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.

DM-PhyClus: a Bayesian phylogenetic algorithm for infectious disease transmission cluster inference.

BACKGROUND: Conventional phylogenetic clustering approaches rely on arbitrary cutpoints applied a posteriori to phylogenetic estimates. Although in practice, Bayesian and bootstrap-based clustering tend to lead to similar estimates, they often produce conflicting measures of confidence in clusters. The current study proposes a new Bayesian phylogenetic clustering algorithm, which we refer to as DM-PhyClus (Dirichlet-Multinomial Phylogenetic Clustering), that identifies sets of sequences resulting from quick transmission chains, thus yielding easily-interpretable clusters, without using any ad hoc distance or confidence requirement. RESULTS: Simulations reveal that DM-PhyClus can outperform conventional clustering methods, as well as the Gap procedure, a pure distance-based algorithm, in terms of mean cluster recovery. We apply DM-PhyClus to a sample of real HIV-1 sequences, producing a set of clusters whose inference is in line with the conclusions of a previous thorough analysis. CONCLUSIONS: DM-PhyClus, by eliminating the need for cutpoints and producing sensible inference for cluster configurations, can facilitate transmission cluster detection. Future efforts to reduce incidence of infectious diseases, like HIV-1, will need reliable estimates of transmission clusters. It follows that algorithms like DM-PhyClus could serve to better inform public health strategies.

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.

HIV-1 Resistance to Dolutegravir Is Affected by Cellular Histone Acetyltransferase Activity.

Integrase strand transfer inhibitors (INSTIs) are the newest class of antiretrovirals to be approved for the treatment of HIV infection. Canonical resistance to these competitive inhibitors develops through substitutions in the integrase active site that disrupt drug-protein interactions. However, resistance against the newest integrase inhibitor, dolutegravir (DTG), is associated with an R263K substitution at the C terminus of integrase that causes resistance through an unknown mechanism. The integrase C-terminal domain is involved in many processes over the course of infection and is posttranslationally modified via acetylation of three lysine residues that are important for enzyme activity, integrase multimerization, and protein-protein interactions. Here we report that regulation of the acetylation of integrase is integral to the replication of HIV in the presence of DTG and that the R263K mutation specifically disrupts this regulation, likely due to enhancement of interactions with the histone deacetylase I complex, as suggested by coimmunoprecipitation assays. Although no detectable differences in the levels of cell-free acetylation of the wild-type (WT) and mutated R263K enzymes were observed, the inhibition of cellular histone acetyltransferase enzymes sensitized the NL4.3WT virus to DTG, while NL4.3R263K was almost completely unaffected. When levels of endogenous acetylation were manipulated in virus-producing cells, inhibitors of acetylation enhanced the replication of NL4.3R263K, whereas inhibition of deacetylation greatly diminished the replication of NL4.3WT Taken together, these results point to a pivotal role of acetylation in the resistance mechanism of HIV to some second-generation integrase strand transfer inhibitors, such as DTG.IMPORTANCE This is, to our knowledge, the first report of the influence of posttranslational modifications on HIV drug resistance. Both viral replication and resistance to second-generation integrase strand transfer inhibitors of both WT and INSTI-resistant HIV strains were differentially affected by acetylation, likely as a result of altered interactions between integrase and the cellular deacetylation machinery. Many "shock and kill" strategies to eradicate HIV manipulate endogenous levels of acetylation in order to reactivate latent HIV. However, our results suggest that some drug-resistant viruses may differentially respond to such stimulation, which may complicate the attainment of this goal. Our future work will further illuminate the mechanisms involved.

HIV drug resistance against strand transfer integrase inhibitors.

Integrase strand transfer inhibitors (INSTIs) are the newest class of antiretroviral drugs to be approved for treatment and act by inhibiting the essential HIV protein integrase from inserting the viral DNA genome into the host cell's chromatin. Three drugs of this class are currently approved for use in HIV-positive individuals: raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG), while cabotegravir (CAB) and bictegravir (BIC) are currently in clinical trials. RAL and EVG have been successful in clinical settings but have relatively low genetic barriers to resistance. Furthermore, they share a high degree of cross-resistance, which necessitated the development of so-called second-generation drugs of this class (DTG, CAB, and BIC) that could retain activity against these resistant variants. In vitro selection experiments have been instrumental to the clinical development of INSTIs, however they cannot completely recapitulate the situation in an HIV-positive individual. This review summarizes and compares all the currently available information as it pertains to both in vitro and in vivo selections with all five INSTIs, and the measured fold-changes in resistance of resistant variants in in vitro assays. While the selection of resistance substitutions in response to RAL and EVG bears high similarity in patients as compared to laboratory studies, there is less concurrence regarding the "second-generation" drugs of this class. This highlights the unpredictability of HIV resistance to these inhibitors, which is of concern as CAB and BIC proceed in their clinical development.

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.

Purification of Zika virus RNA-dependent RNA polymerase and its use to identify small-molecule Zika inhibitors.

Background: The viral RNA-dependent RNA polymerase (RdRp) enzymes of the Flaviviridae family are essential for viral replication and are logically important targets for development of antiviral therapeutic agents. Zika virus (ZIKV) is a rapidly re-emerging human pathogen for which no vaccine or antiviral agent is currently available. Methods: To facilitate development of ZIKV RdRp inhibitors, we have established an RdRp assay using purified recombinant ZIKV NS5 polymerase. Results: We have shown that both the hepatitis C virus (HCV) nucleoside inhibitor sofosbuvir triphosphate and a pyridoxine-derived non-nucleoside small-molecule inhibitor, DMB213, can act against ZIKV RdRp activity at IC 50 s of 7.3 and 5.2 µM, respectively, in RNA synthesis reactions catalysed by recombinant ZIKV NS5 polymerase. Cell-based assays confirmed the anti-ZIKV activity of sofosbuvir and DMB213 with 50% effective concentrations (EC 50 s) of 8.3 and 4.6 µM, respectively. Control studies showed that DMB213 did not inhibit recombinant HIV-1 reverse transcriptase and showed only very weak inhibition of HIV-1 integrase strand-transfer activity. The S604T substitution in motif B of the ZIKV RdRp, which corresponds to the S282T substitution in motif B of HCV RdRp, which confers resistance to nucleotide inhibitors, also conferred resistance to sofosbuvir triphosphate, but not to DMB213. Enzyme assays showed that DMB213 appears to be competitive with natural nucleoside triphosphate (NTP) substrates. Conclusions: Recombinant ZIKV RdRp assays can be useful tools for the screening of both nucleos(t)ide compounds and non-nucleotide metal ion-chelating agents that interfere with ZIKV replication.

Correcting covariate-dependent measurement error with non-zero mean.

There are many settings in which the distribution of error in a mismeasured covariate varies with the value of another covariate. Take, for example, the case of HIV phylogenetic cluster size, large values of which are an indication of rapid HIV transmission. Researchers wish to find behavioral correlates of HIV phylogenetic cluster size; however, the distribution of its measurement error depends on the correctly measured variable, HIV status, and does not have a mean of zero. Further, it is not feasible to obtain validation data or repeated measurements. We propose an extension of simulation-extrapolation, an estimation technique for bias reduction in the presence of measurement error that does not require validation data and can accommodate errors whose distribution depends on other, error-free covariates. The proposed extension performs well in simulation, typically exhibiting less bias and variability than either regression calibration or multiple imputation for measurement error. We apply the proposed method to data from the province of Quebec in Canada to examine the association between HIV phylogenetic cluster size and the number of reported sex partners. Copyright © 2017 John Wiley & Sons, Ltd.

Does antiretroviral treatment change HIV-1 codon usage patterns in its genes: a preliminary bioinformatics study.

BACKGROUND: Codon usage bias has been described for various organisms and is thought to contribute to the regulation of numerous biological processes including viral infections. HIV-1 codon usage has been previously shown to be different from that of other viruses and man. It is evident that the antiretroviral drugs used to restrict HIV-1 replication also select for resistance variants. We wanted to test whether codon frequencies in HIV-1 sequences from treatment-experienced patients differ from those of treatment-naive individuals due to drug pressure affecting codon usage bias. RESULTS: We developed a JavaScript to determine the codon frequencies of aligned nucleotide sequences. Irrespective of subtypes, using HIV-1 pol sequences from 532 treatment-naive and 52 treatment-experienced individuals, we found that pol sequences from treatment-experienced patients had significantly increased AGA (arginine; p = 0.0002***) and GGU (glycine; p = 0.0001***), and decreased AGG (arginine; p = 0.0001***) codon frequencies. The same pattern was not observed when subtypes B and C sequences were analyzed separately. Additionally, irrespective of subtypes, using HIV-1 gag sequences from 524 treatment-naive and 54 treatment-experienced individuals, gag sequences from treatment-experienced patients had significantly increased CUA (leucine; p < 0.0001***), CAG (glutamine; p = 0.0006***), AUC (isoleucine; p < 0.0001***) and UCU (serine; p = 0.0005***), and decreased AUA (isoleucine; p = 0.0003***) and CAA (glutamine; p = 0.0006***) codon frequencies. CONCLUSION: Using pol and gag genes derived from the same HIV-1 genome, we show that antiretroviral therapy changed certain HIV-1 codon frequencies in a subtype specific way.

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.

Beyond Condoms: Risk Reduction Strategies Among Gay, Bisexual, and Other Men Who Have Sex With Men Receiving Rapid HIV Testing in Montreal, Canada.

Gay, bisexual, and other men who have sex with men (MSM) have adapted their sexual practices over the course of the HIV/AIDS epidemic based on available data and knowledge about HIV. This study sought to identify and compare patterns in condom use among gay, bisexual, and other MSM who were tested for HIV at a community-based testing site in Montreal, Canada. Results showed that while study participants use condoms to a certain extent with HIV-positive partners and partners of unknown HIV status, they also make use of various other strategies such as adjusting to a partner's presumed or known HIV status and viral load, avoiding certain types of partners, taking PEP, and getting tested for HIV. These findings suggest that MSM who use condoms less systematically are not necessarily taking fewer precautions but may instead be combining or replacing condom use with other approaches to risk reduction.

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.

Evolution of a novel pathway leading to dolutegravir resistance in a patient harbouring N155H and multiclass drug resistance.

OBJECTIVES: Dolutegravir has been recently approved for treatment-naive and -experienced HIV-infected subjects, including integrase inhibitor (INI)-experienced patients. Dolutegravir is a second-generation INI that can overcome many prior raltegravir and elvitegravir failures. Here, we report the evolution of resistance to dolutegravir in a highly treatment-experienced patient harbouring the major N155H mutation consequent to raltegravir treatment failure. METHODS: Genotypic and phenotypic analyses were done on longitudinal samples to determine viral resistance to INIs. Integrase amino acid sequence interactions with raltegravir and dolutegravir were assessed by molecular modelling and docking simulations. RESULTS: Five mutations (A49P, L68FL, T97A, E138K and L234V) were implicated in emergent dolutegravir resistance, with a concomitant severe compromise in viral replicative capacity. Molecular modelling and docking simulations revealed that dolutegravir binding to integrase was affected by these acquired dolutegravir mutations. CONCLUSIONS: Our findings identify a novel mutational pathway involving integrase mutations A49P and L234V, leading to dolutegravir resistance in a patient with the N155H raltegravir mutation.

HIV-1 group O integrase displays lower enzymatic efficiency and higher susceptibility to raltegravir than HIV-1 group M subtype B integrase.

HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase coding region. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on enzyme function and susceptibility to integrase inhibitors. Accordingly, we cloned and purified integrase proteins from each of HIV-1 group O clades A and B, an HIV-O divergent strain, and HIV-1 group M (HIV-M, subtype B), used as a reference. To assess enzymatic function of HIV-O integrase, we carried out strand transfer and 3' processing assays with various concentrations of substrate (DNA target and long terminal repeats [LTR], respectively) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of various concentrations of several INSTIs. The inhibition constant (Ki) and 50% effective concentration (EC50) values were calculated for HIV-O integrases and HIV-O viruses, respectively, and compared with those of HIV-M. The results showed that HIV-O integrase displayed lower activity in strand transfer assays than did HIV-M enzyme, whereas 3' processing activities were similar to those of HIV-M. HIV-O integrases were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. Molecular modeling suggests that two key polymorphic residues that are close to the integrase catalytic site, 74I and 153A, may play a role in these differences.