The new integrase strand transfer inhibitor (INSTI) dolutegravir (DTG) displays limited cross-resistance with older drugs of this class and selects for the R263K substitution in treatment-experienced patients. We performed tissue culture selections with DTG, using viruses resistant to older INSTIs and infectivity and resistance assays, and showed that the presence of the E92Q or N155H substitution was compatible with the emergence of R263K, whereas the G140S Q148R, E92Q N155H, G140S, Y143R, and Q148R substitutions were not.A lthough considerable progress has been made in HIV therapy, drug resistance has emerged for every available drug class, threatening long-term care and the potential for a cure (1). The first two integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG), can be compromised by primary substitutions that generate resistance at the expense of fitness (2), the majority of which occur in the catalytic core domain of the integrase protein (IN). Although the newer INSTI dolutegravir (DTG) has not yielded resistance when used in initial therapy (3), our group selected an R263K substitution in HIV-1 IN in tissue culture with DTG (4). We showed that R263K, located in the C-terminal portion of the protein, confers moderate resistance to DTG and EVG, while also decreasing the biochemical activity of IN by ϳ30% (5). The R263K substitution was also found in two of four INSTInaïve treatment-experienced participants who failed DTG therapy in the SAILING clinical trial (of 354 patients) but otherwise continued to perform well (6). This suggests that R263K is a signature substitution for DTG.In contrast, 7/51 individuals who previously failed RALbased regimens and who possessed primary resistance mutations subsequently failed DTG-based therapy in the VIKING trial without R263K (7), suggesting that the substitutions that confer resistance to RAL or EVG might be incompatible with R263K.(This work was largely performed by K. Anstett in partial fulfillment of the requirements for a Ph.D. degree at McGill University.)To test this hypothesis, biochemical analyses of the strand transfer efficiencies of integrase proteins containing the E92Q, Y143R, Q148R, and N155H RAL or EVG resistance substitutions, both alone and in combination with R263K, were performed (Fig. 1). Site-directed mutagenesis was performed as described previously (4, 5) to introduce E92Q, Y143R, Q148R, and N155H into the wild-type (pET15b wt ) and mutant (pET15b R263K ) integrase subtype B expression vectors (primer sequences available upon request). Recombinant integrase proteins were expressed and purified and strand transfer assays were performed as published (4, 5). Table 1 summarizes the enzyme kinetics values K m and V max for each protein tested.Every single mutation had a significant negative impact on strand transfer activity, and this effect was exacerbated when these substitutions were combined with R263K. Among single mutant proteins, the Q148R-and N155H-containing integrases were the most impacted with regar...

This study shows that E157Q may act as a compensatory mutation for R263K. Since E157Q is a natural polymorphism present in 1%-10% of HIV-positive individuals, it may be of particular importance for patients receiving INSTI therapy.

Previously, we reported that a mutant of Tat referred to as Nullbasic inhibits HIV-1 reverse transcription although the mechanism of action is unknown. Here we show that Nullbasic is a reverse transcriptase (RT) binding protein that targets the reverse transcription complex rather than directly inhibiting RT activity. An interaction between Nullbasic and RT was observed by using coimmunoprecipitation and pulldown assays, and a direct interaction was measured by using a biolayer interferometry assay. Mixtures of recombinant 6؋His-RT and Nullbasic-FLAG-V5-6؋His at molar ratios of up to 1:20,000 did not inhibit RT activity in standard homopolymer primer template assays. An analysis of virus made by cells that coexpressed Nullbasic showed that Nullbasic copurified with virus particles, indicating that it was a virion protein. In addition, analysis of reverse transcription complexes (RTCs) isolated from cells infected with wild type or Nullbasic-treated HIV-1 showed that Nullbasic reduced the levels of viral DNA in RTC fractions. In addition, a shift in the distribution of viral DNA and CAp24 to less-dense non-RTC fractions was observed, indicating that RTC activity from Nullbasic-treated virus was impaired. Further analysis showed that viral cores isolated from Nullbasic-treated HIV undergo increased disassembly in vitro compared to untreated HIV-1. To our knowledge, this is the first description of an antiviral protein that inhibits reverse transcription by targeting the RTC and affecting core stability. Like all retroviruses, HIV-1 has a single positive-sense strand of RNA genome that is converted into double-strand proviral DNA by a hallmark process called reverse transcription. Proviral DNA is subsequently integrated into the host chromosomes and is transcribed by RNA polymerase II producing viral mRNA. The mechanisms regulating reverse HIV-1 transcription have been described in detail elsewhere (1). Briefly, the viral mRNA genome annealed to host cell tRNA Lys3 form a ribonucleoprotein complex with viral proteins, including reverse transcriptase (RT), integrase (IN), and nucleocapsid to form a prototypical reverse transcription complex (RTC) (2). The initiation of reverse transcription by the RTC begins shortly after cell infection after cytoplasmic nucleotides become available. Using tRNA Lys3 as a primer, DNA synthesis by RT produces a short strand of DNA called negativestrand strong stop DNA (ϪsssDNA). Degradation of the viral RNA strand by RT RNase H activity liberates ϪsssDNA that is transferred to the 3= end of the viral RNA by annealing of complementary nucleotide sequences, a step called first-strand transfer. The synthesis of the remaining negative-strand DNA can then be completed by RT. The complete synthesis of double-strand proviral DNA follows additional DNA synthesis following additional priming reactions and strand displacement DNA synthesis by RT. Cellular factors, including eEF1A, associate with the RTC and play an important role in the reverse transcription process (3, 4).Many virion ...