scholarly journals Selective Excision of AZTMP by Drug-Resistant Human Immunodeficiency Virus Reverse Transcriptase

2001 ◽  
Vol 75 (10) ◽  
pp. 4832-4842 ◽  
Author(s):  
Paul L. Boyer ◽  
Stefan G. Sarafianos ◽  
Edward Arnold ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Nucleoside Analogs ◽  
Azido Group ◽  
Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Azt Resistance ◽  
Hiv 1

ABSTRACT Two distinct mechanisms can be envisioned for resistance of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to nucleoside analogs: one in which the mutations interfere with the ability of HIV-1 RT to incorporate the analog, and the other in which the mutations enhance the excision of the analog after it has been incorporated. It has been clear for some time that there are mutations that selectively interfere with the incorporation of nucleoside analogs; however, it has only recently been proposed that zidovudine (AZT) resistance can involve the excision of the nucleoside analog after it has been incorporated into viral DNA. Although this proposal resolves some important issues, it leaves some questions unanswered. In particular, how do the AZT resistance mutations enhance excision, and what mechanism(s) causes the excision reaction to be relatively specific for AZT? We have used both structural and biochemical data to develop a model. In this model, several of the mutations associated with AZT resistance act primarily to enhance the binding of ATP, which is the most likely pyrophosphate donor in the in vivo excision reaction. The AZT resistance mutations serve to increase the affinity of RT for ATP so that, at physiological ATP concentrations, excision is reasonably efficient. So far as we can determine, the specificity of the excision reaction for an AZT-terminated primer is not due to the mutations that confer resistance, but depends instead on the structure of the region around the HIV-1 RT polymerase active site and on its interactions with the azido group of AZT. Steric constraints involving the azido group cause the end of an AZT 5′-monophosphate-terminated primer to preferentially reside at the nucleotide binding site, which favors excision.

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

2003 ◽  
Vol 77 (11) ◽  
pp. 6127-6137 ◽  
Author(s):  
Peter R. Meyer ◽  
Suzanne E. Matsuura ◽  
Dianna Zonarich ◽  
Rahul R. Chopra ◽  
Eric Pendarvis ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Azt Resistance ◽  
Hiv 1

ABSTRACT Phosphonoformate (foscarnet) is a pyrophosphate (PPi) analogue and a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), acting through the PPi 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 (PPi or a nucleotide such as ATP) which also interacts with the PPi 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, PPi, and ATP. Alternatively, the binding position or orientation of PPi, 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.

scholarly journals Effects of Specific Zidovudine Resistance Mutations and Substrate Structure on Nucleotide-Dependent Primer Unblocking by Human Immunodeficiency Virus Type 1 Reverse Transcriptase

2002 ◽  
Vol 46 (5) ◽  
pp. 1540-1545 ◽  
Author(s):  
Peter R. Meyer ◽  
Suzanne E. Matsuura ◽  
Adviye A. Tolun ◽  
Ingrid Pfeifer ◽  
Antero G. So ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Azt Resistance ◽  
The Difference

ABSTRACT Nucleotide-dependent unblocking of chain-terminated DNA by human immunodeficiency virus type 1 reverse transcriptase (RT) is enhanced by the presence of mutations associated with 3′-azido-3′-deoxythymidine (AZT) resistance. The increase in unblocking activity was greater for mutant combinations associated with higher levels of in vivo AZT resistance. The difference between mutant and wild-type activity was further enhanced by introduction of a methyl group into the nucleotide substrate and was decreased for a nonaromatic substrate, suggesting that π-π interactions between RT and an aromatic structure may be facilitated by these mutations.

scholarly journals Human immunodeficiency virus type 1 reverse transcriptase genotype and drug susceptibility changes in infected individuals receiving dideoxyinosine monotherapy for 1 to 2 years.

1997 ◽  
Vol 41 (4) ◽  
pp. 757-762 ◽  
Author(s):  
M A Winters ◽  
R W Shafer ◽  
R A Jellinger ◽  
G Mamtora ◽  
T Gingeras ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Nucleoside Analogs ◽  
Population Based ◽  
Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Plasma Rna ◽  
Hiv 1

The genetic mechanisms of human immunodeficiency virus type 1 (HIV-1) resistance to dideoxyinosine (ddI) in vivo have been described based on data from primary HIV-1 isolates. To better define the spectrum of HIV-1 reverse transcriptase (RT) changes occurring during ddI therapy, we determined the genotype and ddI susceptibility of the RT gene of HIV RNA isolated from the plasma of 23 patients who had received 1 to 2 years (mean, 87 +/- 16 weeks) of ddI monotherapy. Population-based sequencing of plasma virus showed that 12 of 23 (52%) patients developed known ddI resistance mutations: L74V (7 patients), K65R (2 patients), L74V with M184V (3 patients), and L74V with K65R (1 patient). Five patients developed one or more known zidovudine resistance mutations (at codons 41, 67, 70, 215, and/or 219) during the study. Other amino acid substitutions were found, but only S68G and L210W occurred in more than one patient. Studies of sensitivity to ddI were performed on population-based recombinant-virus stocks generated by homologous recombination between a plasmid containing an HXB2 clone with the RT gene deleted and RT-PCR products of the RT genes from patients' plasma RNA. The sequences of the virus stocks produced by this procedure were typically identical to the sequence of the input PCR product from plasma RNA. Both an MT-2 cell-based culture assay and a cell-free virion-associated RT inhibition assay showed that viruses possessing an L74V and/or M184V mutation or a K65R mutation had reduced sensitivity to ddI. Viruses without these specific mutations had no change in sensitivity to ddI. The results presented here show that the spectrum of RT mutations in a population of patients on ddI monotherapy is more complex than previously described. The development of multiple mutational patterns, including those that confer resistance to other nucleoside analogs, highlights the complexity of using the currently available nucleoside analogs for antiretroviral therapy.

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

2002 ◽  
Vol 76 (7) ◽  
pp. 3248-3256 ◽  
Author(s):  
Paul L. Boyer ◽  
Stefan G. Sarafianos ◽  
Edward Arnold ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Published Data ◽  
Immunodeficiency Virus ◽  
Azt Resistance ◽  
Hiv 1 ◽  
M184v Mutation

ABSTRACT 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 dideoxy-terminated 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.

scholarly journals The Human Immunodeficiency Virus Type 1 Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutation I132M Confers Hypersensitivity to Nucleoside Analogs

2009 ◽  
Vol 83 (8) ◽  
pp. 3826-3833 ◽  
Author(s):  
Zandrea Ambrose ◽  
Brian D. Herman ◽  
Chih-Wei Sheen ◽  
Shannon Zelina ◽  
Katie L. Moore ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Nucleoside Analogs ◽  
Replication Capacity ◽  
Immunodeficiency Virus ◽  
Viral Replication Capacity ◽  
Rt Inhibitors ◽  
Hiv 1

ABSTRACT We previously identified a rare mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), I132M, which confers high-level resistance to the nonnucleoside RT inhibitors (NNRTIs) nevirapine and delavirdine. In this study, we have further characterized the role of this mutation in viral replication capacity and in resistance to other RT inhibitors. Surprisingly, our data show that I132M confers marked hypersusceptibility to the nucleoside analogs lamivudine (3TC) and tenofovir at both the virus and enzyme levels. Subunit-selective mutagenesis studies revealed that the mutation in the p51 subunit of RT was responsible for the increased sensitivity to the drugs, and transient kinetic analyses showed that this hypersusceptibility was due to I132M decreasing the enzyme's affinity for the natural dCTP substrate but increasing its affinity for 3TC-triphosphate. Furthermore, the replication capacity of HIV-1 containing I132M is severely impaired. This decrease in viral replication capacity could be partially or completely compensated for by the A62V or L214I mutation, respectively. Taken together, these results help to explain the infrequent selection of I132M in patients for whom NNRTI regimens are failing and furthermore demonstrate that a single mutation outside of the polymerase active site and inside of the p51 subunit of RT can significantly influence nucleotide selectivity.

scholarly journals Molecular Mechanisms of Tenofovir Resistance Conferred by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Containing a Diserine Insertion after Residue 69 and Multiple Thymidine Analog-Associated Mutations

2004 ◽  
Vol 48 (3) ◽  
pp. 992-1003 ◽  
Author(s):  
Kirsten L. White ◽  
James M. Chen ◽  
Nicolas A. Margot ◽  
Terri Wrin ◽  
Christos J. Petropoulos ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Insertion Mutation ◽  
Wild Type ◽  
Resistance Mutations ◽  
Thymidine Analog ◽  
Immunodeficiency Virus ◽  
High Level ◽  
Hiv 1

ABSTRACT Two amino acids inserted between residues 69 and 70 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are rare mutations that may develop in viruses containing multiple thymidine analog (zidovudine [AZT], stavudine)-associated mutations and that confer high-level resistance to all currently approved chain-terminating nucleoside and nucleotide RT inhibitors (NRTIs). The two known mechanisms of resistance to NRTIs are decreased incorporation and increased excision. The mechanism used by RT insertion mutants has not been described for tenofovir (TFV), a recently approved agent in this class. A patient-derived HIV-1 strain (strain FS-SSS) that contained an insertion mutation in a background of additional resistance mutations M41L, L74V, L210W, and T215Y was obtained. A second virus (strain FS) was derived from FS-SSS. In strain FS the insertion and T69S were reverted but the other resistance mutations were retained. The FS virus showed strong resistance to AZT but low-level changes in susceptibilities to other NRTIs and TFV. The FS-SSS virus showed reduced susceptibilities to all NRTIs including TFV. Steady-state kinetics demonstrated that the relative binding or incorporation of TFV was slightly decreased for FS-SSS RT compared to those for wild-type RT. However, significant ATP-mediated excision of TFV was detected for both mutant RT enzymes and followed the order FS-SSS RT > FS RT > wild-type RT. The presence of physiological concentrations of the +1 nucleotide inhibited TFV excision by the wild-type RT and slightly inhibited excision by the FS RT, whereas the level of excision by the FS-SSS RT remained high. Computer modeling suggests that the increased mobility of the β3-β4 loop may contribute to the high-level and broad NRTI resistance caused by the T69 insertion mutation.

scholarly journals Human Immunodeficiency Virus Type 1 Recombinant Reverse Transcriptase Enzymes Containing the G190A and Y181C Resistance Mutations Remain Sensitive to Etravirine

2009 ◽  
Vol 53 (11) ◽  
pp. 4667-4672 ◽  
Author(s):  
Hongtao Xu ◽  
Yudong Quan ◽  
Bluma G. Brenner ◽  
Tamara Bar-Magen ◽  
Maureen Oliveira ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Resistance Mutations ◽  
Biochemical Assays ◽  
Immunodeficiency Virus ◽  
Drug Resistant Strains ◽  
Hiv 1

ABSTRACT Etravirine (ETR) is a second-generation nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) active against common human immunodeficiency virus type 1 (HIV-1) drug-resistant strains. This study was designed to determine the extent to which each of the Y181C or G190A mutations in RT might confer resistance to ETR and other members of the NNRTI family of drugs. Recombinant HIV-1 RT enzymes containing either the Y181C or the G190A mutation, or both mutations in tandem, were purified. Both RNA- and DNA-dependent DNA polymerase assays were performed in order to determine the extent to which each of these mutations might confer resistance in cell-free biochemical assays against each of ETR, efavirenz, and nevirapine. Both the biochemical and the cell-based phenotypic assays confirmed the susceptibility of G190A-containing enzymes and viruses to ETR. The results of this study indicate that the G190A mutation is not associated with resistance to ETR.

scholarly journals Selection of Mutations in the Connection and RNase H Domains of Human Immunodeficiency Virus Type 1 Reverse Transcriptase That Increase Resistance to 3′-Azido-3′-Dideoxythymidine

2007 ◽  
Vol 81 (15) ◽  
pp. 7852-7859 ◽  
Author(s):  
Jessica H. Brehm ◽  
Dianna Koontz ◽  
Jeffrey D. Meteer ◽  
Vinay Pathak ◽  
Nicolas Sluis-Cremer ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Rnase H ◽  
Cross Resistance ◽  
Polymerase Domain ◽  
Immunodeficiency Virus ◽  
Azt Resistance ◽  
Hiv 1

ABSTRACT Recent work indicates that mutations in the C-terminal domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) increase 3′-azido-3′-dideoxythymidine (AZT) resistance. Because it is not known whether AZT selects for mutations outside of the polymerase domain of RT, we carried out in vitro experiments in which HIV-1LAI or AZT-resistant HIV-1LAI (M41L/L210W/T215Y) was passaged in MT-2 cells in increasing concentrations of AZT. The first resistance mutations to appear in HIV-1LAI were two polymerase domain thymidine analog mutations (TAMs), D67N and K70R, and two novel mutations, A371V in the connection domain and Q509L in the RNase H domain, that together conferred up to 90-fold AZT resistance. Thereafter, the T215I mutation appeared but was later replaced by T215F, resulting in a large increase in AZT resistance (∼16,000-fold). Mutations in the connection and RNase H domains were not selected starting with AZT-resistant virus (M41L/L210W/T215Y). The roles of A371V and Q509L in AZT resistance were confirmed by site-directed mutagenesis: A371V and Q509L together increased AZT resistance ∼10- to 50-fold in combination with TAMs (M41L/L210W/T215Y or D67N/K70R/T215F) but had a minimal effect without TAMs (1.7-fold). A371V and Q509L also increased cross-resistance with TAMs to lamivudine and abacavir, but not stavudine or didanosine. These results provide the first evidence that mutations in the connection and RNase H domains of RT can be selected in vitro by AZT and confer greater AZT resistance and cross-resistance to nucleoside RT inhibitors in combination with TAMs in the polymerase domain.

Effects of Human Immunodeficiency Virus Type 1 Resistance to Protease Inhibitors on Reverse Transcriptase Processing, Activity, and Drug Sensitivity

1999 ◽  
Vol 73 (4) ◽  
pp. 3455-3459 ◽  
Author(s):  
Laurence Carron de la Carrière ◽  
Sylvie Paulous ◽  
François Clavel ◽  
Fabrizio Mammano
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Rt Inhibitors ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors often display a reduced replicative capacity as a result of an impairment of protease function. Such fitness-impaired viruses display Gag precursor maturation defects. Here, we report that some protease inhibitor-resistant viruses also display abnormalities in the processing of reverse transcriptase (RT) by the protease. In three recombinant viruses carrying resistant protease sequences from patient plasma, we observed a marked decrease in the amount of mature RT subunits and of particle-associated RT activity compared to their parental pretherapy counterparts. We investigated the possibility that a decrease in the amount of particle-associated mature RT could affect the sensitivity of the corresponding virus to RT inhibitors. We observed a twofold increase of sensitivity to zidovudine (AZT) when a virus which carried AZT mutations was processed by a resistant protease. Interestingly, the presence of AZT-resistance mutations partially rescued the replication defect associated with the mutated protease. The interplay between resistance to protease inhibitors and to RT inhibitors described here may be relevant to the therapeutic control of HIV-1 infection.

scholarly journals A Family of Insertion Mutations between Codons 67 and 70 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confer Multinucleoside Analog Resistance

1999 ◽  
Vol 43 (8) ◽  
pp. 1961-1967 ◽  
Author(s):  
B. A. Larder ◽  
S. Bloor ◽  
S. D. Kemp ◽  
Kurt Hertogs ◽  
R. L. Desmet ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Multidrug Resistance ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nucleoside Analogs ◽  
Immunodeficiency Virus ◽  
Analog Resistance ◽  
Hiv 1

ABSTRACT To investigate the occurrence of multinucleoside analog resistance during therapy failure, we surveyed the drug susceptibilities and genotypes of nearly 900 human immunodeficiency virus type 1 (HIV-1) samples. For 302 of these, the 50% inhibitory concentrations of at least four of the approved nucleoside analogs had fourfold-or-greater increases. Genotypic analysis of the reverse transcriptase (RT)-coding regions from these samples revealed complex mutational patterns, including the previously recognized codon 151 multidrug resistance cluster. Surprisingly, high-level multinucleoside resistance was associated with a diverse family of amino acid insertions in addition to “conventional” point mutations. These insertions were found between RT codons 67 and 70 and were commonly 69Ser-(Ser-Ser) or 69Ser-(Ser-Gly). Treatment history information showed that a common factor for the development of these variants was AZT (3′-azido-3′-deoxythymidine, zidovudine) therapy in combination with 2′,3′-dideoxyinosine or 2′,3′-dideoxycytidine, although treatment patterns varied considerably. Site-directed mutagenesis studies confirmed that 69Ser-(Ser-Ser) in an AZT resistance mutational background conferred simultaneous resistance to multiple nucleoside analogs. The insertions are located in the “fingers” domain of RT. Modelling the 69Ser-(Ser-Ser) insertion into the RT structure demonstrated the profound direct effect that this change is likely to have in the nucleoside triphosphate binding site of the enzyme. Our data highlight the increasing problem of HIV-1 multidrug resistance and underline the importance of continued resistance surveillance with appropriate, sufficiently versatile genotyping technology and phenotypic drug susceptibility analysis.

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