Control of HIV-1 mRNP Biogenesis in T Cells
Katherine A. Jones, Salk Institute for Biological Studies
Expression of the integrated HIV-1 proviral genome is regulated at many levels, including transcription, splicing, and nuclear export of viral RNAs. HIV-1 Tat enhances RNAPII transcription elongation by binding to cyclin T1 (CycT1), a regulatory subunit of CDK9 in the P-TEFb (positive-acting transcription elongation factor) complex. Co-operative binding of Tat and P-TEFb to the HIV-1 TAR RNA element promotes phosphorylation of RNAPII C-terminal domain (CTD) at the Ser-2 position, which is required for productive transcription elongation, as well as for splicing and downstream RNA processing events. In resting CD4+ T cells, viral transcription is blocked due to the absence of host cell transcription factors (NF-kB, NFAT, P-TEFb), which are strongly up-regulated in activated T cells. Productive virus replication also requires that partially-spliced and unspliced intron-containing viral RNAs be shielded from nucleolytic destruction and efficiently exported. Although non-productive transcription is a major cause of viral latency, low levels of multiply-spliced Tat and Rev mRNAs detected in resting CD4+ T cells have been shown to be aberrantly retained in the nucleus, suggesting that nuclear export is also blocked in resting T cells.
Our lab has recently identified a new link between RNAPII transcription elongation and downstream events: splicing, mRNA surveillance (i.e., degradation of improperly-formed pre-mRNAs by the nuclear exosome), and nuclear export. We discovered that the human Spt6 transcription elongation factor assembles a complex on the Ser2P RNAPII CTD that is important for mRNA processing and efficient nuclear export. Interestingly, the Spt6 SH2 domain interacts directly with Ser2-P RNAPII CTD and assembles a complex with Iws1 ("interacts with Spt6") and the REF/ALY nuclear export adaptor protein. This complex is not required for HIV-1 Tat-induced transcription elongation, but rather is needed for proper HIV-1 mRNA processing and efficient nuclear export of bulk HeLa polyadenylated mRNAs . Moreover, disruption of this complex results a significant delay in gene expression. Our studies also suggest that REF/ALY associates with the Rrp6 subunit of the nuclear exosome, a multisubunit 3' to 5' exoribonuclease complex that travels with actively elongating RNAPII complexes to degrade improperly processed nascent RNAs. We propose that the Spt6:Iws1 complex loads nuclear export and mRNA surveillance factors onto the RNAPII CTD during HIV-1 transcription elongation in a Tat:P-TEFb-dependent manner, and that RNA export factors are transferred from the CTD to the nascent RNA co-transcriptionally. Our recent data indicate that assembly of this complex is controlled by Tat and T cell activation.
Here we will examine the affect of Tat and P-TEFb on the assembly of the Spt6:Iws1 complex and assess whether this complex is required to load REF/ALY onto nascent HIV-1 RNA in vitro. We will determine whether this complex mediates export of early viral mRNAs, and determine how its assembly is up-regulated in activated T cells. We will assess whether Iws1 is required for the association of REF/ALY with poly(A)+ and viral mRNAs in vivo, and for recruitment of splicing, 3'-processing factors, nuclear export, and mRNA surveillance factors to the HIV-1 genome in vivo. Taken together, these findings will help elaborate the role of complex in HIV-1 mRNP complex assembly and export of early viral mRNAs.