Cross-Resistance Profile Determination of Two Second-Generation HIV-1 Integrase Inhibitors Using a Panel of Recombinant Viruses Derived from Raltegravir-Treated Clinical Isolates

et al. 2014

Studies on the in vitro susceptibility of SIV to integrase strand transfer inhibitors (INSTIs) have been rare. In order to determine the susceptibility of SIVmac239 to INSTIs and characterize the genetic pathways that might lead to drug resistance, we inserted various integrase (IN) mutations that had been selected with HIV under drug pressure with raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG) into the IN gene of SIV. We evaluated the effects of these mutations on SIV susceptibility to INSTIs and on viral infectivity. Sequence alignments of SIVmac239 IN with various HIV-1 isolates showed a high degree of homology and conservation of each of the catalytic triad and the key residues involved in drug resistance. Each of the G118R, Y143R, Q148R, R263K, and G140S/Q148R mutations, when introduced into SIV, impaired infectiousness and replication fitness compared to wild-type virus. Using TZM-bl cells, we demonstrated that the Q148R and N155H mutational pathways conferred resistance to EVG (36-and 62-fold, respectively), whereas R263K also displayed moderate resistance to EVG (12-fold). In contrast, Y143R, Q148R, and N155H all yielded low levels of resistance to RAL. The combination of G140S/Q148R conferred highlevel resistance to both RAL and EVG (>300-and 286-fold, respectively). DTG remained fully effective against all site-directed mutants except G118R and R263K. Thus, HIV INSTI mutations, when inserted into SIV, resulted in a similar phenotype. These findings suggest that SIV and HIV may share similar resistance pathways profiles and that SIVmac239 could be a useful nonhuman primate model for studies of HIV resistance to INSTIs. IMPORTANCEThe goal of our project was to establish whether drug resistance against integrase inhibitors in SIV are likely to be the same as those responsible for drug resistance in HIV. Our data answer this question in the affirmative and show that SIV can probably serve as a good animal model for studies of INSTIs and as an early indicator for possible emergent mutations that may cause treatment failure. An SIV-primate model remains an invaluable tool for investigating questions related to the potential role of INSTIs in HIV therapy, transmission, and pathogenesis, and the present study will facilitate each of the above.

Section: Discussionmentioning

confidence: 99%

Effect of HIV-1 Integrase Resistance Mutations When Introduced into SIVmac239 on Susceptibility to Integrase Strand Transfer Inhibitors

Hassounah

Mésplède

et al. 2014

“…E92Q is a nonpolymorphic mutation that has been selected in patients receiving either EVG (40)(41)(42)(43)(44) or RAL (39,40,45) and is associated with virological failure on EVG-based regimens (45). The A54A/V, A265A/V, and V277V/M partial substitutions were selected under INSTI pressure, but only the A265 position is conserved among HIV integrases of different subtypes.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Drug Resistance Profiles of Integrase Strand Transfer Inhibitors in Simian Immunodeficiency Virus SIVmac239

Hassounah

Liu

et al. 2015

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 K m = and V max =/K m = 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. IMPORTANCEOur 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.T he limitations of current highly active antiretroviral therapy (HAART) include the incidence of drug resistance observed in patients, issues of drug adherence, and numbers of newly acquired infections. The emergence of drug-resistant viruses can lead to increases in viral load and treatment failure, and such viruses can be transmitted to both healthy individuals as well as to untreated HIV-positive individuals, therefore threatening the long-term effica...

“…Secondgeneration INSTIs have been developed, which possess a more robust resistance profile than RAL and EVG (3,21,27,33,67). These include MK-2048 (3, 4, 20, 67) and dolutegravir (DTG) (3,20,33,45,46,61,67).Selection studies have shown that MK-2048 can select a G118R resistance mutation (3), and during similar studies with DTG, changes were observed at positions E92, L101, T124, S153, and G193 (33,57,59). However, fold changes (FC) in susceptibility were moderate (FC, Ͻ2.5) for all of these substitutions; the substitutions at the well-characterized polymorphic positions L101 and T124 did not increase DTG or RAL FC (33, 68).…”

mentioning

confidence: 99%

“…Three main resistance pathways have been identified for RAL, involving initial mutations of the N155, Q148, and Y143 residues within IN (11). Both N155 and Q148 confer cross-resistance to EVG (19,41,47,67), while Y143 has been reported to be specific for RAL (44). Numerous secondary mutations confer low levels of resistance against both drugs (reviewed in reference 47).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Characterization of the R263K Mutation in HIV-1 Integrase That Confers Low-Level Resistance to the Second-Generation Integrase Strand Transfer Inhibitor Dolutegravir

Mésplède

Han

et al. 2012

214

306

Integrase (IN) strand transfer inhibitors (INSTIs) have been developed to inhibit the ability of HIV-1 integrase to irreversibly link the reverse-transcribed viral DNA to the host genome. INSTIs have proven their high efficiency in inhibiting viral replication in vitro and in patients. However, first-generation INSTIs have only a modest genetic barrier to resistance, allowing the virus to escape these powerful drugs through several resistance pathways. Second-generation INSTIs, such as dolutegravir (DTG, S/GSK1349572), have been reported to have a higher resistance barrier, and no novel drug resistance mutation has yet been described for this drug. Therefore, we performed in vitro selection experiments with DTG using viruses of subtypes B, C, and A/G and showed that the most common mutation to emerge was R263K. Further analysis by site-directed mutagenesis showed that R263K does confer low-level resistance to DTG and decreased integration in cell culture without altering reverse transcription. Biochemical cell-free assays performed with purified IN enzyme containing R263K confirmed the absence of major resistance against DTG and showed a slight decrease in 3= processing and strand transfer activities compared to the wild type. Structural modeling suggested and in vitro IN-DNA binding assays show that the R263K mutation affects IN-DNA interactions.T he high mutation rate of HIV-1 reverse transcriptase (RT) allows the virus to escape pressure through adaptive mutations that include drug resistance mutations that limit the effectiveness of antiretroviral drugs (5,31,66,69,70). The use of multiple drugs in combination can hamper this process by restraining viral replication, limiting the emergence of resistant strains. The addition of integrase inhibitors to the arsenal of drugs against HIV-1 is important since these inhibitors are active against viruses resistant to other drug classes (16,49,63).The HIV-1 integrase enzyme catalyzes two reactions. The first is 3= processing, which consists of cleavage of a dinucleotide at both 3= ends of the reverse-transcribed linear viral DNA and results in the exposure of reactive hydroxyl groups. The second step termed "strand transfer" is carried out through a nucleophilic attack by exposed 3= hydroxyl groups on host genomic DNA (26, 47). Even though 3= processing may be a suitable therapeutic target, the integrase inhibitors developed so far are integrase strand transfer inhibitors (INSTIs) that preferentially inhibit strand transfer while only modestly affecting 3= processing (18,24,26). Raltegravir (RAL) was the first INSTI to be approved for therapy in 2007 (64) and is safe and efficient in both treatment-naïve and treatment-experienced subjects (11,17,23,35,49,62,63). Elvitegravir (EVG) is another INSTI currently in advanced clinical trials (10,12,77).Although first-generation INSTIs strongly inhibit HIV-1 replication, they possess only a modest genetic barrier to resistance. Three main resistance pathways have been identified for RAL, involving initial mutations of the N1...