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.

The Role of Phylogenetics in Unravelling Patterns of HIV Transmission towards Epidemic Control: The Quebec Experience (2002-2020).

Phylogenetics has been advanced as a structural framework to infer evolving trends in the regional spread of HIV-1 and guide public health interventions. In Quebec, molecular network analyses tracked HIV transmission dynamics from 2002-2020 using MEGA10-Neighbour-joining, HIV-TRACE, and MicrobeTrace methodologies. Phylogenetics revealed three patterns of viral spread among Men having Sex with Men (MSM, n = 5024) and heterosexuals (HET, n = 1345) harbouring subtype B epidemics as well as B and non-B subtype epidemics (n = 1848) introduced through migration. Notably, half of new subtype B infections amongst MSM and HET segregating as solitary transmissions or small cluster networks (2-5 members) declined by 70% from 2006-2020, concomitant to advances in treatment-as-prevention. Nonetheless, subtype B epidemic control amongst MSM was thwarted by the ongoing genesis and expansion of super-spreader large cluster variants leading to micro-epidemics, averaging 49 members/cluster at the end of 2020. The growth of large clusters was related to forward transmission cascades of untreated early-stage infections, younger at-risk populations, more transmissible/replicative-competent strains, and changing demographics. Subtype B and non-B subtype infections introduced through recent migration now surpass the domestic epidemic amongst MSM. Phylodynamics can assist in predicting and responding to active, recurrent, and newly emergent large cluster networks, as well as the cryptic spread of HIV introduced through migration.

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.

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.

Durable suppression of HIV-1 with resistance mutations to integrase inhibitors by dolutegravir following drug washout.

OBJECTIVES: Dolutegravir (DTG) has achieved better long-term suppression of HIV-1 replication than other integrase strand transfer inhibitors (INSTIs), such as raltegravir (RAL) and elvitegravir (EVG). In in-vitro drug washout experiments, we previously showed that removal of DTG from pretreated MT-2 cells infected with wild-type HIV-1 showed slower rebound in viral replication as compared to removal of RAL. Now, we performed DTG, EVG and RAL washout experiments to compare the recovery of viral integration and production of 2-long terminal repeat (LTR) circles using wild-type HIV-1 clones, R263K viruses with low-level resistance to DTG and viruses with G140S/Q148H mutations showing cross-resistance against all currently approved INSTIs. DESIGN AND METHODS: MT-2 cells infected with wild-type, R263K or G140S/Q148H HIV-1 clones were treated with DTG, RAL or EVG for 3 days. Viral rebound following drug washout was assessed, monitoring viral integration and 2-LTR circle production by qPCR. RESULTS: Viral integration did not resume for up to 8 days after DTG washout from the wild-type or R263K infections but increased soon after washout of either RAL or EVG. With the G140S/Q148H virus, levels of integration were not significantly affected by the presence of either RAL or EVG. With DTG, integration was much lower at 3 days after infection than for the no-drug control. At 8 days after DTG washout, viral integration resumed but remained relatively low. CONCLUSION: DTG antiretroviral activity in tissue culture is more durable than that of either RAL or EVG after drug washout and this is true for both wild-type and drug-resistant viruses.

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.

Evaluation of Sofosbuvir (ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methyluridine) as an inhibitor of Dengue virus replication.

We evaluated Sofosbuvir (SOF), the anti-hepatitis C virus prodrug of ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methyluridine-5'-monophosphate, for potential inhibitory activity against DENV replication. Both cell-based and biochemical assays, based on use of purified DENV full-length NS5 enzyme, were studied. Cytopathic effect protection and virus yield reduction assays confirmed that SOF possessed anti-DENV activity in cell culture with a 50% effective concentration (EC50) of 4.9 µM and 1.4 µM respectively. Real-time RT-PCR verified that SOF inhibits generation of viral RNA with an EC50 of 9.9 µM. Purified DENV NS5 incorporated the active triphosphate form (SOF-TP) into nascent RNA, causing chain-termination. Relative to the natural UTP, the incorporation efficiency of SOF-TP was low (discrimination value = 327.5). In a primer extension assay, SOF-TP was active against DENV NS5 wild-type polymerase activity with an IC50 of 14.7 ± 2.5 µM. The S600T substitution in the B Motif of DENV polymerase conferred 4.3-fold resistance to SOF-TP; this was due to decreased incorporation efficiency rather than enhanced excision of the incorporated SOF nucleotide. SOF has antiviral activity against DENV replication. The high discrimination value in favor of UTP in enzyme assays may not necessarily preclude antiviral activity in cells. SOF may be worthy of evaluation against severe DENV infections in humans.

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.

Identification of resveratrol analogs as potent anti-dengue agents using a cell-based assay.

Dengue virus (DENV) causes a variety of difficult-to-treat diseases that threaten almost half of the world's population. Currently, no effective vaccine or antiviral therapy is available. We have examined a series of synthetic resveratrol analogs to identify potential anti-DENV agents. Here, we demonstrate that two resveratrol analogs, PNR-4-44 and PNR-5-02, possess potent anti-DENV activity with EC50 values in the low nanomolar range. These two resveratrol analogs were shown to mainly target viral RNA translation and viral replication, but PNR-5-02 is also likely to target cellular factors inside host cells. Although the precise molecular mechanism(s) mediating anti-DENV activities have not been elucidated, further structure-guided design might lead to the development of newer improved resveratrol derivatives that might have therapeutic value. J. Med. Virol. 89:397-407, 2017. © 2016 Wiley Periodicals, Inc.

The M184I/V and K65R nucleoside resistance mutations in HIV-1 prevent the emergence of resistance mutations against dolutegravir.

OBJECTIVE: Recommended treatments for newly diagnosed HIV-positive individuals now focus on the integrase strand transfer inhibitors, raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG). In treatment-naive individuals, cases of RAL-based and EVG-based virological failure, although rare, are associated with the occurrence of resistance mutations in integrase and/or reverse transcriptase coding sequences. In such cases, common resistance substitutions in reverse transcriptase that were associated with nucleos(t)ide reverse transcriptase inhibitors included M184I/V and K65R and these occurred together with various mutations in integrase. In some instances, these mutations in reverse transcriptase preceded the emergence of mutations in integrase. In contrast, no resistance substitutions in either integrase or reverse transcriptase have been observed to date in viruses isolated from treatment-naive individuals who experienced treatment failure with DTG-based regimens. DESIGN: The objective of this study was to determine the effects of the M184I/V and K65R substitutions in reverse transcriptase on the ability of HIV-1 to become resistant against RAL, EVG or DTG. METHODS: We performed tissue culture selection experiments using reverse transcriptase inhibitor-resistant viruses containing resistance substitutions at positions K65R, M184I or M184V in the presence of increasing concentrations of RAL, EVG or DTG and monitored changes in integrase sequences by genotyping. RESULTS: Selections using EVG and RAL led to the emergence of resistance mutations in integrase. In contrast, only the wild-type virus was able to acquire resistance mutations for DTG. CONCLUSION: Resistance mutations against nucleos(t)ide reverse transcriptase inhibitors antagonized the development of HIV-1 resistance against DTG but not RAL or EVG.

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.

Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase.

The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 µM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 µM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor.

The dolutegravir R263K resistance mutation in HIV-1 integrase is incompatible with the emergence of resistance against raltegravir.

OBJECTIVE: Although the integrase inhibitor dolutegravir (DTG) has demonstrated greater resilience than other antiretroviral drugs at withstanding the emergence of HIV-1 resistance mutations, such substitutions can develop, albeit rarely, in treatment-experienced integrase inhibitor-naïve individuals. The most common substitution in integrase under those circumstances is R263K whereas another substitution that was selected against DTG in tissue culture was G118R. The objective of this study was to determine the effects of these DTG-specific resistance substitutions on the ability of HIV-1 to become resistant against either of two other integrase inhibitors, raltegravir (RAL) and elvitegravir (EVG). DESIGN AND METHODS: We performed tissue culture selection experiments using DTG-resistant viruses containing integrase substitutions at positions R263K, H51Y/R263K, E138K/R263K, G118R and H51Y/G118R in the presence of increasing concentrations of either RAL or EVG. Changes in integrase sequences were monitored by genotyping. RESULTS: The presence of the R263K substitution delayed the emergence of resistance against RAL whereas the simultaneous presence of either the H51Y or E138K secondary substitutions in combination with R263K somewhat mitigated this inhibitory effect. In contrast, resistance against EVG appeared earlier than in wild-type virus in viruses containing the R263K and E138K/R263K DTG-associated resistance substitutions. CONCLUSION: The DTG-resistant R263K substitution antagonized the development of HIV-1 resistance against RAL while partially facilitating the occurrence of resistance against EVG.

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 Combination of the R263K and T66I Resistance Substitutions in HIV-1 Integrase Is Incompatible with High-Level Viral Replication and the Development of High-Level Drug Resistance.

UNLABELLED: The R263K substitution in integrase has been selected in tissue culture with dolutegravir (DTG) and has been reported for several treatment-experienced individuals receiving DTG as part of salvage therapy. The R263K substitution seems to be incompatible with the presence of common resistance mutations associated with raltegravir (RAL), a different integrase strand transfer inhibitor (INSTI). T66I is a substitution that is common in individuals who have developed resistance against a different INSTI termed elvitegravir (EVG), but it is not known whether these two mutations might be compatible in the context of resistance against DTG or what impact the combination of these substitutions might have on resistance against INSTIs. E138K is a common secondary substitution observed with various primary resistance substitutions in RAL- and EVG-treated individuals. Viral infectivity, replicative capacity, and resistance against INSTIs were measured in cell-based assays. Strand transfer and 3' processing activities were measured biochemically. The combination of the R263K and T66I substitutions decreased HIV-1 infectivity, replicative capacity, and strand transfer activity. The addition of the E138K substitution partially compensated for these deficits and resulted in high levels of resistance against EVG but not against DTG or RAL. These findings suggest that the presence of the T66I substitution will not compromise the activity of DTG and may also help to prevent the additional generation of the R263K mutation. Our observations support the use of DTG in second-line therapy for individuals who experience treatment failure with EVG due to the T66I substitution. IMPORTANCE: The integrase strand transfer inhibitors (INSTIs) elvitegravir and dolutegravir are newly developed inhibitors against human immunodeficiency virus type 1 (HIV-1). HIV drug-resistant mutations in integrase that can arise in individuals treated with elvitegravir commonly include the T66I substitution, whereas R263K is a signature resistance substitution against dolutegravir. In order to determine how different combinations of integrase resistance mutations can influence the outcome of therapy, we report here the effects of the T66I, E138K, and R263K substitutions, alone and in combination, on viral replicative capacity and resistance to integrase inhibitors. Our results show that the addition of R263K to the T66I substitution diminishes viral replicative capacity and strand transfer activity while not compromising susceptibility to dolutegravir. This supports the use of dolutegravir in second-line therapy for patients failing elvitegravir therapy who harbor the T66I resistance substitution.

Dolutegravir maintains a durable effect against HIV replication in tissue culture even after drug washout.

OBJECTIVES: Of the currently approved HIV integrase strand transfer inhibitors (INSTIs), dolutegravir has shown greater efficacy than raltegravir at suppressing HIV-1 replication in treatment-experienced individuals. Biochemical experiments have also shown that dolutegravir has a longer dissociative half-life when bound to HIV integrase than does raltegravir. In order to study the intracellular efficacy of various INSTIs, we asked whether drug removal from INSTI-treated HIV-1-infected cells would result in different times to viral rebound. In addition, we assessed the role of the R263K substitution within the integrase ORF that is associated with low-level resistance to dolutegravir. METHODS: HIV-infected MT-2 cells were treated with dolutegravir, raltegravir or a third experimental INSTI (MK-2048) and the drugs were washed out after varying times. Viral replication was monitored by measuring reverse transcriptase (RT) activity in the culture fluids. RESULTS: We observed a significantly slower increase in RT activity after the removal of dolutegravir compared with raltegravir or MK-2048. The incubation time before the drug was removed also had an impact on the level of RT activity independently of the drug and virus used. The R263K substitution did not significantly impact on levels of RT activity after drug washout, suggesting that dolutegravir remained tightly bound to the integrase enzyme despite the presence of this mutation. CONCLUSIONS: These results suggest that the residency time of INSTIs on integrase is a key factor in the activity of these drugs and that the anti-HIV activity of dolutegravir persists more effectively than that of other INSTIs after drug washout.

Identification of a dibenzocyclooctadiene lignan as a HIV-1 non-nucleoside reverse transcriptase inhibitor.

BACKGROUND: Due to resistance to all classes of anti-HIV drugs and drug toxicity, there is a need for the discovery and development of new anti-HIV drugs. METHODS: HIV-1 inhibitors were identified and biologically characterized for mechanism of action. RESULTS: We identified a dibenzocyclooctadiene lignan, termed HDS2 that possessed anti-HIV activity against a wide variety of viral strains with EC50 values in the 1-3 µM range. HDS2 was shown to act as an NNRTI by qPCR and in vitro enzyme assays. CONCLUSIONS: This compound provides a new scaffold for further optimization of activity through structure-guided design.