Dolutegravir-Selected HIV-1 Containing the N155H and R263K Resistance Substitutions Does Not Acquire Additional Compensatory Mutations under Drug Pressure That Lead to Higher-Level Resistance and Increased Replicative Capacity

Abstract

We have previously shown that the addition of the raltegravir/elvitegavir (RAL/EVG) primary resistance mutation N155H to the R263K dolutegravir (DTG) resistance mutation partially compensated for the fitness cost imposed by R263K while also slightly increasing DTG resistance in vitro (K. Anstett, T. Mesplede, M. Oliveira, V. Cutillas, and M. A. Wainberg, J Virol 89:4681-4684, 2015, doi:10.1128/JVI.03485-14). Since many patients failing RAL/EVG are given DTG as part of rescue therapy, and given that the N155H substitution often is found in combination with other compensatory resistance mutations in such individuals, we investigated the effects of multiple such substitutions within integrase (IN) on each of integrase function, HIV-1 infectivity, and levels of drug resistance. To this end, each of the L74M, E92Q, T97A, E157Q, and G163R substitutions were introduced into NL4.3 subtype B HIV-1 vectors harboring N155H and R263K in tandem [termed NL4.3 IN(N155H/R263K) ]. Relevant recombinant integrase enzymes also were expressed, and purified and biochemical assays of strand transfer efficiency as well as viral infectivity and drug resistance studies were performed. We found that the addition of T97A, E157Q, or G163R somewhat improved the affinity of IN N155H/R263K for its target DNA substrate, while the presence of L74M or E92Q had a negative effect on this process. However, viral infectivity was significantly decreased from that of NL4.3 IN(N155H/R263K) after the addition of each tertiary mutation, and no increases in levels of DTG resistance were observed. This work shows that the compensatory mutations that evolve after N155H under continued DTG or RAL/EVG pressure in patients are unable to improve either enzyme efficiency or viral infectivity in an N155H/R263K background. IMPORTANCEIn contrast to other drugs, dolutegravir has not selected for resistance in HIV-positive individuals when used in first-line therapy. We had previously shown that HIV containing the primary raltegravir/elvitegravir resistance substitution N155H could select for R263K under dolutegravir pressure and that this virus was fit and displayed low-level resistance to dolutegravir (Anstett et al., J Virol 89:4681-4684). Therefore, the current study aimed to uncover whether accessory mutations that appear after N155H in response to raltegravir/elvitegravir were compatible with N155H and R263K. We found, however, that the addition of a third mutation negatively impacted both the enzyme and the virus in terms of activity and infectivity without large shifts in integrase inhibitor resistance. Thus, it is unlikely that these substitutions would be selected under dolutegravir pressure. These data support the hypothesis that primary resistance against DTG cannot evolve through RAL/EVG resistance pathways and that the selection of R263K leads HIV into an evolutionary dead-end. Integrase strand transfer inhibitors (INSTIs) constitute the newest class of drugs approved for the treatment of HIV. They act by inhibiting the HIV enzyme integrase (IN) ...

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

“…Single or double SIV and HIV IN mutations were generated using the QuikChange II XL site-directed mutagenesis (SDM) kit (Stratagene). Primers used to generate mutations in SIVmac239 and HIV-1 pNL4-3 have been previously described (11,12,21,22,25,(48)(49)(50)(51). Following mutagenesis, the plasmids were digested with DpnI for 4 h at 37°C and transformed using MAX Efficiency Stbl2 competent cells [F Ϫ mcrA Δ(mcrBC-hsdRMS-mrr) recA1 endA1lon gyrA96 thi supE44 relA1 -Δ(lac-proAB) (Invitrogen)] for SIV and Escherichia coli strain XL10-Gold ultracompetent cells, Tetr _(mcrA)183 _(mcrCB-hsdSMR-mrr) 173endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F ϭ proAB lacIqZ_M15Tn10 (Tetr) Amy Camr (Stratagene)] for HIV-1 SDM products.…”

Might dolutegravir be part of a functional cure for HIV?

“…While the latter had no effects on resistance to DTG, the addition of H51Y to R263K increased resistance to DTG to ϳ8-fold, while dramatically decreasing viral replication capacity by ϳ90% and enzyme strand transfer activity by ϳ80% (Mesplède et al 2013). Other studies have shown that R263K in combination with M50I, G118R, H51Y, E138K, T66I, N155H, or M184I/V slightly increased resistance to DTG but did not restore viral replication capacity (Quashie et al 2013aWares et al 2014;Anstett et al 2015;Liang et al 2015). Interestingly, no additional compensatory mutations have been identified for DTG, even in cell culture selection experiments conducted over more than 4 years (Wainberg and Han 2015).…”

Impact of HIV-1 Integrase L74F and V75I Mutations in a Clinical Isolate on Resistance to Second-Generation Integrase Strand Transfer Inhibitors

“…The first-generation INSTIs raltegravir (RAL) and elvitegravir (EVG) display broad crossresistance, whereas second-generation INSTIs of carbamoyl pyridine analogues, dolutegravir (DTG) and cabotegravir (CAB), demonstrate superior activity against firstgeneration INSTI-resistant HIV-1 variants (4, 9-13). Although a DTG resistance mutation, R263K, has been reported, and its resistance mechanism has been well studied (10,12,14,15), other potential DTG resistance mutations and their mechanisms are not fully understood.In a clinical case (Fig. 1A), the plasma HIV-1 RNA level rebounded to 2,900 copies/ml 1 year after starting antiretroviral therapy (ART) that included tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and RAL.…”