HIV transcription is tightly controlled both at the level of transcription initiation and elongation. There is no specific host repressor that directs a provirus to become latent. Instead, the switch between productive transcription and latency is due to the manipulation of a powerful feedback mechanism fueled by the viral trans-activator protein, Tat.
The HIV LTR which includes multiple upstream DNA regulatory elements that serve as binding sites for cellular transcription initiation factors. The core promoter, which includes all the elements required for transcription in productive infections, is a powerful and highly optimized promoter comprised of three tandem SP1 binding sites [7
], an efficient TATA element [8
] and a highly active initiator sequence [9
]. SP1 functions as an essential upstream activator; deletion of one or more of the SP1 sites effectively blocks both Tat-dependent and basal transcription [10
]. In addition, the HIV LTR carries a unique “enhancer” sequence which contains two tandem NF-κB binding motifs [11
]. Members of both the NF-κB family and NFAT can bind to the HIV NF-κB motifs and is stimulated by cooperative interactions with Sp1 [12
]. In contrast to the elements of the core promoter, mutation of the NF-κB sites results in only a modest inhibition of virus growth in most cell lines [13
], however, signaling through the viral enhancer is essential in order to re-activate latent proviruses.
The key feature that distinguishes the HIV LTR from cellular promoters is that is auto-regulated by Tat. Detailed biochemical investigations over the last 25 years have shown that Tat exclusively stimulates transcription elongation. In the absence of Tat, transcription initiation is normal but only short abortive transcripts are produced. Tat directs the cellular transcriptional elongation factor P-TEFb [14
] to nascent RNA polymerases by binding to the HIV TAR element, an RNA stem-loop structure found at the 5′ end of all viral transcripts (). P-TEFb is a protein kinase complex comprised of a regulatory Cyclin T1 (CycT1) subunit and the catalytic CDK-9 subunit. This enzyme potently stimulates HIV elongation by targeting both positive and negative elongation factors. Phosphorylation of the negative elongation factor NELF removes a powerful block to elongation [15
], while phosphorylation of the C-terminal domains (CTD) of RNAP II [16
] and Spt5 [17
] results in the direct activation of polymerase processivity.
Reactivation of latent proviruses
In the culmination of over 2 decades of research on P-TEFb by David Price and his colleagues, the crystal structure of a Tat:pTEFb complex was determined earlier this year [19••
]. The structure shows that Tat forms extensive contacts both with the CycT1 subunit of P-TEFb and also with the T-loop of the Cdk9 subunit. Importantly, Tat induces significant conformational changes in CDK9 providing an explanation for how it is able to constitutively activate the enzyme [19••
The strong amplification of transcription stimulated by Tat, coupled with the disproportionate decline in transcription that ensues when Tat levels become restricted, gives the HIV promoter a “bipolar” character (). Insightful studies by Weinberger et al. [20
] and Burnett et al. [22•
] have emphasized how stochastic fluctuations in Tat gene expression can act as a molecular switch. Small changes in initiation rates, which can be experimentally mimicked by introducing mutations into the NF-κB and Sp1 binding sites, are sufficient to restrict Tat production and lead to enhanced rates of viral entry into latency [22•
]. This switching mechanism crucially depends on the auto-regulation of Tat. When Tat is expressed in trans from an ectopic promoter, HIV proviruses become constitutively active and are unable to enter latency [23
Autoregulation of HIV transcription by Tat
The idea that the switch between active transcription and latency is regulated by Tat expression levels is also supported by the observation that introduction of mutations that attenuate Tat activity leads to an increased frequency of viruses entering latency [23
]. Similarly, viruses recovered from the latently-infected CD4+ T cells of patients are enriched for HIV-1 Tat variants with impaired transactivation activity [24
In summary, changes in the cellular environment that restrict transcription initiation are able to reduce Tat availability and force the virus into latency, but the virus remains poised to resume its replication in response to triggers that stimulate transcription initiation and restore Tat levels.
How is this subtle balance achieved?