The human immunodeficiency virus type 1 (HIV-1) potent transactivator Tat protein mediates pleiotropic effects on various cell functions. Post-translational modifications of Tat affect its activity during viral transcription. Tat binds to TAR and subsequently is acetylated on lysine residues by histone acetyltransferases (HATs). Novel protein-protein interaction domains on acetylated Tat (AcTat) are then established, which are necessary for both sustained transcriptional activation of the HIV-1 promoter and viral transcription elongation. In this study, we investigated the identity of proteins that preferentially bound acetylated Tat. Using a proteomic approach, we identified a number of proteins that preferentially bound AcTat, among which p32, a cofactor of splicing factor ASF/SF-2, was identified. We found that p32 was recruited to the HIV-1 genome, which lead us to hypothesize that acetylation of Tat inhibits HIV-1 splicing needed for the production of full-length transcripts. Using Tat from different clades, harboring a different number of acetylation sites, as well as Tat mutated at lysine residues, we demonstrated that Tat acetylation affected splicing in vivo. Using confocal microscopy, we found that p32 and Tat colocalize in vivo in HIV-1-infected cells.

In two independent studies, p32 has been shown to inhibit splicing by inhibiting phosphorylation of the ASF/SF-2 [42] and to bind CDK13, a kinase involved in the regulation of splicing [30, 43]. We hypothesized that AcTat interacts with both p32 and CDK13 and inhibits HIV-1 mRNA splicing by affecting ASF/SF-2 phosphorylation. In fact, we demonstrated that Tat interacts with CDK13 both in vivo and in vitro. We also found that CDK13 regulates HIV-1 mRNA splicing and phosphorylates the splicing factor ASF/SF-2. In addition, CDK13 was shown to act as a restriction factor, in that its overexpression suppresses virus production. Using siRNA against CDK13, we showed that silencing CDK13 leads to a significant increase in virus production. The model supported in this study is illustrated by the fact that AcTat interacts with p32 and CDK13 in a trimeric complex and inhibits HIV-1 splicing by affecting phosphorylation of the splicing factor ASF/SF-2.