Long Dissociation of Bictegravir from HIV-1 Integrase-DNA Complexes.

The HIV integrase (IN) strand transfer inhibitor (INSTI) bictegravir (BIC) has a long dissociation half-life (t1/2) from wild-type IN-DNA complexes: BIC 163 hr > dolutegravir (DTG) 96 hr > raltegravir (RAL) 10 hr > elvitegravir (EVG) 3.3 hr. In cells, BIC had more durable antiviral activity against wild-type HIV after drug washout than RAL or EVG. BIC also had a longer t1/2 and maintained longer antiviral activity after drug washout than DTG with the clinically relevant resistance IN mutant G140S+Q148H. Structural analyses indicate that BIC makes more contacts with the IN-DNA complex than DTG mainly via its bicyclic ring system which may contribute to more prolonged residence time and resilience against many resistance mutations.

Doravirine responses to HIV-1 viruses bearing mutations to NRTIs and NNRTIs under in vitro selective drug pressure.

OBJECTIVES: The NNRTI doravirine has been recently approved for the first-line treatment of HIV-infected patients, eliciting favourable responses against viruses bearing the K103N, Y181C and G190A mutations. This study used in vitro drug selections to elaborate the breadth of doravirine responses against viruses bearing NNRTI and NRTI resistance-associated mutations (RAMs). METHODS: WT clinical isolates (n = 6) and viruses harbouring common NRTI and NNRTI RAMs (n = 6) were serially passaged in escalating concentrations of doravirine, doravirine/islatravir, doravirine/lamivudine and rilpivirine over 24 weeks. Genotypic analysis ascertained the appearance and accumulation of NNRTI RAMs. Phenotypic drug susceptibility assays assessed resistance conferred by acquired NNRTI RAMs. RESULTS: For WT viruses, doravirine pressure led to the appearance of V108I or V106A/I/M RAMs after 8 weeks, conferring low-level (∼2-fold) resistance. After 24 weeks, the accumulation of three to six secondary RAMs, including F227L, M230L, L234I and/or Y318, resulted in high-level (>100-fold) resistance to doravirine. Notably, viruses with these doravirine RAMs remained susceptible to rilpivirine and efavirenz. This contrasted with rilpivirine where acquisition of E138K, L100I and/or K101E resulted in >50-fold cross-resistance to all NNRTIs. Doravirine selection of viruses bearing common NRTI and NNRTI RAMs showed delayed acquisition of RAMs compared with WT virus. Pairing doravirine with islatravir or lamivudine attenuated the development of NNRTI RAMs. CONCLUSIONS: Doravirine showed favourable resistance profiles against viruses harbouring NRTI and NNRTI RAMs. The high barrier to resistance to doravirine coupled with the long intracellular half-life of islatravir may provide the opportunity for long-acting treatment options.

Cell culture selections reveal favourable drug resistance profiles for doravirine and islatravir.

BACKGROUND: The newer generation NNRTIs, including doravirine and rilpivirine, were designed to show high potency and overcome K103N, Y181C and G190A resistance. OBJECTIVES: To assess emergent resistance to doravirine and rilpivirine, alone and paired with lamivudine or islatravir through in vitro drug selections. METHODS: Subtype B (n = 3), non-B subtype (n = 3), and pNL4.3 viral isolates were passaged in cord blood mononuclear cells with progressively increasing concentrations of drug(s). Genotypic analysis compared the acquisition and accumulation of drug resistance mutations at weeks 8 and 24 following drug pressure. Cell-based phenotypic assays assessed cross-resistance patterns to NNRTIs by acquired resistance mutations. RESULTS: Doravirine pressure resulted in the acquisition of V108I (6/7) and V106A/I/M (5/7) mutations at weeks 8, followed by F227L (4/7), Y318F (4/7), M230L (2/7) or L234I (2/7) by weeks 24. In contrast, rilpivirine resulted in E138K (5/7) followed by L100I (3/7), K101E (1/7), or M230L (1/7). Doravirine resistance pathways retained susceptibility to rilpivirine, whereas rilpivirine resistance conferred intermediate resistance (12-152-fold) to doravirine. Dual selections with islatravir or lamivudine delayed and diminished emergent resistance to doravirine, resulting in V108I (9/15) with fewer or no other changes at weeks 24. There was a lesser delay in emergent resistance to rilpivirine when combined with islatravir or lamivudine. The M184V mutation did not arise in dual selections with islatravir or lamivudine. CONCLUSIONS: Doravirine showed a more robust resistance profile compared with other NNRTIs. The long intracellular half-life of islatravir and delayed acquisition of resistance in dual selections provide an opportunity for long-acting treatment options.

The S230R Integrase Substitution Associated With Virus Load Rebound During Dolutegravir Monotherapy Confers Low-Level Resistance to Integrase Strand-Transfer Inhibitors.

Background: Dolutegravir (DTG) is an integrase strand-transfer inhibitor (INSTI) used for treatment of human immunodeficiency virus (HIV)-infected individuals. Owing to its high genetic barrier to resistance, DTG has been clinically investigated as maintenance monotherapy to maintain viral suppression and to reduce complication and healthcare costs. Our study aims to explain the underlying mechanism related to the emergence of a S230R substitution in patients who experienced virologic failure while using DTG monotherapy. Methods: We evaluated the effect of the S230R substitution in regard to integrase enzyme activity, viral infectivity, replicative capacity, and susceptibility to different INSTIs by biochemical and cell-based assays. Results: The S230R substitution conferred a 63% reduction in enzyme efficiency. S230R virus was 1.29-fold less infectious than wild-type virus but could replicate in PM1 cells without significant delay. Resistance levels against DTG, cabotegravir, raltegravir, and elvitegravir in tissue culture were 3.85-, 3.72-, 1.52-, and 1.21-fold, respectively, in virus with the S230R substitution. Conclusions: Our data indicate that the S230R substitution is comparable to the previously reported R263K substitution in some respects. Virologic failure during DTG monotherapy can occur through the development of the S230R or R263K mutation, without the need for high-level DTG resistance.

Selective resistance profiles emerging in patient-derived clinical isolates with cabotegravir, bictegravir, dolutegravir, and elvitegravir.

BACKGROUND: Integrase strand transfer inhibitors (INSTIs) are recommended for first-line HIV therapy based on their relatively high genetic barrier to resistance. Although raltegravir (RAL) and elvitegravir (EVG) resistance profiles are well-characterized, resistance patterns for dolutegravir (DTG), bictegravir (BIC), and cabotegravir (CAB) remain largely unknown. Here, in vitro drug selections compared the development of resistance to DTG, BIC, CAB, EVG and RAL using clinical isolates from treatment-naïve primary HIV infection (PHI) cohort participants (n = 12), and pNL4.3 recombinant strains encoding patient-derived Integrase with (n = 5) and without (n = 5) the E157Q substitution. RESULTS: Patient-derived viral isolates were serially passaged in PHA-stimulated cord blood mononuclear cells in the presence of escalating concentrations of INSTIs over the course of 36-46 weeks. Drug resistance arose more rapidly in primary clinical isolates with EVG (12/12), followed by CAB (8/12), DTG (8/12) and BIC (6/12). For pNL4.3 recombinant strains encoding patient-derived integrase, the comparative genetic barrier to resistance was RAL > EVG > CAB > DTG and BIC. The E157Q substitution in integrase delayed the advent of resistance to INSTIs. With EVG, T66I/A, E92G/V/Q, T97A or R263K (n = 16, 3, 2 and 1, respectively) arose by weeks 8-16, followed by 1-4 accessory mutations, conferring high-level resistance (> 100-fold) by week 36. With DTG and BIC, solitary R263K (n = 27), S153F/Y (n = 7) H51Y (n = 2), Q146 R (n = 3) or S147G (n = 1) mutations conferred low-level (< 3-fold) resistance at weeks 36-46. Similarly, most CAB selections (n = 18) resulted in R263K, S153Y, S147G, H51Y, or Q146L solitary mutations. However, three CAB selections resulted in Q148R/K followed by secondary mutations conferring high-level cross-resistance to all INSTIs. EVG-resistant viruses (T66I/R263K, T66I/E157Q/R263K, and S153A/R263K) retained residual susceptibility when switched to DTG, BIC or CAB, losing T66I by week 27. Two EVG-resistant variants developed resistance to DTG, BIC and CAB through the additional acquisition of E138A/Q148R and S230N, respectively. One EVG-resistant variant (T66I) acquired L74M/G140S/S147G, L74M/E138K/S147G and H51Y with DTG CAB and BIC, respectively. CONCLUSIONS: Second generation INSTIs show a higher genetic barrier to resistance than EVG and RAL. The potency of CAB was lower than BIC and DTG. The development of Q148R/K with CAB can result in high-level cross-resistance to all INSTIs.

Antiviral Activity of Bictegravir and Cabotegravir against Integrase Inhibitor-Resistant SIVmac239 and HIV-1.

Animal models are essential to study novel antiretroviral drugs, resistance-associated mutations (RAMs), and treatment strategies. Bictegravir (BIC) is a novel potent integrase strand transfer inhibitor (INSTI) that has shown promising results against HIV-1 infection in vitro and in vivo and against clinical isolates with resistance against INSTIs. BIC has a higher genetic barrier to the development of resistance than two clinically approved INSTIs, termed raltegravir and elvitegravir. Another clinically approved INSTI, dolutegravir (DTG) also possesses a high genetic barrier to resistance, while a fourth compound, termed cabotegravir (CAB), is currently in late phases of clinical development. Here we report the susceptibilities of simian immunodeficiency virus (SIV) and HIV-1 integrase (IN) mutants containing various RAMs to BIC, CAB, and DTG. BIC potently inhibited SIV and HIV-1 in single cycle infection with 50% effective concentrations (EC50s) in the low nM range. In single cycle SIV infections, none of the E92Q, T97A, Y143R, or N155H substitutions had a significant effect on susceptibility to BIC (≤4-fold increase in EC50), whereas G118R and R263K conferred ∼14-fold and ∼6-fold increases in EC50, respectively. In both single and multiple rounds of HIV-1 infections, BIC remained active against the Y143R, N155H, R263K, R263K/M50I, and R263K/E138K mutants (≤4-fold increase in EC50). In multiple rounds of infection, the G140S/Q148H combination of substitutions decreased HIV-1 susceptibility to BIC 4.8-fold compared to 16.8- and 7.4-fold for CAB and DTG, respectively. BIC possesses an excellent resistance profile in regard to HIV and SIV and could be useful in nonhuman primate models of HIV infection.

M184I/V substitutions and E138K/M184I/V double substitutions in HIV reverse transcriptase do not significantly affect the antiviral activity of EFdA.

BACKGROUND: 4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a potent nucleoside analogue inhibitor of HIV that has an unusually long half-life. Cell culture selections with either EFdA or lamivudine using both subtype B and non-B clinical isolates selected the M184I/V substitutions in reverse transcriptase (RT). Unlike lamivudine, however, EFdA retained significant activity against viruses containing the M184I/V substitutions. In other clinical trials that evaluated rilpivirine together with emtricitabine in first-line therapy, the emergence of both the M184I/V and E138K mutations in RT was demonstrated. Moreover, the M184I/V and E138K substitutions were shown to be compensatory for each other with regard to both efficiency of RT activity and viral replicative capacity. This creates concern that E138K might emerge as a compensatory mutation for M184I/V in the aftermath of the use of EFdA. OBJECTIVES: We wished to determine whether the E138K mutation in HIV RT together with M184I/V would compromise the activity of EFdA. METHODS: Recombinant viruses containing the M184I/V and/or E138K substitutions were generated by site-directed mutagenesis and evaluated in tissue culture for susceptibility to various nucleoside compounds, including EFdA. RESULTS: Susceptibility to EFdA was retained in M184I/V viruses that also contained the E138K substitution. Moreover, the E138K substitution was not generated in these studies under selection pressure with EFdA. CONCLUSIONS: These findings help to alleviate concern that EFdA may not be active against viruses that contain both the M184I/V and E138K substitutions in RT.

HIV-1 strains belonging to large phylogenetic clusters show accelerated escape from integrase inhibitors in cell culture compared with viral isolates from singleton/small clusters.

Objectives: Viral phylogenetics revealed two patterns of HIV-1 spread among MSM in Quebec. While most HIV-1 strains ( n = 2011) were associated with singleton/small clusters (cluster size 1-4), 30 viral lineages formed large networks (cluster size 20-140), contributing to 42% of diagnoses between 2011 and 2015. Herein, tissue culture selections ascertained if large cluster lineages possessed higher replicative fitness than singleton/small cluster isolates, allowing for viral escape from integrase inhibitors. Methods: Primary HIV-1 isolates from large 20+ cluster ( n = 11) or singleton/small cluster ( n = 6) networks were passaged in vitro in escalating concentrations of dolutegravir, elvitegravir and lamivudine for 24-36 weeks. Sanger and deep sequencing assessed genotypic changes under selective drug pressure. Results: Large cluster HIV-1 isolates selected for resistance to dolutegravir, elvitegravir and lamivudine faster than HIV-1 strains forming small clusters. With dolutegravir, large cluster HIV-1 variants acquired solitary R263K ( n = 7), S153Y ( n = 1) or H51Y ( n = 1) mutations as the dominant quasi-species within 8-12 weeks as compared with small cluster lineages where R263K ( n = 1/6), S153Y (1/6) or WT species (4/6) were observed after 24 weeks. Interestingly, dolutegravir-associated mutations compromised viral replicative fitness, precluding escalations in concentrations beyond 5-10 nM. With elvitegravir, large cluster variants more rapidly acquired first mutations (T66I, A92G, N155H or S147G) by week 8 followed by sequential accumulation of multiple mutations leading to viral escape (>10 µM) by week 24. Conclusions: Further studies are needed to understand virological features of large cluster viruses that may favour their transmissibility, replicative competence and potential to escape selective antiretroviral drug pressure.

Development of a G118R mutation in HIV-1 integrase following a switch to dolutegravir monotherapy leading to cross-resistance to integrase inhibitors.

OBJECTIVES: Dolutegravir shows a high barrier to resistance with no previously reported cases of acquired integrase mutations during first-line therapy. In this study, rapid development of the G118R mutation arose following a switch from first-line elvitegravir/cobicistat/tenofovir disoproxil fumarate/emtricitabine to dolutegravir monotherapy. The G118R mutation also arose in a treatment-experienced patient switched to dolutegravir monotherapy. The genetic basis for G118R selection and potential phenotypic outcome was ascertained. PATIENT AND METHODS: Genotypic analysis was performed on patients with virological failure (<1000 copies/mL) on dolutegravir-containing regimens. The Los Alamos database was queried for glycine codon 118 polymorphisms. Cell culture selections and phenotypic drug susceptibility assays assessed resistance via the G118R pathway. RESULTS: We report on two patients who developed viral failure while on dolutegravir monotherapy. Both patients had been on a current or previous regimen containing integrase inhibitors. Virological failure (<1000 copies/mL) emerged early within 2 months following the dolutegravir switch. The appearance of G118R in these two cases and subtype C and CRF02_AG in vitro selections were related to a rare GGA natural polymorphism at codon 118 (1.5% prevalence), facilitating a GGA to AGA transition. Cell culture selections were used to assess the in vitro progression of the G118R pathway leading to cross-resistance to all integrase inhibitors. CONCLUSIONS: Although resistance to dolutegravir is typically rare, genetic polymorphisms and monotherapy can facilitate the acquisition of G118R.

Characterization of the Drug Resistance Profiles of Integrase Strand Transfer Inhibitors in Simian Immunodeficiency Virus SIVmac239.

UNLABELLED: We previously showed that the simian immunodeficiency virus SIVmac239 is susceptible to human immunodeficiency virus (HIV) integrase (IN) strand transfer inhibitors (INSTIs) and that the same IN drug resistance mutations result in similar phenotypes in both viruses. Now we wished to determine whether tissue culture drug selection studies with SIV would yield the same resistance mutations as in HIV. Tissue culture selection experiments were performed using rhesus macaque peripheral blood mononuclear cells (PBMCs) infected with SIVmac239 viruses in the presence of increasing concentrations of dolutegravir (DTG), elvitegravir (EVG), and raltegravir (RAL). We now show that 22 weeks of selection pressure with DTG yielded a mutation at position R263K in SIV, similar to what has been observed in HIV, and that selections with EVG led to emergence of the E92Q substitution, which is a primary INSTI resistance mutation in HIV associated with EVG treatment failure. To study this at a biochemical level, purified recombinant SIVmac239 wild-type (WT) and E92Q, T97A, G118R, Y143R, Q148R, N155H, R263K, E92Q T97A, E92Q Y143R, R263K H51Y, and G140S Q148R recombinant substitution-containing IN enzymes were produced, and each of the characteristics strand transfer, 3'-processing activity, and INSTI inhibitory constants was assessed in cell-free assays. The results show that the G118R and G140S Q148R substitutions decreased Km' and Vmax'/Km' for strand transfer compared to those of the WT. RAL and EVG showed reduced activity against both viruses and against enzymes containing Q148R, E92Q Y143R, and G140S Q148R. Both viruses and enzymes containing Q148R and G140S Q148R showed moderate levels of resistance against DTG. This study further confirms that the same mutations associated with drug resistance in HIV display similar profiles in SIV. IMPORTANCE: Our goal was to definitively establish whether HIV and simian immunodeficiency virus (SIV) share similar resistance pathways under tissue culture drug selection pressure with integrase strand transfer inhibitors and to test the effect of HIV-1 integrase resistance-associated mutations on SIV integrase catalytic activity and resistance to integrase strand transfer inhibitors. Clinically relevant HIV integrase resistance-associated mutations were selected in SIV in our tissue culture experiments. Not only do we report on the characterization of SIV recombinant integrase enzyme catalytic activities, we also provide the first research anywhere on the effect of mutations within recombinant integrase SIV enzymes on drug resistance.

The Gap Procedure: for the identification of phylogenetic clusters in HIV-1 sequence data.

BACKGROUND: In the context of infectious disease, sequence clustering can be used to provide important insights into the dynamics of transmission. Cluster analysis is usually performed using a phylogenetic approach whereby clusters are assigned on the basis of sufficiently small genetic distances and high bootstrap support (or posterior probabilities). The computational burden involved in this phylogenetic threshold approach is a major drawback, especially when a large number of sequences are being considered. In addition, this method requires a skilled user to specify the appropriate threshold values which may vary widely depending on the application. RESULTS: This paper presents the Gap Procedure, a distance-based clustering algorithm for the classification of DNA sequences sampled from individuals infected with the human immunodeficiency virus type 1 (HIV-1). Our heuristic algorithm bypasses the need for phylogenetic reconstruction, thereby supporting the quick analysis of large genetic data sets. Moreover, this fully automated procedure relies on data-driven gaps in sorted pairwise distances to infer clusters, thus no user-specified threshold values are required. The clustering results obtained by the Gap Procedure on both real and simulated data, closely agree with those found using the threshold approach, while only requiring a fraction of the time to complete the analysis. CONCLUSIONS: Apart from the dramatic gains in computational time, the Gap Procedure is highly effective in finding distinct groups of genetically similar sequences and obviates the need for subjective user-specified values. The clusters of genetically similar sequences returned by this procedure can be used to detect patterns in HIV-1 transmission and thereby aid in the prevention, treatment and containment of the disease.

Basis for early and preferential selection of the E138K mutation in HIV-1 reverse transcriptase.

E138K, a G→A mutation in HIV-1 reverse transcriptase (RT), is preferentially selected by etravirine (ETR) and rilpivirine over other substitutions at position E138 that offer greater drug resistance. We hypothesized that there was a mutational bias for the E138K substitution and designed an allele-specific PCR to monitor the emergence of E138A/G/K/Q/R/V during ETR selection experiments. We also performed competition experiments using mutated viruses and quantified the prevalence of E138 minority species in drug-naive patients. E138K, as well as E138G, consistently emerged first during ETR selection experiments, followed by E138A and E138Q; E138R was never selected. Surprisingly, E138K was identified as a tiny minority in 23% of drug-naive subtype B patients, a result confirmed by ultradeep sequencing (UDS). This result could reflect a low fitness cost of E138K; however, E138K was one of the least fit substitutions at codon E138, even after taking into account the deoxynucleoside triphosphate pools of the cells used in competition experiments. Further UDS analysis revealed other minority species in a pattern consistent with the mutational bias of HIV RT. There was no evidence of APOBEC3-hypermutation in these selection experiments or in patients. Our results confirm the mutational bias of HIV-1 in patients and highlight the importance of G→A mutations in HIV-1 drug resistance evolution.

In vitro and structural evaluation of PL-100 as a potential second-generation HIV-1 protease inhibitor.

OBJECTIVES: HIV-1 protease inhibitors (PIs) are key components of HIV therapy. PL-100 is a novel lysine sulphonamide that demonstrates potent antiviral activity against multiresistant HIV-1 strains as well as a higher genetic barrier for development of resistance mutations compared with first-generation PIs. In the present study, we compared the antiviral activity of PL-100 against HIV-1 subtype B with that of darunavir. METHODS: We used tissue culture experiments to evaluate the in vitro development of resistance to PL-100 and tested the antiviral activity of several clinically approved PIs against PL-100-selected resistant variants. Structural modelling was also used to compare the binding of PL-100 and darunavir to the HIV-1 protease (PR) enzyme. RESULTS: PL-100-resistant variants that emerged within 8-48 weeks showed low- to high-level resistance (3.5- to 21.6-fold) to PL-100, but commonly retained susceptibility to darunavir, which, in contrast, did not select for resistance mutations over a period of 40 weeks. Structural modelling demonstrated that binding of PL-100 was predominantly based on polar interactions and delocalized hydrophobic interactions through its diphenyl groups, while darunavir has numerous interactions with PR that include hydrogen bonding to PR backbone oxygens at amino acid positions A28, D29 and D30 via di-tetrahydrofuran (di-THF) groups. CONCLUSIONS: Hydrogen-bonding contacts and the di-THF group in darunavir, as well as the hydrophobic nature of PL-100, contribute to PI binding and a high genetic barrier for resistance. Redesigning the structure of PL-100 to include a di-THF group might improve it.

The preferential selection of K65R in HIV-1 subtype C is attenuated by nucleotide polymorphisms at thymidine analogue mutation sites.

OBJECTIVES: We recently reported the preferential selection of the K65R resistance mutation in subtype C HIV-1 compared with subtype B and showed the underlying mechanism to be dependent on subtype C-specific silent nucleotide polymorphisms, i.e. genomic mutations that change the genotype but not the phenotype. The number of clinical reports demonstrating elevated numbers of K65R nevertheless suggests the existence of factors limiting the increased incidence of K65R mutations. Thus, we investigated the contributions of subtype C-specific silent nucleotide polymorphisms at thymidine analogue mutation (TAM) sites 70, 210 and/or 219 that might reduce the previously described preferential selection of K65R in subtype C HIV-1 associated with subtype C-specific nucleotide polymorphisms at sites 64/65. METHODS: Cell culture drug selections were performed with various drugs in MT2 cells. RESULTS: The use of nucleoside/nucleotide reverse transcriptase inhibitors [N(t)RTIs] as single drugs or in combination confirmed the more frequent selection of K65R by multiple N(t)RTIs in a subtype B virus that contained the 64/65 nucleotide polymorphisms of subtype C than in a wild-type subtype B virus. This effect was attenuated in the presence of several silent TAM nucleotide polymorphisms, except when stavudine was employed in the selection protocol. CONCLUSIONS: These results further demonstrate that stavudine can preferentially select for K65R in subtype C virus and also provide a basis for understanding the importance of silent nucleotide polymorphisms in regard to altered HIV drug resistance profiles.

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.

The role of polymorphisms at position 89 in the HIV-1 protease gene in the development of drug resistance to HIV-1 protease inhibitors.

OBJECTIVES: Relatively little is known about the development of resistance to protease inhibitors (PIs) in non-B subtypes. In subtype B viruses, L89 is commonly found at position 89 in the HIV protease (PR) gene, whereas M89 is commonly observed as a polymorphism in other subtypes. We compared the frequencies of substitutions at position 89 in PR in tissue culture selections and in clinical databases of PI-naive and PI-experienced populations. METHODS: Representative subtype A/CRF01_AE (n = 2 and 3) and subtype C (n = 5) isolates were cultured in MT-2 cells and cord blood mononuclear cells (CBMCs), respectively, under increasing drug pressure with PIs, and drug resistance mutations were identified. RESULTS: The M89 natural polymorphism in non-B subtypes commonly led to the appearance of an M89T mutation in selections with atazanavir in subtypes A/AE and C, and was accompanied by other previously recognized atazanavir mutations. The M89T mutation contributed to phenotypic resistance to atazanavir and cross-resistance to lopinavir and nelfinavir, but not to other PIs. A shift from a L89 natural polymorphism to L89I/M arose in two of five subtype C selections with PIs. M89I/V/T mutations were acquired by 10%-11% of individuals harbouring non-B subtypes who were failing PI-based regimens, but were rarely observed in drug-naive persons and in patients failing non-PI-based regimens. CONCLUSIONS: The M/L89 natural polymorphism present in non-B subtypes may lead to the M89T mutational pathway conferring resistance to atazanavir, lopinavir and nelfinavir.

Compensation by the E138K mutation in HIV-1 reverse transcriptase for deficits in viral replication capacity and enzyme processivity associated with the M184I/V mutations.

Recently, several phase 3 clinical trials (ECHO and THRIVE) showed that E138K and M184I were the most frequent mutations to emerge in patients who failed therapy with rilpivirine (RPV) together with two nucleos(t)ide reverse transcriptase inhibitors, emtricitabine (FTC) and tenofovir (TDF). To investigate the basis for the copresence of E138K and M184I, we generated recombinant mutated and wild-type (WT) reverse transcriptase (RT) enzymes and HIV-1(NL4-3) infectious clones. Drug susceptibilities were determined in cord blood mononuclear cells (CBMCs). Structural modeling was performed to analyze any impact on deoxynucleoside triphosphate (dNTP) binding. The results of phenotyping showed that viruses containing both the E138K and M184V mutations were more resistant to each of FTC, 3TC, and ETR than viruses containing E138K and M184I. Viruses with E138K displayed only modest resistance to ETR, little resistance to efavirenz (EFV), and no resistance to either FTC or 3TC. E138K restored viral replication capacity (RC) in the presence of M184I/V, and this was confirmed in cell-free RT processivity assays. RT enzymes containing E138K, E138K/184I, or E138K/184V exhibited higher processivity than WT RT at low dNTP concentrations. Steady-state kinetic analysis demonstrated that the E138K mutation resulted in decreased K(m)s for dNTPs. In contrast, M184I/V resulted in an increased K(m) for dNTPs compared to those for WT RT. These results indicate that the E138K mutation compensates for both the deficit in dNTP usage and impairment in replication capacity by M184I/V. Structural modeling shows that the addition of E138K to M184I/V promotes tighter dNTP binding.

Transmission clustering drives the onward spread of the HIV epidemic among men who have sex with men in Quebec.

Phylodynamic analysis and epidemiologic data identified 3 patterns of spread of primary human immunodeficiency virus type 1 infection (PHI) among men who have sex with men (2001-2009): 420 unique PHIs, 102 small clusters (2-4 PHIs per cluster, n = 280), and 46 large clusters (5-31 PHIs per cluster, n = 450). Large clusters disproportionately increased from 25.2% of PHIs in 2005 to 39.1% in 2009 (χ(2) = 33.9, P < .001). Scalar expansion of large clusters over 11 months (interquartile range, 3.5-25.5 months) correlated with cluster membership size (r(2) = 0.174, F = 4.424, P = .047). PHI cohort data revealed variations in social networks and risk behaviors among the 3 groups, suggesting the need for tailored prevention measures.

Phylogenetic Insights on Patterns of HIV-1 Spread and the Design of Epidemic Control Measures.

Phylogenetics provides a unique structural framework to track the spread of viral diseases, such as HIV-1 (human immunodeficiency virus type 1), the causative agent of AIDS (acquired immunodeficiency syndrome) [...].

Characterization of the E138K resistance mutation in HIV-1 reverse transcriptase conferring susceptibility to etravirine in B and non-B HIV-1 subtypes.

We have selected for resistance to etravirine (ETR) and efavirenz (EFV) in tissue culture using three subtype B, three subtype C, and two CRF02_AG clinical isolates, grown in cord blood mononuclear cells. Genotypic analysis was performed at baseline and at various weeks of selection. Phenotypic resistance in regard to ETR, EFV, and nevirapine (NVP) was evaluated at weeks 25 to 30 for all ETR-selected viruses and in viral clones that contained specific resistance mutations that were inserted by site-directed mutagenesis into pNL-4.3 and AG plasmids. The results show that ETR selected mutations at positions V90I, K101Q, E138K, V179D/E/F, Y181C, V189I, G190E, H221H/Y, and M230L and that E138K was the first of these to emerge in most instances. The time to the emergence of resistance was longer in the case of ETR (18 weeks) compared to EFV (11 weeks), and no differences in the patterns of emergent mutations could be documented between the B and non-B subtypes. Viral clones containing E138K displayed low-level phenotypic resistance to ETR (3.8-fold) and modestly impaired replication capacity (2-fold) compared to wild-type virus. ETR-selected virus showed a high degree of cross-resistance to NVP but not to EFV. We identified K101Q, E138K, V179E, V189I, G190E, and H221Y as mutations not included among the 17 currently recognized resistance-associated mutations for ETR.