CDK13, a New Potential Human Immunodeficiency Virus Type 1 Inhibitory Factor Regulating Viral mRNA Splicing

Abstract

The human immunodeficiency virus type 1 (HIV-1) Tat is a 14-kDa viral protein that acts as a potent transactivator by binding to the transactivation-responsive region, a structured RNA element located at the 5 end of all HIV-1 transcripts. Tat transactivates viral gene expression by inducing the phosphorylation of the C-terminal domain of RNA polymerase II through several Tat-activated kinases and by recruiting chromatinremodeling complexes and histone-modifying enzymes to the HIV-1 long terminal repeat. Histone acetyltransferases, including p300 and hGCN5, not only acetylate histones but also acetylate Tat at lysine positions 50 and 51 in the arginine-rich motif. Acetylated Tat at positions 50 and 51 interacts with a specialized protein module, the bromodomain, and recruits novel factors having this particular domain, such as P/CAF and SWI/SNF. In addition to having its effect on transcription, Tat has been shown to be involved in splicing. In this study, we demonstrate that Tat interacts with cyclin-dependent kinase 13 (CDK13) both in vivo and in vitro. We also found that CDK13 increases HIV-1 mRNA splicing and favors the production of the doubly spliced protein Nef.In addition, we demonstrate that CDK13 acts as a possible restriction factor, in that its overexpression decreases the production of the viral proteins Gag and Env and subsequently suppresses virus production. Using small interfering RNA against CDK13, we show that silencing of CDK13 leads to a significant increase in virus production. Finally, we demonstrate that CDK13 mediates its effect on splicing through the phosphorylation of ASF/SF2.Human immunodeficiency virus type 1 (HIV-1) is a complex retrovirus that depends on alternative splicing to generate mRNAs encoding viral proteins essential for viral replication. More than 40 different alternatively spliced HIV-1 mRNAs are produced through the differential combination of four 5Ј splice donor sites (D1 to D4) and eight 3Ј splice acceptor sites (A 1 , A 2 , A 3 , A 4a , A 4b , A 4c , A 5 , and A 7 ) (49,51,58). Splicing of HIV-1 mRNA generates three classes of RNA: unspliced (US) (9 kb), including Gag and polymerase; singly spliced (4 kb), including Env, Vpu, Vif, and Vpr; and doubly spliced (2 kb), including Tat, Rev, and Nef. The expression of these different viral mRNA species is temporally regulated. While multiply spliced viral RNA species are abundant during early stages of infection, US and singly spliced viral RNA appear at the later stages of infection (32, 33).HIV-1 is highly dependent on the host splicing machinery in order to process viral transcripts into the different mRNA isoforms present in infected cells (reviewed in reference 59). Serine/ arginine-rich (SR) proteins are among the splicing factors that are essential for the operation and regulation of viral and cellular splicing (29,52). They belong to a family of a dozen different proteins characterized by one or two RNA recognition motifs at their N termini and an arginine/serine-rich (RS) motif at their C termini (12). SR prote...

Characterization of Human Cyclin-Dependent Kinase 12 (CDK12) and CDK13 Complexes in C-Terminal Domain Phosphorylation, Gene Transcription, and RNA Processing

“…In addition, both the splicing factor SRSF1 and its inhibitory subunit, C1QBP (28), were enriched in both the CDK12 and CDK13 purifications compared to HEK293T cells. This is consistent with previous studies (21,22) that demonstrated that CDK13 could bind and phosphorylate SRSF1 and C1QBP. Moreover, we found numerous other RNA processing proteins that were significantly enriched in CDK12 and CDK13 purifications compared to HEK293T cells (Fig.…”

“…These RNA processing proteins may interact more specifically with CDK12 complexes, or they may dissociate during size-exclusion chromatography. Together, these data support previous reports of CDK12 and/or CDK13 functioning in RNA processing (18,21).…”

“…CDK12 has been shown to regulate the splicing pattern of an E1a minigene in P19 cells (18), neurexin V in glial cells (19), and 3=-end processing of MYC in HeLa cells (20). CDK13 was demonstrated to interact with the splicing factor SRSF1 and to regulate the alternative splicing of HIV (21,22). However, the relative roles of CDK12 and CDK13 in these processes are largely unknown.…”

Molecular mechanisms of HIV latency

“…CycH/CDK7 and P-TEFb are not sufficient for optimal cotranscriptional processing of viral transcripts, which is necessary for HIV gene expression; additional transcriptional CDKs, including CycK/CDK13 and CycL/CDK11, are required (66)(67)(68). Both of these complexes increase levels of Ser2P at HIV coding and 3′ end sequences (69,70).…”

“…Both of these complexes increase levels of Ser2P at HIV coding and 3′ end sequences (69,70). Levels of CDK13 affect HIV splicing, and high and low levels of CDK13 increase and decrease splicing of HIV mRNA, respectively (66). Only the unspliced transcripts lead to the production of infectious viral particles.…”