The S230R Integrase Substitution Associated With Virus Load Rebound During Dolutegravir Monotherapy Confers Low-Level Resistance to Integrase Strand-Transfer Inhibitors.

Background: Dolutegravir (DTG) is an integrase strand-transfer inhibitor (INSTI) used for treatment of human immunodeficiency virus (HIV)-infected individuals. Owing to its high genetic barrier to resistance, DTG has been clinically investigated as maintenance monotherapy to maintain viral suppression and to reduce complication and healthcare costs. Our study aims to explain the underlying mechanism related to the emergence of a S230R substitution in patients who experienced virologic failure while using DTG monotherapy. Methods: We evaluated the effect of the S230R substitution in regard to integrase enzyme activity, viral infectivity, replicative capacity, and susceptibility to different INSTIs by biochemical and cell-based assays. Results: The S230R substitution conferred a 63% reduction in enzyme efficiency. S230R virus was 1.29-fold less infectious than wild-type virus but could replicate in PM1 cells without significant delay. Resistance levels against DTG, cabotegravir, raltegravir, and elvitegravir in tissue culture were 3.85-, 3.72-, 1.52-, and 1.21-fold, respectively, in virus with the S230R substitution. Conclusions: Our data indicate that the S230R substitution is comparable to the previously reported R263K substitution in some respects. Virologic failure during DTG monotherapy can occur through the development of the S230R or R263K mutation, without the need for high-level DTG resistance.

The ability of an arginine to tryptophan substitution in Saccharomyces cerevisiae tRNA nucleotidyltransferase to alleviate a temperature-sensitive phenotype suggests a role for motif C in active site organization.

We report that the temperature-sensitive (ts) phenotype in Saccharomyces cerevisiae associated with a variant tRNA nucleotidyltransferase containing an amino acid substitution at position 189 results from a reduced ability to incorporate AMP and CMP into tRNAs. We show that this defect can be compensated for by a second-site suppressor converting residue arginine 64 to tryptophan. The R64W substitution does not alter the structure or thermal stability of the enzyme dramatically but restores catalytic activity in vitro and suppresses the ts phenotype in vivo. R64 is found in motif A known to be involved in catalysis and nucleotide triphosphate binding while E189 lies within motif C previously thought only to connect the head and neck domains of the protein. Although mutagenesis experiments indicate that residues R64 and E189 do not interact directly, our data suggest a critical role for residue E189 in enzyme structure and function. Both R64 and E189 may contribute to the organization of the catalytic domain of the enzyme. These results, along with overexpression and deletion analyses, show that the ts phenotype of cca1-E189F does not arise from thermal instability of the variant tRNA nucleotidyltransferase but instead from the inability of a partially active enzyme to support growth only at higher temperatures.