Subunit-Selective Mutational Analysis and Tissue Culture Evaluations of the Interactions of the E138K and M184I Mutations in HIV-1 Reverse Transcriptase

The emergence of HIV-1 drug resistance remains a major obstacle in antiviral therapy. M184I/V and E138K are signature mutations of clinical relevance in HIV-1 reverse transcriptase (RT) for the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine (3TC) and emtricitabine (FTC) and the second-generation (new) nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV), respectively, and the E138K mutation has also been shown to be selected by etravirine in cell culture. The E138K mutation was recently shown to compensate for the low enzyme processivity and viral fitness associated with the M184I/V mutations through enhanced deoxynucleoside triphosphate (dNTP) usage, while the M184I/V mutations compensated for defects in polymerization rates associated with the E138K mutations under conditions of high dNTP concentrations. The M184I mutation was also shown to enhance resistance to RPV and ETR when present together with the E138K mutation. These mutual compensatory effects might also enhance transmission rates of viruses containing these two mutations. Therefore, we performed tissue culture studies to investigate the evolutionary dynamics of these viruses. Through experiments in which E138K-containing viruses were selected with 3TC-FTC and in which M184I/V viruses were selected with ETR, we demonstrated that ETR was able to select for the E138K mutation in viruses containing the M184I/V mutations and that the M184I/V mutations consistently emerged when E138K viruses were selected with 3TC-FTC. We also performed biochemical subunit-selective mutational analyses to investigate the impact of the E138K mutation on RT function and interactions with the M184I mutation. We now show that the E138K mutation decreased rates of polymerization, impaired RNase H activity, and conferred ETR resistance through the p51 subunit of RT, while an enhancement of dNTP usage as a result of the simultaneous presence of both mutations E138K and M184I occurred via both subunits.

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The impact of HIV-1 reverse transcriptase polymorphisms on responses to first-line nonnucleoside reverse transcriptase inhibitor-based therapy in HIV-1-infected adults

AIDS ◽

10.1097/qad.0b013e3283636179 ◽

2013 ◽

Vol 27 (14) ◽

pp. 2245-2253 ◽

Cited By ~ 18

Author(s):

Nicola E. Mackie ◽

David T. Dunn ◽

David Dolling ◽

Lucy Garvey ◽

Linda Harrison ◽

...

Keyword(s):

Reverse Transcriptase ◽

Transcriptase Inhibitor ◽

First Line ◽

Nonnucleoside Reverse Transcriptase Inhibitor ◽

Reverse Transcriptase Inhibitor ◽

The Impact ◽

Hiv 1

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Role of the K101E Substitution in HIV-1 Reverse Transcriptase in Resistance to Rilpivirine and Other Nonnucleoside Reverse Transcriptase Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01536-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5649-5657 ◽

Cited By ~ 9

Author(s):

Hong-Tao Xu ◽

Susan P. Colby-Germinario ◽

Wei Huang ◽

Maureen Oliveira ◽

Yingshan Han ◽

...

Keyword(s):

Viral Replication ◽

Reverse Transcriptase ◽

Salt Bridge ◽

Transcriptase Inhibitor ◽

Drug Susceptibility ◽

Replication Capacity ◽

Nonnucleoside Reverse Transcriptase Inhibitor ◽

Viral Replication Capacity ◽

The Impact ◽

Hiv 1

ABSTRACTResistance to the recently approved nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 reverse transcriptase (RT), together with an M184I substitution that is associated with resistance to coutilized emtricitabine (FTC). Previous biochemical and virological studies have shown that compensatory interactions between substitutions E138K and M184I can restore enzyme processivity and the viral replication capacity. Structural modeling studies have also shown that disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study was designed to investigate the impact of K101E, alone or in combination with E138K and/or M184I, on drug susceptibility, viral replication capacity, and enzyme function. We show here that K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, but not E138K, were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR and RPV, but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV resistance compared to that obtained with E138K alone but restored susceptibility to NVP and DLV. The K101E substitution can compensate for deficits in viral replication capacity and enzyme processivity associated with M184I, while M184I can compensate for the diminished efficiency of DNA polymerization associated with K101E. The coexistence of K101E and E138K does not impair either viral replication or enzyme fitness. We conclude that K101E can play a significant role in resistance to RPV.

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Effects of the W153L Substitution in HIV Reverse Transcriptase on Viral Replication and Drug Resistance to Multiple Categories of Reverse Transcriptase Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02729-14 ◽

2014 ◽

Vol 58 (8) ◽

pp. 4515-4526 ◽

Cited By ~ 2

Author(s):

Hong-Tao Xu ◽

Susan P. Colby-Germinario ◽

Maureen Oliveira ◽

Daniel Rajotte ◽

Richard Bethell ◽

...

Keyword(s):

Viral Replication ◽

Reverse Transcriptase ◽

Rnase H ◽

Hiv Reverse Transcriptase ◽

Compound A ◽

Enzymatic Function ◽

Biochemical Assays ◽

Rt Inhibitors ◽

The Impact ◽

Hiv 1

ABSTRACTA W153L substitution in HIV-1 reverse transcriptase (RT) was recently identified by selection with a novel nucleotide-competing RT inhibitor (NcRTI) termed compound A that is a member of the benzo[4,5]furo[3,2,d]pyrimidin-2-one NcRTI family of drugs. To investigate the impact of W153L, alone or in combination with the clinically relevant RT resistance substitutions K65R (change of Lys to Arg at position 65), M184I, K101E, K103N, E138K, and Y181C, on HIV-1 phenotypic susceptibility, viral replication, and RT enzymatic function, we generated recombinant RT enzymes and viruses containing each of these substitutions or various combinations of them. We found that W153L-containing viruses were impaired in viral replicative capacity and were hypersusceptible to tenofovir (TFV) while retaining susceptibility to most nonnucleoside RT inhibitors. The nucleoside 3TC retained potency against W153L-containing viruses but not when the M184I substitution was also present. W153L was also able to reverse the effects of the K65R substitution on resistance to TFV, and K65R conferred hypersusceptibility to compound A. Biochemical assays demonstrated that W153L alone or in combination with K65R, M184I, K101E, K103N, E138K, and Y181C impaired enzyme processivity and polymerization efficiency but did not diminish RNase H activity, providing mechanistic insights into the low replicative fitness associated with these substitutions. We show that the mechanism of the TFV hypersusceptibility conferred by W153L is mainly due to increased efficiency of TFV-diphosphate incorporation. These results demonstrate that compound A and/or derivatives thereof have the potential to be important antiretroviral agents that may be combined with tenofovir to achieve synergistic results.

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Drug Resistance-Associated Mutations in Antiretroviral Treatment-Experienced Patients in Kuwait

Medical Principles and Practice ◽

10.1159/000488108 ◽

2018 ◽

Vol 27 (2) ◽

pp. 152-157

Author(s):

Wassim Chehadeh ◽

Osama Albaksami ◽

Sonia Elezebeth John ◽

Widad Al-Nakib

Keyword(s):

Reverse Transcriptase ◽

Transcriptase Inhibitor ◽

Antiretroviral Drugs ◽

First Line ◽

Genotypic Resistance ◽

Nonnucleoside Reverse Transcriptase Inhibitor ◽

High Level ◽

Interpretation Algorithm ◽

Hiv 1 ◽

Level Resistance

Objectives: To investigate the prevalence of nonpolymorphic resistance-associated mutations (RAM) in HIV-1 patients on first-line antiretroviral therapy in Kuwait. Subjects and Methods: Total RNA was isolated from plasma samples of 42 patients who received a first-line nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimen. HIV-1 protease and reverse transcriptase genetic regions were then amplified by nested reverse transcription-polymerase chain reaction and directly sequenced. The HIV-1 subtype was identified using the Bayesian phylogenetic method, and RAM were identified using the Stanford University genotypic resistance interpretation algorithm. Results: The HIV-1 viral load at sampling ranged from < 20 to 8.25 × 104 copies/ml. CRF01_AE, C, and B were the most predominant HIV-1 subtypes. Nonpolymorphic mutations associated with resistance to antiretroviral drugs were detected in 11 (26.2%) of the 42 patients; 5 (11.9%) patients had mutations associated with a high-level resistance to nucleoside reverse transcriptase inhibitors (NRTI), 4 (9.5%) patients had mutations associated with resistance to NNRTI, 1 (2.4%) patient had mutations associated with resistance to both NRTI and NNRTI, and 1 (2.4%) patient had mutations potentially associated with low-level resistance to both protease inhibitors and NNRTI. All patients with RAM had a detectable plasma HIV-1 RNA level. Conclusion: Our results indicate the development of RAM during an NNRTI-based regimen and highlight the importance of considering other regimens to avoid treatment failure.

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L74V increases the reverse transcriptase content of HIV-1 virions with non-nucleoside reverse transcriptase drug-resistant mutations L100I+K103N and K101E+G190S, which results in increased fitness

Journal of General Virology ◽

10.1099/vir.0.050914-0 ◽

2013 ◽

Vol 94 (7) ◽

pp. 1597-1607 ◽

Cited By ~ 3

Author(s):

Jiong Wang ◽

Dongge Li ◽

Robert A. Bambara ◽

Hongmei Yang ◽

Carrie Dykes

Keyword(s):

Reverse Transcriptase ◽

Resistance Mutation ◽

Transcriptase Inhibitor ◽

Rnase H ◽

Drug Resistant ◽

Resistance Mutations ◽

Nnrti Resistance ◽

Reverse Transcriptase Inhibitor ◽

Subsequent Addition ◽

Hiv 1

The fitness of non-nucleoside reverse transcriptase inhibitor (NNRTI) drug-resistant reverse transcriptase (RT) mutants of HIV-1 correlates with the amount of RT in the virions and the RNase H activity of the RT. We wanted to understand the mechanism by which secondary NNRTI-resistance mutations, L100I and K101E, and the nucleoside resistance mutation, L74V, alter the fitness of K103N and G190S viruses. We measured the amount of RT in virions and the polymerization and RNase H activities of mutant RTs compared to wild-type, K103N and G190S. We found that L100I, K101E and L74V did not change the polymerization or RNase H activities of K103N or G190S RTs. However, L100I and K101E reduced the amount of RT in the virions and subsequent addition of L74V restored RT levels back to those of G190S or K103N alone. We conclude that fitness changes caused by L100I, K101E and L74V derive from their effects on RT content.

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HIV-1 drug resistance mutations in children after failure of first-line nonnucleoside reverse transcriptase inhibitor-based antiretroviral therapy

HIV Medicine ◽

10.1111/j.1468-1293.2010.00828.x ◽

2010 ◽

Vol 11 (9) ◽

pp. 565-572 ◽

Cited By ~ 19

Author(s):

T Puthanakit ◽

G Jourdain ◽

S Hongsiriwon ◽

P Suntarattiwong ◽

K Chokephaibulkit ◽

...

Keyword(s):

Drug Resistance ◽

Antiretroviral Therapy ◽

Reverse Transcriptase ◽

Transcriptase Inhibitor ◽

Resistance Mutations ◽

First Line ◽

Drug Resistance Mutations ◽

Nonnucleoside Reverse Transcriptase Inhibitor ◽

Reverse Transcriptase Inhibitor ◽

Hiv 1

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Insertions in the Reverse Transcriptase Increase both Drug Resistance and Viral Fitness in a Human Immunodeficiency Virus Type 1 Isolate Harboring the Multi-Nucleoside Reverse Transcriptase Inhibitor Resistance 69 Insertion Complex Mutation

Journal of Virology ◽

10.1128/jvi.76.20.10546-10552.2002 ◽

2002 ◽

Vol 76 (20) ◽

pp. 10546-10552 ◽

Cited By ~ 30

Author(s):

Miguel E. Quiñones-Mateu ◽

Mahlet Tadele ◽

Mariona Parera ◽

Antonio Mas ◽

Jan Weber ◽

...

Keyword(s):

Reverse Transcriptase ◽

Transcriptase Inhibitor ◽

Viral Fitness ◽

Wild Type ◽

Sequence Context ◽

Recombinant Viruses ◽

Immunodeficiency Virus ◽

Reverse Transcriptase Inhibitor ◽

Hiv 1

ABSTRACT Recent studies have shown that the accumulation of multiple mutations associated with nucleoside reverse transcriptase inhibitor (NRTI) resistance may be grouped as multi-NRTI resistance (MNR) complexes. In this study, we have examined the viral fitness of recombinant viruses carrying the reverse transcriptase (RT) of a human immunodeficiency virus type 1 (HIV-1) primary isolate harboring mutations comprising the MNR 69 insertion complex. Different RT mutants were prepared in the sequence context of either the wild-type RT sequence of the HIV-1BH10 isolate or the sequence found in a clinical HIV-1 isolate with the MNR 69 insertion mutation. As expected, in the presence of zidovudine, recombinant viruses harboring the MNR RT from the patient were more fit than wild-type viruses. However, in the absence of drug, the virus with the RT from the original clinical isolate (SS) was more fit than (i) the wild-type virus with an engineered serine insertion between residues 69 and 70 (T69SSS) and (ii) the recombinant virus with the MNR RT where the insertion was removed (2S0S). These results suggest that RT insertions, in the right sequence context (i.e., additional mutations contained in the MNR 69 insertion complex), enhance NRTI resistance and may improve viral fitness. Thus, comparing complex mutation patterns with viral fitness may help to elucidate the role of uncharacterized drug resistance mutations in antiretroviral treatment failure.

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