Crystal Structures of Zidovudine- or Lamivudine-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptases Containing Mutations at Codons 41, 184, and 215

Chamberlain, Philip P.; Ren, Jingshan; Nichols, C.E.; Douglas, L. A.; Lennerstrand, Johan; Larder, B. A.; Stuart, David I.; Stammers, D.K.

doi:10.1128/jvi.76.19.10015-10019.2002

Cited by 58 publications

(42 citation statements)

References 25 publications

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“…This hypothesis is challenged by kinetic data as well as by mutagenesis studies that fail to show an effect of 215Y on RT affinity for ATP and suggest instead that this mutation optimizes the orientation of ATP for the primer excision reaction (29,35). Our finding of higher rates of primer unblocking in the 67N/70R/219Q enzyme compared to the 41L/210W/215Y enzyme is consistent with the latter model that shows that the 67N/70R/219Q cluster in the absence of changes at the 215 position is also well positioned to make direct contact with ATP which facilitates a catalytically efficient alignment with the primer terminus (8).…”

Section: Discussionsupporting

confidence: 80%

The L74V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Counteracts Enhanced Excision of Zidovudine Monophosphate Associated with Thymidine Analog Resistance Mutations

Miranda

Götte

Kuritzkes

2005

Antimicrob Agents Chemother

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Thymidine analog mutations (TAMs) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) confer resistance to zidovudine (AZT) by increasing the rate of ATP-dependent phosphorolysis of the terminal nucleotide monophosphate (primer unblocking). By contrast, the L74V mutation, which confers resistance to didanosine, sensitizes HIV-1 to AZT and partially restores AZT susceptibility when present together with one or more TAMs. To compare rates of primer unblocking in RTs carrying different clusters of TAMs and to explore the biochemical mechanism by which L74V affects AZT susceptibility, ATP-mediated rescue of AZT-blocked DNA synthesis was assayed using a series of purified recombinant RTs. Rates of primer unblocking were higher in the 67N/70R/219Q RT than in the 41L/210W/215Y enzyme and were similar to rates observed with an RT carrying six TAMs (41L/67N/70R/210W/215Y/219Q). The presence of 74V in an otherwise wild-type RT reduced the rate of primer unblocking to a degree similar to that observed with the M184V mutation for lamivudine resistance, which also sensitizes HIV-1 to AZT. Introduction of 74V into RTs carrying TAMs partially counteracted the effect of TAMs on the rate of primer unblocking. The effect of 74V was less marked than that of the 184V mutation in the 67N/70R/219Q and 41L/210W/215Y RTs but similar in the RT carrying six TAMs. These results demonstrate that L74V enhances AZT susceptibility by reducing the extent of its removal by ATP-dependent phosphorolysis and provides further evidence for a common mechanism by which mutations conferring resistance to didanosine and lamivudine sensitize HIV-1 to AZT.Nucleoside analogs are prodrugs that inhibit the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). Once phosphorylated by cellular kinases to their active triphosphate forms, these drugs compete with the natural deoxynucleoside triphosphates for incorporation by RT into the nascent reverse transcript. Because these inhibitors lack a 3Ј-OH they function as chain terminators, blocking further DNA polymerization. Nucleoside reverse transcriptase inhibitors (NRTIs) are a cornerstone of antiretroviral therapy and have contributed importantly to the dramatic reduction in HIV-related morbidity and mortality in the developed world (34, 50). However, the high prevalence of drug resistance limits the clinical benefits of NRTIs in many patients.In the case of zidovudine (AZT), resistance emerges in a stepwise manner by accumulation of thymidine analog resistance mutations (TAMs) at RT codons 41, 67, 70, 210, 215, and 219 (3, 19, 23). The combined presence of three to six TAMs results in high-level (Ͼ500-fold) AZT resistance and contributes significantly to cross-resistance to other nucleoside RT inhibitors (46). Data from several studies suggest that these mutations usually are found in two distinct clusters: those linked to a T215Y mutation, and those linked to the K70R mutation (18,28). When present together with other TAMs, the 215Y mutation most often occurs as a ...

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Section: Discussionsupporting

confidence: 80%

The L74V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Counteracts Enhanced Excision of Zidovudine Monophosphate Associated with Thymidine Analog Resistance Mutations

Miranda

Götte

Kuritzkes

2005

Antimicrob Agents Chemother

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“…So far, no crystal structure has been reported for RT that contains bound PP i , ATP, or foscarnet; however, since PP i is derived from the ␤ and ␥ phosphates of the incoming dNTP, it is possible to infer which residues are likely to interact with PP i from the crystal structure of the RT-dTTP-primer-template complex (3,6,16,32,46). If this inference is correct, then RT must adopt a closed configuration to allow residue 65 to interact with PP i and, by extension, with ATP or foscarnet (compare Fig.…”

Section: Resultsmentioning

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

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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...

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“…Using a large sample size (66,224 genotypes), we found that K65R shows a strong negative association with specific TAMs, including M41L, D67N, L210W, T215F/Y, and K219Q/E. TAMs facilitate the excision reaction by increasing binding of ATP through substitutions at 210 and 215 (6,48). M41L and D67N may enhance the effect of T215F/Y by altering the interaction of the 3Ј-azido group of AZT with the dNTP binding pocket, favoring excision (33).…”

Section: Discussionmentioning

confidence: 99%

The K65R Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Exhibits Bidirectional Phenotypic Antagonism with Thymidine Analog Mutations

Parikh

Bacheler²,

Koontz

et al. 2006

J Virol

117

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The K65R mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is selected in vitro by many D-nucleoside analog RT inhibitors (NRTI) but has been rarely detected in treated patients. In recent clinical trials, the K65R mutation has emerged frequently in patients experiencing virologic failure on antiretroviral combinations that do not include 3-azidothymidine (AZT). The reason for this change is uncertain. To gain insight, we examined trends in the frequency of K65R in a large genotype database, the association of K65R with thymidine analog mutations (TAMs) and other NRTI mutations, and the viral susceptibility profile of HIV-1 with K65R alone and in combination with TAMs. Among >60,000 clinical samples submitted for genotype analysis that contained one or more NRTI resistance mutations, the frequency of K65R increased from 0.4% in 1998 to 3.6% in 2003. Among samples with K65R, a strong negative association was evident with the TAMs M41L, D67N, L210W, T215Y/F, and K219Q/E (P < 0.005) but not with other NRTI mutations, including the Q151M complex. This suggested that K65R and TAMs are antagonistic. To test this possibility, we generated recombinant HIV-1 encoding K65R in two different TAM backgrounds: M41L/L210W/ T215Y and D67N/K70R/T215F/K219Q. K65R reduced AZT resistance from >50-fold to <2.5-fold in both backgrounds. In addition, TAMs antagonized the phenotypic effect of K65R, reducing resistance to tenofovir, abacavir, 2,3-dideoxycytidine, dideoxyinosine, and stavudine. In conclusion, K65R and TAMs exhibit bidirectional phenotypic antagonism. This antagonism likely explains the negative association of these mutations in genotype databases, the rare emergence of K65R with antiretroviral therapies that contain AZT, and its more frequent emergence with combinations that exclude AZT.

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Crystal Structures of Zidovudine- or Lamivudine-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptases Containing Mutations at Codons 41, 184, and 215

Cited by 58 publications

References 25 publications

The L74V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Counteracts Enhanced Excision of Zidovudine Monophosphate Associated with Thymidine Analog Resistance Mutations

The L74V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Counteracts Enhanced Excision of Zidovudine Monophosphate Associated with Thymidine Analog Resistance Mutations

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

The K65R Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Exhibits Bidirectional Phenotypic Antagonism with Thymidine Analog Mutations

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