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...
V106M may be a signature mutation in clade C patients treated with EFV and may have the potential to confer high-level multi-NNRTI resistance.
Recently, several phase 3 clinical trials (ECHO and THRIVE) showed that E138K and M184I were the most frequent mutations to emerge in patients who failed therapy with rilpivirine (RPV) together with two nucleos(t)ide reverse transcriptase inhibitors, emtricitabine (FTC) and tenofovir (TDF). To investigate the basis for the copresence of E138K and M184I, we generated recombinant mutated and wild-type (WT) reverse transcriptase (RT) enzymes and HIV-1 NL4-3 infectious clones. Drug susceptibilities were determined in cord blood mononuclear cells (CBMCs). Structural modeling was performed to analyze any impact on deoxynucleoside triphosphate (dNTP) binding. The results of phenotyping showed that viruses containing both the E138K and M184V mutations were more resistant to each of FTC, 3TC, and ETR than viruses containing E138K and M184I. Viruses with E138K displayed only modest resistance to ETR, little resistance to efavirenz (EFV), and no resistance to either FTC or 3TC. E138K restored viral replication capacity (RC) in the presence of M184I/V, and this was confirmed in cell-free RT processivity assays. RT enzymes containing E138K, E138K/ 184I, or E138K/184V exhibited higher processivity than WT RT at low dNTP concentrations. Steady-state kinetic analysis demonstrated that the E138K mutation resulted in decreased K m s for dNTPs. In contrast, M184I/V resulted in an increased K m for dNTPs compared to those for WT RT. These results indicate that the E138K mutation compensates for both the deficit in dNTP usage and impairment in replication capacity by M184I/V. Structural modeling shows that the addition of E138K to M184I/V promotes tighter dNTP binding.
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