Nathan Osman

Viral fitness cost prevents HIV-1 from evading dolutegravir drug pressure

BackgroundClinical studies have shown that integrase strand transfer inhibitors can be used to treat HIV-1 infection. Although the first-generation integrase inhibitors are susceptible to the emergence of resistance mutations that impair their efficacy in therapy, such resistance has not been identified to date in drug-naïve patients who have been treated with the second-generation inhibitor dolutegravir. During previous in vitro selection study, we identified a R263K mutation as the most common substitution to arise in the presence of dolutegravir with H51Y arising as a secondary mutation. Additional experiments reported here provide a plausible explanation for the absence of reported dolutegravir resistance among integrase inhibitor-naïve patients to date.ResultsWe now show that H51Y in combination with R263K increases resistance to dolutegravir but is accompanied by dramatic decreases in both enzymatic activity and viral replication.ConclusionsSince H51Y and R263K may define a unique resistance pathway to dolutegravir, our results are consistent with the absence of resistance mutations in antiretroviral drug-naive patients treated with this drug.

The M50I polymorphic substitution in association with the R263K mutation in HIV-1 subtype B integrase increases drug resistance but does not restore viral replicative fitness

BackgroundFirst-generation integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL) and elvitegravir (EVG), have been clinically proven to be effective antiretrovirals for the treatment of HIV-positive patients. However, their relatively low genetic barrier for resistance makes them susceptible to the emergence of drug resistance mutations. In contrast, dolutegravir (DTG) is a newer INSTI that appears to have a high genetic barrier to resistance in vivo. However, the emergence of the resistance mutation R263K followed by the polymorphic substitution M50I has been observed in cell culture. The M50I polymorphism is also observed in 10-25% of INSTI-naïve patients and has been reported in combination with R263K in a patient failing treatment with RAL.ResultsUsing biochemical cell-free strand-transfer assays and resistance assays in TZM-bl cells, we demonstrate that the M50I polymorphism in combination with R263K increases resistance to DTG in tissue culture and in biochemical assays but does not restore the viral fitness cost associated with the R263K mutation.ConclusionsSince the combination of the R263K mutation and the M50I polymorphism results in a virus with decreased viral fitness and limited cross-resistance, the R263K resistance pathway may represent an evolutionary dead-end. Although this hypothesis has not yet been proven, it may be more advantageous to treat HIV-positive individuals with DTG in first-line than in second or third-line therapy.

Biochemical Analysis of the Role of G118R-Linked Dolutegravir Drug Resistance Substitutions in HIV-1 Integrase

ABSTRACT Drug resistance mutations (DRMs) have been reported for all currently approved anti-HIV drugs, including the latest integrase strand transfer inhibitors (INSTIs). We previously used the new INSTI dolutegravir (DTG) to select a G118R integrase resistance substitution in tissue culture and also showed that secondary substitutions emerged at positions H51Y and E138K. Now, we have characterized the impact of the G118R substitution, alone or in combination with either H51Y or E138K, on 3′ processing and integrase strand transfer activity. The results show that G118R primarily impacted the strand transfer step of integration by diminishing the ability of integrase-long terminal repeat (LTR) complexes to bind target DNA. The addition of H51Y and E138K to G118R partially restored strand transfer activity by modulating the formation of integrase-LTR complexes through increasing LTR DNA affinity and total DNA binding, respectively. This unique mechanism, in which one function of HIV integrase partially compensates for the defect in another function, has not been previously reported. The G118R substitution resulted in low-level resistance to DTG, raltegravir (RAL), and elvitegravir (EVG). The addition of either of H51Y or E138K to G118R did not enhance resistance to DTG, RAL, or EVG. Homology modeling provided insight into the mechanism of resistance conferred by G118R as well as the effects of H51Y or E138K on enzyme activity. The G118R substitution therefore represents a potential avenue for resistance to DTG, similar to that previously described for the R263K substitution. For both pathways, secondary substitutions can lead to either diminished integrase activity and/or increased INSTI susceptibility.

Addition of E138K to R263K in HIV integrase increases resistance to dolutegravir, but fails to restore activity of the HIV integrase enzyme and viral replication capacity

BACKGROUND The results of several clinical trials suggest that the integrase inhibitor dolutegravir may be less prone than other drugs to the emergence of HIV drug resistance mutations in treatment-naive patients. We have shown that the R263K mutation commonly emerged during tissue culture selection studies with dolutegravir and conferred low levels of resistance to this drug while simultaneously diminishing both HIV replication capacity and integrase enzymatic activity. E138K has been identified as a secondary mutation for dolutegravir in selection studies and has also been observed as a secondary mutation in the clinic for the integrase inhibitors raltegravir and elvitegravir. METHODS We used biochemical cell-free strand-transfer assays and tissue culture assays to characterize the effects of the E138K/R263K combination of mutations on resistance to dolutegravir, integrase enzyme activity and HIV-1 replication capacity. RESULTS We show here that the addition of the E138K substitution to R263K increased the resistance of HIV-1 to dolutegravir but failed to restore viral replication capacity, integrase strand-transfer activity and integration within cellular DNA. We also show that the addition of E138K to R263K did not increase the resistance to raltegravir or elvitegravir. The addition of the E138K substitution to R263K was also less detrimental to integrase strand-transfer activity and integration than a different secondary mutation at position H51Y that had also been selected in culture. CONCLUSIONS The E138K substitution failed to restore the defect in viral replication capacity that is associated with R263K, confirming previous selection studies that failed to identify compensatory mutation(s) for the latter primary mutation. This study suggests that the R263K resistance pathway may represent an evolutionary dead end for HIV in treatment-naive individuals who are treated with dolutegravir and will need to be confirmed by the long-term use of dolutegravir in the clinic.

Differential Effects of the G118R, H51Y, and E138K Resistance Substitutions in Different Subtypes of HIV Integrase

ABSTRACT Dolutegravir (DTG) is the latest antiretroviral (ARV) approved for the treatment of human immunodeficiency virus (HIV) infection. The G118R substitution, previously identified with MK-2048 and raltegravir, may represent the initial substitution in a dolutegravir resistance pathway. We have found that subtype C integrase proteins have a low enzymatic cost associated with the G118R substitution, mostly at the strand transfer step of integration, compared to either subtype B or recombinant CRF02_AG proteins. Subtype B and circulating recombinant form AG (CRF02_AG) clonal viruses encoding G118R-bearing integrases were severely restricted in their viral replication capacity, and G118R/E138K-bearing viruses had various levels of resistance to dolutegravir, raltegravir, and elvitegravir. In cell-free experiments, the impacts of the H51Y and E138K substitutions on resistance and enzyme efficiency, when present with G118R, were highly dependent on viral subtype. Sequence alignment and homology modeling showed that the subtype-specific effects of these mutations were likely due to differential amino acid residue networks in the different integrase proteins, caused by polymorphic residues, which significantly affect native protein activity, structure, or function and are important for drug-mediated inhibition of enzyme activity. This preemptive study will aid in the interpretation of resistance patterns in dolutegravir-treated patients. IMPORTANCE Recognized drug resistance mutations have never been reported for naive patients treated with dolutegravir. Additionally, in integrase inhibitor-experienced patients, only R263K and other previously known integrase resistance substitutions have been reported. Here we suggest that alternate resistance pathways may develop in non-B HIV-1 subtypes and explain how “minor” polymorphisms and substitutions in HIV integrase that are associated with these subtypes can influence resistance against dolutegravir. This work also highlights the importance of phenotyping versus genotyping when a strong inhibitor such as dolutegravir is being used. By characterizing the G118R substitution, this work also preemptively defines parameters for a potentially important pathway in some non-B HIV subtype viruses treated with dolutegravir and will aid in the inhibition of such a virus, if detected. The general inability of strand transfer-related substitutions to diminish 3′ processing indicates the importance of the 3′ processing step and highlights a therapeutic angle that needs to be better exploited.

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.

The R263K substitution in HIV-1 subtype C is more deleterious for integrase enzymatic function and viral replication than in subtype B.

Objectives:Dolutegravir is an integrase strand-transfer inhibitor that has shown unprecedented robustness against the emergence of HIV drug-resistant strains in treatment-naive individuals. The R263K substitution in integrase was identified through culture selection as a resistance-associated substitution for dolutegravir and was recently detected in two treatment-experienced participants in the SAILING clinical trial, who experienced dolutegravir-based treatment failure, one of whom was infected by a subtype C virus. The objective of this study was to characterize the R263K substitution in HIV-1 subtype C integrase. Design and methods:We used cell-free strand transfer assays and tissue culture experiments to characterize the R263K substitution in HIV-1 subtype C integrase in comparison with subtype B. Results:Cell-free biochemical assays showed that the R263K substitution diminished subtype C integrase strand-transfer activity by decreasing the affinity of integrase for target DNA. Similarly, both viral infectiousness and replication capacity were reduced by the R263K substitution in tissue culture. Decrease in enzyme activity and viral infectiousness exceeded 35 and 50%, respectively – significantly more than in HIV-1 subtype B. R263K in HIV-1 subtype C also conferred low levels of resistance against dolutegravir and high levels of cross-resistance against elvitegravir, but not raltegravir. Conclusions:The R263K substitution is more deleterious to integrase strand-transfer activity and viral infectiousness in HIV-1 subtype C than in subtype B. Our results suggest that cross-resistance may prevent treatment-experienced individuals who are experiencing treatment failure with dolutegravir from being subsequently treated with elvitegravir.

Evaluation of Sofosbuvir (β-D-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine) as an inhibitor of Dengue virus replication #

We evaluated Sofosbuvir (SOF), the anti-hepatitis C virus prodrug of β-d-2′-deoxy-2′-α-fluoro-2′-β-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.

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

ABSTRACT 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.