HIV-1 Integrase C-terminal Domain Mutations Regulate ALLINI Sensitivity
HIV-1 integrase (IN) performs two essential functions in virus replication: catalyzing the integration of reverse-transcribed viral cDNA into host DNA and promoting proper virion maturation. The latter is mediated by IN binding to the genomic RNA (gRNA) through basic residues in its C-terminal domain (CTD), thereby ensuring correct repackaging of the gRNA inside the remodeled capsid lattice. Allosteric integrase inhibitors (ALLINIs) bind within a V-shaped pocket at the interface of the catalytic core domain (CCD) of IN dimers, promote the engagement of the CTDs from adjacent dimers, and thereby occlude gRNA binding. However, virological evidence supporting a role for the IN-CTD in ALLINI mechanism of action is lacking, as mutations in CTD residues implicated in ALLINI-induced multimerization typically yield non-infectious viruses. Here we demonstrate that IN-CTD mutants bearing compensatory substitutions that restore infectivity exhibit markedly altered sensitivity to ALLINIs. Specifically, we show that R269A/K273A substitutions within the IN-CTD confer ~100-fold enhanced sensitivity to ALLINIs. This correlates with a loss of IN-gRNA binding at low ALLINI concentrations, while exerting minimal effects on IN multimerization in vitro and in virions. In contrast, we found that the IN I268T substitution which arose as a suppressor mutation of I268A confers partial resistance to ALLINI-induced inhibition of gRNA binding. Collectively, these findings provide the first direct genetic evidence that CTD residues, despite being distal to the primary drug binding site at the CCD dimer, modulate sensitivity to ALLINIs and highlights IN-gRNA binding as a central target of ALLINIs.