Background:  HIV-1 integrase is involved in the nuclear translocation of the pre-integration complex and integration of proviral DNA into the host cell chromosome. We recently described that the transcriptional co-activator LEDGF/p75 (p75) strongly interacts with integrase.

Methods:  We silenced p75 expression using different siRNA, and used random and directed mutagenesis to select mutants of integrase deficient for interaction with p75, and constructed HIV-1 harboring such point mutations. We then performed single and multiple rounds of HIV-1 infection experiments, and, via Q-PCR, analyzed early and late reverse transcription and integration steps of the viral cycle. Nuclear localization studies of integrase, integrase activity, and in vitro nuclear import assays were also performed.

Results:  In multiple rounds infections of Hela CD4⁺ cells we found that replication of HIV-1 was strongly inhibited when the expression of p75 was silenced. HIV replication was restored after p75 back complementation. In a single-round assay, silencing of p75 inhibited infection by a VSV-G pseudotyped NL4-3Denv virus. Mutant viruses harboring integrase point mutations, which disrupt interaction of integrase with p75 without impairing integrase enzymatic activities, were completely replication defective in T-cell lines. Q-PCR assays showed that these mutants had normal early and late reverse transcription activities, but integration was specifically impaired. In an in vitro nuclear import assay, recombinant integrase was imported with the same efficiency in cells treated with p75 siRNA or control siRNA. Integrase mutants deficient for interaction with LEDGF and fused to GFP displayed a diffuse staining, and did not bind condensed chromosomes in mitotic cells.

Conclusions:  Altogether, these results indicate that p75 is a major cellular co-factor involved in integration and replication of HIV-1. From these experiments, we can conclude that p75 is probably not directly responsible for the active nuclear import of integrase, but rather targets integrase to the chromosome, thereby influencing the retention of integrase in the nucleus. Interaction of integrase with p75 could play a role in the selection of target sites for integration of HIV-1.

Keywords: Integrase; Hiv co-factors; Chromosome targeting