Two Cases of Viral Re-suppression After M184V + R263K Selection on Dolutegravir/Lamivudine Without Treatment Modification.

Dolutegravir/lamivudine (DTG/3TC) has a high genetic barrier against the development of human immunodeficiency virus drug resistance. We report 2 cases of R263K + M184V mutations during DTG/3TC failure followed by viral suppression after adherence intervention without treatment change that we attribute to residual drug activity, reduced viral fitness, and robust immune competence.

The G118R plus R263K Combination of Integrase Mutations Associated with Dolutegravir-Based Treatment Failure Reduces HIV-1 Replicative Capacity and Integration.

Human immunodeficiency virus (HIV) treatment with antiretroviral regimens containing integrase strand transfer inhibitors such as dolutegravir (DTG) and bictegravir (BIC) offers high levels of protection against the development of drug resistance mutations. Despite this, resistance to DTG and BIC can occur through the development of the R263K integrase substitution. Failure with DTG has also been associated with the emergence of the G118R substitution. G118R and R263K are usually found separately but have been reported together in highly treatment-experienced persons who experienced treatment failure with DTG. We used cell-free strand transfer and DNA binding assays and cell-based infectivity, replicative capacity, and resistance assays to characterize the G118R plus R263K combination of integrase mutations. R263K reduced DTG and BIC susceptibility ~2-fold, in agreement with our previous work. Single-cycle infectivity assays showed that G118R and G118R plus R263K conferred ~10-fold resistance to DTG. G118R alone conferred low levels of resistance to BIC (3.9-fold). However, the G118R plus R263K combination conferred high levels of resistance to BIC (33.7-fold), likely precluding the use of BIC after DTG failure with the G118R plus R263K combination. DNA binding, viral infectivity, and replicative capacity of the double mutant were further impaired, compared to single mutants. We propose that impaired fitness helps to explain the scarcity of the G118R plus R263K combination of integrase substitutions in clinical settings and that immunodeficiency likely contributes to its development.

Progressive emergence of an S153F plus R263K combination of integrase mutations in the proviral DNA of one individual successfully treated with dolutegravir.

OBJECTIVES: The development of HIV drug resistance against the integrase strand transfer inhibitor dolutegravir is rare. We report here the transient detection, by near full-genome ultradeep sequencing, of minority HIV-1 subtype B variants bearing the S153F and R263K integrase substitutions in the proviral DNA from blood cells of one patient who successfully initiated dolutegravir-based ART, over 24 weeks. Our objective was to study the effects of these substitutions. METHODS: Strand transfer and DNA-binding activities of recombinant integrase proteins were measured in cell-free assays. Cell-based resistance, infectivity and replicative capacities were measured using molecular clones. Structural modelling was performed to understand experimental results. RESULTS: R263K emerged first, followed by the addition of S153F at Week 12. By Week 24, both mutations remained present, but at lower prevalence. We confirmed the coexistence of S153F and R263K on single viral genomes. Combining S153F or S153Y with R263K decreased integration and viral replicative capacity and conferred high levels of drug resistance against all integrase inhibitors. Alone, S153Y and S153F did little to infectivity or dolutegravir resistance. We identified altered DNA binding as a mechanism of resistance. The patient remained with undetectable viral loads at all timepoints. CONCLUSIONS: Drug-resistant minority variants have often been reported under suppressive ART. Our study adds to these observations by unravelling a progression towards higher levels of resistance through a novel pathway despite continuous undetectable viral loads. Poorly replicative HIV drug-resistant minority proviral variants did not compromise viral suppression in one individual treated with dolutegravir.

Dolutegravir Monotherapy of Simian Immunodeficiency Virus-Infected Macaques Selects for Several Patterns of Resistance Mutations with Variable Virological Outcomes.

Drug resistance remains a major concern for human immunodeficiency virus (HIV) treatment. To date, very few resistance mutations have emerged in first-line combination therapy that includes the integrase strand transfer inhibitor (INSTI) dolutegravir (DTG). In vitro, DTG selects for several primary mutations that induce low-level DTG resistance; secondary mutations, while increasing the level of resistance, however, further impair replication fitness, which raised the idea that DTG monotherapy may be feasible. The simian immunodeficiency virus (SIV) rhesus macaque model of HIV infection can be useful to explore this concept. Nine macaques were infected with virulent SIVmac251 and started on DTG monotherapy during either acute (n = 2) or chronic infection (n = 7). Within 4 weeks of treatment, all animals demonstrated a reduction in viremia of 0.8 to 3.5 log RNA copies/ml plasma. Continued treatment led to overall sustained benefits, but the outcome after 10 to 50 weeks of treatment was highly variable and ranged from viral rebound to near pretreatment levels to sustained suppression, with viremia being 0.5 to 5 logs lower than expected based on pretreatment viremia. A variety of mutations previously described to confer low-level resistance of HIV-1 to DTG or other INSTI were detected, and these were sometimes followed by mutations believed to be compensatory. Some mutations, such as G118R, previously shown to severely impair the replication capacity in vitro, were associated with more sustained virological and immunological benefits of continued DTG therapy, while other mutations, such as E92Q and G140A/Q148K, were associated with more variable outcomes. The observed variability of the outcomes in macaques warrants avoidance of DTG monotherapy in HIV-infected people.IMPORTANCE A growing number of anti-HIV drug combinations are effective in suppressing virus replication in HIV-infected persons. However, to reduce their cost and risk for toxicity, there is considerable interest in simplifying drug regimens. A major concern with single-drug regimens is the emergence of drug-resistant viral mutants. It has been speculated that DTG monotherapy may be a feasible option, because DTG may have a higher genetic barrier for the development of drug resistance than other commonly used antiretrovirals. To explore treatment initiation with DTG monotherapy, we started SIV-infected macaques on DTG during either acute or chronic infection. Although DTG initially reduced virus replication, continued treatment led to the emergence of a variety of viral mutations previously described to confer low-level resistance of HIV-1 to DTG, and this was associated with variable clinical outcomes. This unpredictability of mutational pathways and outcomes warns against using DTG monotherapy as initial treatment for HIV-infected people.

Diverse Types of Diterpenoids with an Aromatized C Ring from the Twigs of Podocarpus imbricatus.

An ethanol extract of the powdered twigs of Podocarpus imbricatus afforded 14 new diterpenoids (1-14), which all share an aromatized C ring. These isolates belong to five diterpenoid types that include abietanes (1-3), semperviranes (4-9), totaranes (10-12), a C-17 norabietane (13), and an icetexane (14). Their structures were assigned mainly by analysis of the spectroscopic data, and the absolute configuration of 1 was determined by X-ray crystallography. A biosynthetic pathway for five of the biogenetically related types of diterpenoids was proposed. Compound 7 showed moderate inhibitory activity against Zika virus with an IC50 value of 2.5 µM.

Antimalarial drugs and their metabolites are potent Zika virus inhibitors.

Studies aimed at repurposing existing drugs revealed that some antimalarial compounds possess anti-Zika virus (anti-ZIKV) activity. Here, we further tested 14 additional antimalarial drugs and their metabolites or analogs for anti-ZIKV activity using a phenotypic screening approach. We identified four compounds with varying anti-ZIKV activity, including a metabolite of amodiaquine termed desethylamodiaquine (DAQ) and N-desethylchloroquine (DECQ), a metabolite of chloroquine, which both exhibited low micromolar effective concentrations against three different ZIKV strains. Two other compounds termed dihydroartemisinin (DHA) and quinidine (QD) exhibited only partial inhibition of ZIKV replication. Characterization of the inhibitory mechanisms of DAQ and DECQ showed that both drugs target the entry step as well as postentry events of the viral replication cycle. These hits represent attractive starting points for future optimization of new anti-ZIKV drug candidates derived from antimalarial drugs and their analogs.

HIV-1 Resistance Dynamics in Patients With Virologic Failure to Dolutegravir Maintenance Monotherapy.

Background: A high genetic barrier to resistance to the integrase strand transfer inhibitor (INSTI) dolutegravir has been reported in vitro and in vivo. We describe the dynamics of INSTI resistance-associated mutations (INSTI-RAMs) and mutations in the 3'-polypurine tract (3'-PPT) in relation to virologic failure (VF) observed in the randomized Dolutegravir as Maintenance Monotherapy for HIV-1 study (DOMONO, NCT02401828). Methods: From 10 patients with VF, plasma samples were collected before the start of cART and during VF, and were used to generate Sanger sequences of integrase, the 5' terminal bases of the 3' long terminal repeat (LTR), and the 3'-PPT. Results: Median human immunodeficiency virus RNA load at VF was 3490 copies/mL (interquartile range 1440-4990 copies/mL). INSTI-RAMs (S230R, R263K, N155H, and E92Q+N155H) were detected in 4 patients, no INSTI-RAMs were detected in 4 patients, and sequencing of the integrase gene was unsuccessful in 2 patients. The time to VF ranged from 4 weeks to 72 weeks. In 1 patient, mutations developed in the highly conserved 3'-PPT. No changes in the terminal bases of the 3'-LTR were observed. Conclusions: The genetic barrier to resistance is too low to justify dolutegravir maintenance monotherapy because single INSTI-RAMs are sufficient to cause VF. The large variation in time to VF suggests that stochastic reactivation of a preexisting provirus containing a single INSTI-RAM is the mechanism for failure. Changes in the 3'-PPT point to a new dolutegravir resistance mechanism in vivo. Clinical Trials Registration: NCT02401828.

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.

Dolutegravir reshapes the genetic diversity of HIV-1 reservoirs.

Objectives: Better understanding of the dynamics of HIV reservoirs under ART is a critical step to achieve a functional HIV cure. Our objective was to assess the genetic diversity of archived HIV-1 DNA over 48 weeks in blood cells of individuals starting treatment with a dolutegravir-based regimen. Methods: Eighty blood samples were prospectively and longitudinally collected from 20 individuals (NCT02557997) including: acutely (n = 5) and chronically (n = 5) infected treatment-naive individuals, as well as treatment-experienced individuals who switched to a dolutegravir-based regimen and were either virologically suppressed (n = 5) or had experienced treatment failure (n = 5). The integrase and V3 loop regions of HIV-1 DNA isolated from PBMCs were analysed by pyrosequencing at baseline and weeks 4, 24 and 48. HIV-1 genetic diversity was calculated using Shannon entropy. Results: All individuals achieved or maintained viral suppression throughout the study. A low and stable genetic diversity of archived HIV quasispecies was observed in individuals starting treatment during acute infection. A dramatic reduction of the genetic diversity was observed at week 4 of treatment in the other individuals. In these patients and despite virological suppression, a recovery of the genetic diversity of the reservoirs was observed up to 48 weeks. Viral variants bearing dolutegravir resistance-associated substitutions at integrase position 50, 124, 230 or 263 were detected in five individuals (n = 5/20, 25%) from all groups except those who were ART-failing at baseline. None of these substitutions led to virological failure. Conclusions: These data demonstrate that the genetic diversity of the HIV-1 reservoir is reshaped following the initiation of a dolutegravir-based regimen and strongly suggest that HIV-1 can continue to replicate despite successful treatment.

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.

JAK-STAT Signaling Pathways and Inhibitors Affect Reversion of Envelope-Mutated HIV-1.

HIV can spread by both cell-free and cell-to-cell transmission. Here, we show that many of the amino acid changes in Env that are close to the CD4 binding pocket can affect HIV replication. We generated a number of mutant viruses that were unable to infect T cells as cell-free viruses but were nevertheless able to infect certain T cell lines as cell-associated viruses, which was followed by reversion to the wild type. However, the activation of JAK-STAT signaling pathways caused the inhibition of such cell-to-cell infection as well as the reversion of multiple HIV Env mutants that displayed differences in their abilities to bind to the CD4 receptor. Specifically, two T cell activators, interleukin-2 (IL-2) and phorbol 12-myristate 13-acetate (PMA), both capable of activation of JAK-STAT pathways, were able to inhibit cell-to-cell viral transmission. In contrast, but consistent with the above result, a number of JAK-STAT and mTOR inhibitors actually promoted HIV-1 transmission and reversion. Hence, JAK-STAT signaling pathways may differentially affect the replication of a variety of HIV Env mutants in ways that differ from the role that these pathways play in the replication of wild-type viruses.IMPORTANCE Specific alterations in HIV Env close to the CD4 binding site can differentially change the ability of HIV to mediate infection for cell-free and cell-associated viruses. However, such differences are dependent to some extent on the types of target cells used. JAK-STAT signaling pathways are able to play major roles in these processes. This work sheds new light on factors that can govern HIV infection of target cells.

The antimalarial drug amodiaquine possesses anti-ZIKA virus activities.

Zika virus (ZIKV) outbreak has emerged as a global health threat, particularly in tropical areas, over the past few years. No antiviral therapy or vaccine is available at present. For these reasons, repurposing clinically approved drugs against ZIKV infection may provide rapid and cost-effective global health benefits. Here, we explored this strategy and screened eight FDA-approved drugs for antiviral activity against ZIKV using a cell-based assay. Our results show that the antimalarial drug amodiaquine has anti-ZIKV activity with EC50 at low micromolar concentrations in cell culture. We further characterized amodiaquine antiviral activity against ZIKV and found that it targets early events of the viral replication cycle. Altogether, our results suggest that amodiaquine may be efficacious for the treatment of ZIKV infection.

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.

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.

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.

Effect on HIV-1 viral replication capacity of DTG-resistance mutations in NRTI/NNRTI resistant viruses.

BACKGROUND: Recommended regimens for HIV-positive individuals include the co-administration of dolutegravir (DTG) with two reverse transcriptase inhibitors (RTIs). Although rare, emerging resistance against DTG is often associated with the R263K substitution in integrase. In-vitro-selected R263K was associated with impaired viral replication capacity, DNA integration, and integrase strand-transfer activity, especially when accompanied by the secondary mutation H51Y. Given the reduced fitness of RTI-resistant viruses, we investigated potential impacts on viral replication of combining R263K and H51Y/R263K with major RTI-resistance substitutions including K65R, L74V, K103N, E138K, and M184I/V. RESULTS: We combined the R263K or H51Y/R263K with RTI-resistance mutations into the proviral plasmid pNL4.3 and measured the resulting viral infectiousness, replication capacity, and ability to integrate viral DNA into host cells. Infectiousness was determined by luciferase assay in TZM-bl cells. Replicative capacity was monitored over 7 days and viral DNA integration was studied by real-time Alu-qPCR in PM1 cells. We found that viral infectiousness, replication capacities and integration levels were greatly reduced in triple mutants, i.e. H51Y/R263K plus a RT mutation, and moderately reduced in double mutants, i.e. R263K plus a RT mutation, compared to wild-type and single RT-mutant viruses. CONCLUSIONS: Our findings help to explain the absence of RTI mutations in individuals who experienced DTG-treatment failure.