Low level of HIV-1C integrase strand transfer inhibitor resistance mutations among recently diagnosed ART-naive Ethiopians.

With the widespread use of Integrase strand transfer inhibitors (INSTIs), surveillance of HIV-1 pretreatment drug resistance is critical in optimizing antiretroviral treatment efficacy. However, despite the introduction of these drugs, data concerning their resistance mutations (RMs) is still limited in Ethiopia. Thus, this study aimed to assess INSTI RMs and polymorphisms at the gene locus coding for Integrase (IN) among viral isolates from ART-naive HIV-1 infected Ethiopian population. This was a cross-sectional study involving isolation of HIV-1 from plasma of 49 newly diagnosed drug-naive HIV-1 infected individuals in Addis-Ababa during the period between June to December 2018. The IN region covering the first 263 codons of blood samples was amplified and sequenced using an in-house assay. INSTIs RMs were examined using calibrated population resistance tool version 8.0 from Stanford HIV drug resistance database while both REGA version 3 online HIV-1 subtyping tool and the jumping profile Hidden Markov Model from GOBICS were used to examine HIV-1 genetic diversity. Among the 49 study participants, 1 (1/49; 2%) harbored a major INSTIs RM (R263K). In addition, blood specimens from 14 (14/49; 28.5%) patients had accessory mutations. Among these, the M50I accessory mutation was observed in a highest frequency (13/49; 28.3%) followed by L74I (1/49; 2%), S119R (1/49; 2%), and S230N (1/49; 2%). Concerning HIV-1 subtype distribution, all the entire study subjects were detected to harbor HIV-1C strain as per the IN gene analysis. This study showed that the level of primary HIV-1 drug resistance to INSTIs is still low in Ethiopia reflecting the cumulative natural occurrence of these mutations in the absence of selective drug pressure and supports the use of INSTIs in the country. However, continues monitoring of drug resistance should be enhanced since the virus potentially develop resistance to this drug classes as time goes by.

Changes to the HIV long terminal repeat and to HIV integrase differentially impact HIV integrase assembly, activity, and the binding of strand transfer inhibitors.

Human immunodeficiency virus (HIV) integrase enzyme is required for the integration of viral DNA into the host cell chromosome. Integrase complex assembly and subsequent strand transfer catalysis are mediated by specific interactions between integrase and bases at the end of the viral long terminal repeat (LTR). The strand transfer reaction can be blocked by the action of small molecule inhibitors, thought to bind in the vicinity of the viral LTR termini. This study examines the contributions of the terminal four bases of the nonprocessed strand (G(2)T(1)C(-1)A(-2)) of the HIV LTR on complex assembly, specific strand transfer activity, and inhibitor binding. Base substitutions and abasic replacements at the LTR terminus provided a means to probe the importance of each nucleotide on the different functions. An approach is described wherein the specific strand transfer activity for each integrase/LTR variant is derived by normalizing strand transfer activity to the concentration of active sites. The key findings of this study are as follows. 1) The G(2):C(2) base pair is necessary for efficient assembly of the complex and for maintenance of an active site architecture, which has high affinity for strand transfer inhibitors. 2) Inhibitor-resistant enzymes exhibit greatly increased sensitivity to LTR changes. 3) The strand transfer and inhibitor binding defects of a Q148R mutant are due to a decreased affinity of the complex for magnesium. 4) Gln(148) interacts with G(2), T(1), and C(-1) at the 5' end of the viral LTR, with these four determinants playing important and overlapping roles in assembly, strand transfer catalysis and high affinity inhibitor binding.

Nucleophile selection for the endonuclease activities of human, ovine, and avian retroviral integrases.

Retroviral integrases catalyze four endonuclease reactions (processing, joining, disintegration, and nonspecific alcoholysis) that differ in specificity for the attacking nucleophile and target DNA sites. To assess how the two substrates of this enzyme affect each other, we performed quantitative analyses, in three retroviral systems, of the two reactions that use a variety of nucleophiles. The integrase proteins of human immuno- deficiency virus type 1, visna virus, and Rous sarcoma virus exhibited distinct preferences for water or other nucleophiles during site-specific processing of viral DNA and during nonspecific alcoholysis of nonviral DNA. Although exogenous alcohols competed with water as the nucleophile for processing, the alcohols stimulated nicking of nonviral DNA. Moreover, different nucleophiles were preferred when the various integrases acted on different DNA targets. In contrast, the nicking patterns were independent of whether integrase was catalyzing hydrolysis or alcoholysis and were not influenced by the particular exogenous alcohol. Thus, although the target DNA influenced the choice of nucleophile, the nucleophile did not affect the choice of target sites. These results indicate that interaction with target DNA is the critical step before catalysis and suggest that integrase does not reach an active conformation until target DNA has bound to the enzyme.