Transcriptional and posttranscriptional regulation of HIV-1 gene expression

Abstract

Control of HIV-1 gene expression depends on two viral regulatory proteins, Tat and Rev. Tat stimulates transcription elongation by directing the cellular transcriptional elongation factor P-TEFb to nascent RNA polymerases. Rev is required for the transport from the nucleus to the cytoplasm of the unspliced and incompletely spliced mRNAs that encode the structural proteins of the virus. Molecular studies of both proteins have revealed how they interact with the cellular machinery to control transcription from the viral LTR and regulate the levels of spliced and unspliced mRNAs. The regulatory feedback mechanisms driven by HIV-1 Tat and Rev ensure that HIV-1 transcription proceeds through distinct phases. In cells that are not fully activated, limiting levels of Tat and Rev act as potent blocks to premature virus production.

Figure 1.

Tat and its interactions with P-TEFb. (A) Autoregulation of HIV-1 transcription by Tat. Tat binds to the TAR RNA element encoded in the HIV-1 leader sequence and recruits P-TEFb and other elongation factors to the transcription complex. Small changes in initiation efficiency, caused by epigenetic silencing or reductions in NF-κB levels in the cell, reduce Tat levels and inhibit transcription, driving the HIV-1 provirus into latency. Reinitiation by NF-κB stimulates Tat production and restores full transcription efficiency. Thus, positive feedback by Tat results in a bistable switch. (B) Recognition of TAR RNA by Tat and P-TEFb. The diagram on the left shows the bases in TAR that are recognized by Tat in the TAR bulge region and by CycT1 in the TAR loop region (red bases). The structures at right show the conformational changes induced by Tat binding (Aboul-ela et al. 1995). (C) Structure of the Tat:P-TEFb complex. Note that Tat folds on the outer surface of the CycT1 cyclin domain. The amino-terminal “activation” domain of Tat binds to the CDK9 T-loop, a region of the molecule that is essential for its enzymatic activity (Tahirov et al. 2010).

Figure 2.

Transactivation mechanism. (A) NF-κB and Tat-activated transcription. Initiation is strongly induced by NF-κB, which acts primarily to remove chromatin restrictions near the promoter through recruitment of histone acetyltransferases. After the transcription through the TAR element, both NELF and the Tat/P-TEFb complex (including CDK9 and CycT1 and the accessory elongation factors including ELL2) are recruited to the elongation complex via binding interactions with TAR RNA. This activates the CDK9 kinase and leads to hyperphosphorylation of the CTD of RNA polymerase II, Spt5, and NELF-E. The phosphorylation of NELF-E leads to its release. The presence of hyperphosphorylated RNAP II and Spt5 allows enhanced transcription of the full HIV-1 genome. (B) Control of P-TEFb by 7SK and Tat. The majority of the P-TEFb in cells is found in a transcriptionally inactive snRNP complex containing 7SK RNA, HEXIM, and the RNA binding proteins MePCE and LARP7. Tat disrupts this complex by displacing HEXIM and forming a stable complex with P-TEFb. Prior to recruitment to the transcription complex, a larger complex is formed between P-TEFb and transcription elongation factors from the mixed lineage leukemia (MLL) family, including ELL2. (Figure is adapted from Karn 2011; reprinted, with permission, from Wolters Kluwer Health © 2011.)

Figure 3.

Locations of splice sites, exons, and splicing elements in the HIV-1 genome. (Top) Schematic diagram of HIV-1 genome. The dark blue rectangles indicate open reading frames and are labeled with the gene names. The LTRs are shown at each edge of the genome: U3-gray, R-black, U5-light blue. Full-length RNA transcripts begin at the 5′-end of the R region of the 5′-LTR (left) and 3′processing and poly(A) addition takes place at the 3′-end of the R region in the 3′-LTR (right). (Middle) Locations of 5′ss (red bars) and 3′ss (black bars) in the HIV-1 genome. The location of the RRE is shown by the red rectangle. The exons present in the incompletely spliced ∼4-kb and ∼1.8-kb mRNA species corresponding to the HIV-1 genes are shown as cyan rectangles. Noncoding exon 1 is present in all spliced HIV-1 mRNA species. Either both or one of the small noncoding exons 2 and 3 shown are included in a fraction of the mRNA species. The exon compositions of the RNA species are also shown. RNA species designated by an “I” are incompletely spliced mRNA species. Brackets indicate that mRNA isoforms containing neither exon 2 nor 3, only exon 2 or 3, or both exons 2 and 3. The locations of the AUG codons used to initiate protein synthesis are shown as purple bars within the exons. (Bottom) Locations of the known splicing regulatory elements in HIV-1. Splicing enhancers are designated by green bars and splicing silencers are designated by red bars. (Figure is adapted from Stoltzfus 2009; reproduced, with permission, from Elsevier © 2009.)

Figure 4.

Early and late phases of HIV-1 mRNA expression. Full-length unspliced ∼9-kb, incompletely spliced ∼4-kb mRNA, and completely spliced ∼1.8-kb mRNAs are expressed at both early and late times. (A) In the absence of Rev or when Rev is below the threshold necessary for it to function, the ∼9-kb and ∼4-kb mRNAs are confined to the nucleus and either spliced or degraded. Completely spliced ∼1.8-kb mRNAs are constitutively exported to the cytoplasm and translated to yield Rev, Tat, and Nef. (B) When the levels of Rev (shown as a pink oval) in the nucleus exceed the threshold necessary for function, the ∼9-kb and ∼4-kb mRNAs are exported to the cytoplasm and translated. The Rev-response element (RRE) is shown as a red rectangle. (Figure adapted from Pollard and Malim 1998; reprinted, with permission, from Annual Review of Microbiology © 1998.)

Figure 5.

Rev:RRE interactions and the Rev nuclear import/export cycle. (A) Rev binds to the RRE through its arginine-rich domain (ARD). In this model developed by Daugherty et al. (2010), the crystal structures of a Rev dimer are combined with the NMR structures of the Rev high affinity site. Notice the distortion of the RNA helix at the site of Rev binding. (B) Rev oligomerizes on the RRE and forms a complex with Crm1. The full-length RRE folds into an elongated RNA-stem loop structure with the high affinity binding site for Rev at the apex (Mann et al. 1994; Watts et al. 2009). (C) Model for the interactions between Rev and the nuclear export complex containing CRM-1 through the Rev nuclear export sequence (NES). The NES is an extended unstructured region emerging from one face of the Rev molecule. The core arginine-rich RNA binding domains interact with the RRE (Daugherty et al. 2010). (D) The Rev nuclear export cycle. Rev and the nuclear export complex containing CRM-1 interacts with nuclear pore proteins and is exported through nuclear pores to the cytoplasm. Once in the cytoplasm, Ran-GTP is converted to Ran-GDP, which is mediated by RanGAP and RanBP1. Crm1 is then transported back into the nucleus and Rev is released from the RRE. Importin-β binds to Rev through the nuclear localization signal in the ARD and interacts with Ran-GDP to facilitate import through the nuclear pore into the nucleus. In the nucleus, Ran-GDP is converted to Ran-GTP in the presence of RCC1. This releases Rev, which can begin another cycle of RRE-dependent Rev export. (Figure adapted from Pollard and Malim 1998; reprinted, with permission, from Annual Review of Microbiology © 1998.)