Viral-host interactions that control HIV-1 transcriptional elongation

Abstract

Regulation of the pause and elongation by RNA polymerase (Pol) II is used widely by metazoans to attain the pattern of gene expression that is essential for optimal cell growth/renewal, differentiation and stress response. Currently, much of what we know about Pol II elongation control comes from pioneering studies of the HIV-1-encoded Tat protein and its host cellular co-factors. The interaction between the two fuels a powerful feedback circuit that activates HIV transcription and prevents the virus from entering latency. One of the key Tat cofactors is the human positive transcription elongation factor b (P-TEFb), which exists in a family of complexes with distinct functions during Tat transactivation. This article reviews recent progresses in HIV transcription research with an emphasis on the intricate control of the various P-TEFb complexes, structural and functional insights into their interactions with Tat, the multifaceted roles of post-translational modifications of Tat and epigenetic control of HIV chromatin in modulating Tat activity and HIV latency. The knowledge from these studies will not only help design better strategies to fight HIV infection and transcriptional latency, but also advance the overall understanding of the mechanism controlling transcriptional elongation in general.

Fig. 1. HIV-encoded Tat protein stimulates production of full-length viral transcripts through binding to HIV TAR RNA stem-loop structure

The genomic structure of the HIV-1 virus is shown with the coding region for the two-exon form of Tat highlighted in blue. Tat, in conjunction with other cellular cofactors (indicated by question marks), binds to the TAR RNA structure that is formed at the 5′ end of the nascent HIV transcript to stimulate the production of the full-length HIV mRNA (i.e. transcriptional elongation) by Pol II. TSS: transcription start site.

Fig. 2. P-TEFb is sequestered in 7SK snRNP and released in response to various signaling events

Under normal conditions, the majority of nuclear P-TEFb is sequestered in the 7SK snRNP, where P-TEFb’s kinase activity is inhibited by HEXIM1 in a 7SK snRNA-dependent fashion. The stability of 7SK RNA is maintained by MePCE and LARP7, which bind to the 5′ and 3′ ends of 7SK, respectively. When cells are subjected to the indicated treatments (highlighted in red), various signal transduction pathways are turned on, leading to changes in posttranslational modifications that include phosphorylation and acetylation on the indicated 7SK snRNP subunits and the release of P-TEFb from 7SK snRNP.

Fig. 3. Tat induces transfer of P-TEFb from 7SK snRNP via possibly Tatcom2 to SEC, where P-TEFb cooperates with ELL2 to synergistically activate HIV LTR transcription

Tat is known to target the 7SK snRNP to capture P-TEFb and release HEXIM1. The Tatcom2 complex, whose composition is similar to that of 7SK snRNP except for the substitution of HEXIM1 with Tat, could be a reaction intermediate before the emergence of HIV TAR RNA. Once TAR is produced, P-TEFb and Tat are transferred onto the TAR structure, and through a still unknown mechanism, nucleate the formation of the multi-subunit SEC complex. Besides P-TEFb, which phosphorylates the Pol II CTD and negative elongation factors NELF and DSIF to antagonize their inhibitory effects, SEC also contains another well-characterized elongation stimulatory factor ELL2, which directly enhances the catalytic activity of Pol II. By acting on the same Pol II enzyme, P-TEFb and ELL2 synergistically activate HIV transcription.

Fig. 4. Brd4 is a potent suppressor of Tat-transactivation and BET bromodomain inhibitor JQ1 efficiently antagonizes this suppressive effect

A. In the absence of JQ1, the promoter-bound Brd4 (through interacting with acetylated histones or Ac) competitively blocks the interaction between P-TEFb and Tat. Likewise, methylation of Tat by SETDB1 and PRMT6 also prevents this interaction. Meanwhile, most of cellular P-TEFb are sequestered in the inactive 7SK snRNP. All of these inhibit the ability of Tat to form on the HIV TAR RNA a functional SEC that is essential for activated viral transcription. B. JQ1 dissociates Brd4 from the HIV promoter and increases the local concentration of active P-TEFb for Tat to assemble into the SEC for efficient phosphorylation of the Pol II CTD, DSIF and NELF and activation of productive elongation. Additionally, JQ1 inhibits the expressions of SETDB1 and PRMT6 while promoting the production of SIRT1, which deacetylates Tat to enhance the Tat-P-TEFb interaction. Finally, JQ1 also disrupts the 7SK snRNP to release P-TEFb, providing another source of P-TEFb for SEC assembly at the HIV promoter.

Fig. 5. HIV Tat and SEC component AFF4 are predicted to make direct contacts on CycT1

Superposition of the crystal structures of the AFF4-P-TEFb complex and the Tat-P-TEFb complex using the CycT1 subunit (yellow) shows the close proximity of AFF4 (blue) and Tat (red). The resulting Tat-AFF4-P-TEFb complex model is shown in two different orientations that are rotated by about 90 degrees (CDK9 is in gray). The side chains of Tat are from only those residues that are known to have an effect on transcription when mutated and that don’t have any identified binding partner or structural function. Lys28, which is well known for its influence on complex stability upon acetylation, is engaged in the AFF4 interaction in this model but exposed to solvent in the Tat-P-TEFb complex. Arg49, which is the last visible Tat residue in the structure, indicates where the RNA binding domain will be located [figure courtesy of Ursula Schulze-Gahmen and Tom Alber of UC Berkeley].

Fig. 6. Post-translational modifications of Tat and their effects on HIV transcription

The modifications (modified residues shown in boxes) can be classified into two types based on their impact on Tat’s transactivation activity: positive (green) and negative (red). The physiological consequences of the modifications during different phases of the HIV and Tat transactivation cycles are displayed in blue. The black arrows accompanied by a plus “+” sign mean that the modifications promote the formation of the indicated complexes. Me1, Me2 and Me3 denote mono-, di-, and tri-methylation, respectively. Ac: acetylation; Pi: phosphorylation; (Ub)n: polyubiquitination. The amino acid sequence of the 86-aa form of HIV-1 Tat is shown at the bottom with the modified residues indicated in boldface type and their positions shown above and below the sequence.