1 AZT resistance.The most widely used clinical therapeutic against the human immunodeficiency syndrome virus type 1 (HIV-1) 1 is the nucleoside analogue 3Ј-azido-3Ј-deoxythymidine (AZT). Unfortunately, the long term utility of AZT therapy in HIV-1-infected individuals is limited by the emergence of drug-resistant strains, which exhibit up to 200-fold decreases in sensitivity to AZT compared with wild type (WT) virus. High level viral resistance to AZT correlates with multiple mutations in HIV-1 reverse transcriptase (RT), namely D67N, K70R, T215Y/F, and K219Q (1), as well as M41L and L210W in some cases (2-4).While the genotype for AZT resistance has been well characterized for more than a decade, the phenotypic mechanism of AZT resistance remained unclear. AZT inhibits primarily by acting as a chain terminator of viral DNA synthesis. In the past year, we and others have shown that phosphorolytic excision of chain-terminating AZT is an important feature of the AZT resistance mechanism. We first demonstrated that the mutant RT shows an enhanced rate of pyrophosphorolysis compared with wild-type drug-sensitive enzyme at physiologically relevant concentrations of PP i (5, 6). It was subsequently shown that nucleotide-dependent phosphorolysis is also enhanced in AZT resistance (7,8). While these two mechanisms use different substrates for the phosphorolytic removal of chain-terminating AZT, the chemistry involved in both mechanisms is identical (6). The net result is the decreased ability of AZT to a...

show abstract

“…Mutations introduced by site-directed mutagenesis at codons 72, 89, 90, 113, 114, 115, 151, 154, 183, and 190 in RT were shown to decrease sensitivity to foscarnet (17,26,27,29,39,42,49).…”

mentioning

confidence: 99%

Relationship between 3′-Azido-3′-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Meyer

Matsuura

Zonarich

et al. 2003

J Virol

View full text Add to dashboard Cite

Phosphonoformate (foscarnet) is a pyrophosphate (PP i ) analogue and a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), acting through the PP i binding site on the enzyme. HIV-1 RT can unblock a chain-terminated DNA primer by phosphorolytic transfer of the terminal residue to an acceptor substrate (PP i or a nucleotide such as ATP) which also interacts with the PP i binding site. Primer-unblocking activity is increased in mutants of HIV-1 that are resistant to the chain-terminating nucleoside inhibitor 3-azido-3-deoxythymidine (AZT). We have compared the primer-unblocking activity for HIV-1 RT containing various foscarnet resistance mutations (K65R, W88G, W88S, E89K, S117T, Q161L, M164I, and the double mutant Q161L/H208Y) alone or in combination with AZT resistance mutations. The level of primer-unblocking activity varied over a 150-fold range for these enzymes and was inversely correlated with foscarnet resistance and directly correlated with AZT resistance. Based on published crystal structures of HIV-1 RT, many of the foscarnet resistance mutations affect residues that do not make direct contact with the catalytic residues of RT, the incoming deoxynucleoside triphosphate (dNTP), or the primer-template. These mutations may confer foscarnet resistance and reduce primer unblocking by indirectly decreasing the binding and retention of foscarnet, PP i , and ATP. Alternatively, the binding position or orientation of PP i , ATP, or the primer-template may be changed in the mutant enzyme complex so that molecular interactions required for the unblocking reaction are impaired while dNTP binding and incorporation are not.Phosphonoformate (foscarnet) inhibits a wide variety of DNA and RNA polymerases including human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (40, 43). Foscarnet competes with pyrophosphate (PP i ) for binding in PP i exchange reactions, suggesting that foscarnet interacts with the site on the enzyme where PP i is formed (7, 38). On structural grounds, the site of interaction for PP i (and, presumably, for foscarnet) should lie within the deoxynucleoside triphosphate (dNTP) binding site since PP i is formed from the ␤ and ␥ phosphates of the dNTP substrate, but foscarnet inhibition of DNA synthesis in vitro is noncompetitive with dNTP substrates (58), indicating that foscarnet and dNTP bind to distinguishable forms of RT. In addition, median-effect analysis comparing foscarnet and 3Ј-azido-3Ј-deoxythymidine (AZT) triphosphate (AZTTP) alone or as mixtures has shown that inhibition by these compounds is mutually exclusive (10, 22, 50), implying that binding of either inhibitor prevents binding of the other to the same enzyme molecule.In intact cells, the inhibition of HIV-1 by foscarnet and AZT is synergistic (10,22). The mechanism of this synergy is unclear, but it has recently been demonstrated that AZT monophosphate (AZTMP) incorporation can be reversed by the primer-unblocking activity of RT, in which the chain-terminating residue is trans...

show abstract

Site-directed Mutagenesis of Arginine 72 of HIV-1 Reverse Transcriptase

Cited by 71 publications

References 36 publications

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

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

Mechanism by Which Phosphonoformic Acid Resistance Mutations Restore 3′-Azido-3′-deoxythymidine (AZT) Sensitivity to AZT-resistant HIV-1 Reverse Transcriptase

Relationship between 3′-Azido-3′-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Contact Info

Product

Resources

About