Acetylated Tat Regulates Human Immunodeficiency Virus Type 1 Splicing through Its Interaction with the Splicing Regulator p32

Abstract

The human immunodeficiency virus type 1 (HIV-1) potent transactivator Tat protein mediates pleiotropic effects on various cell functions. Posttranslational modification of Tat affects its activity during viral transcription. Tat binds to TAR and subsequently becomes acetylated on lysine residues by histone acetyltransferases. Novel protein-protein interaction domains on acetylated Tat 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, suggesting a mechanism by which acetylation of Tat may inhibit 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. Finally, using confocal microscopy, we found that p32 and Tat colocalize in vivo in HIV-1-infected cells.

Differential Isoform Expression and Interaction with the P32 Regulatory Protein Controls the Subcellular Localization of the Splicing Factor U2AF26

“…In addition, P32 plays a role in the regulation of the life cycle of several viruses such as HIV or cytomegalovirus (22,23). Consistent with the large diversity of its functions, P32 is localized to different subcellular compartments, such as mitochondria, to the outer cell membrane, or to the nucleus (24).…”

Construction of a Doxycycline-Dependent Simian Immunodeficiency Virus Reveals a Nontranscriptional Function of Tat in Viral Replication

“…It seems unlikely that this second Tat function involves binding to TAR RNA, since the Tat-TAR binding study demonstrated that the mutations in the SIV-rtTA TAR element prevent Tat binding. Multiple additional roles for Tat have been suggested in HIV-1 biology; for example, Tat has been reported to affect mRNA capping, splicing, and translation (14,18,20,21,23,67,69,78), to stimulate reverse transcription (1,36,37,42,43,71), and to suppress RNA interference (7,35). Moreover, Huang et al demonstrated that HIV-1 variants regulated by the Gal4-VP16 transactivator protein require Tat for infectivity (38), and we recently observed that the introduction of a frameshift mutation in the HIV-1 Tat gene, which prevents Tat production, abolished HIV-rtTA replication.…”

Novel Neuroprotective GSK-3β Inhibitor Restricts Tat-Mediated HIV-1 Replication

“…HLM-1 cells are HeLa-T4 ϩ cells containing one integrated copy of the HIV-1 genome with a Tat-defective mutation at the first AUG of the Tat gene. In the absence of stimulation, HLM-1 is completely negative for virus particle production, and viral transcripts are completely absent (83). Suspensions of J1.1 and ACH2 cells and their uninfected counterparts, Jurkat and CEM cells, as well as the promonocytic U1 cell line and the corresponding uninfected U937 cell line, were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 1% streptomycin/penicillin.…”