The activity of the viral long terminal repeat (LTR) promoter of the HIV-1 is the stage on which the drama of the host–virus interaction is played out, leading to latency or production, as HIV co-opts numerous cellular factors to control the rate of viral transcript production.25
In addition to cellular factors that bind LTR DNA sequences, acting as classical cis regulators, the viral transactivator Tat binds to TAR, the viral leader messenger RNA (mRNA) sequence that serves as a unique target in the regulation of LTR transcription. In most settings, HIV rapidly appropriates the resources of the activated CD4+
T cell to transform it into a factory for virus production.
However, should factors that assist in HIV transcription, the production of the viral Tat activator, and the subsequent action of Tat be even transiently deficient, this presents an opportunity for factors that antagonize HIV expression to exert their effect, dampening HIV expression. Over time, these factors may establish restrictions on HIV expression that are increasingly potent and durable. Here we highlight the basic mechanisms for the establishment and maintenance of viral reservoirs within resting CD4+ T cells, mechanisms that may be targets for therapeutics to disrupt latency and eliminate persistent infection.
It is the down-regulation of expression of the HIV-1 genome that plays a pivotal role in the establishment of the rare latent stage of the life cycle of this pathogenic retrovirus. Latency may be established (1) by direct albeit inefficient, infection of resting memory cells, (2) by the infection of T cells just prior to their natural reversion to a quiescent state, as with memory T cells, or (3) in the case of the naive T cell population, infection of cells that are undergoing differentiation during thymopoiesis. Given the potency of the viral transactivator Tat, and the responsiveness of the HIV promoter to many cellular activating signals, counterregulatory mechanisms that repress transcription appear to be required to allow HIV to establish or maintain a persistent, nonproductive infection.
Low nuclear levels of the coactivating factors nuclear factor (NF)-κB and nuclear factor of activated T cells (NFAT) are a feature of resting CD4+
cells, and may set the stage for the establishment of latency.23
Naturally low levels of the P-TEFb component CycT1 and sequestration of the P-TEFb complex by the HEXIM/7SK RNA complex in resting lymphocytes present an additional restriction to viral expression in this cellular milieu. Posttranscriptional mechanisms may also pose a barrier to expression of provirus in resting cells.26–28
Recent data have enhanced the argument that transcriptional interference mediated by nearby host gene promoters contributes to the quiescence of some HIV proviral genomes,29–31
a concept observed because most viral integrants reside in introns of actively transcribed genes.16
Finally, modeling studies suggest that stochastic, transient deficiencies in the availability of the viral Tat transactivator could allow the viral promoter to slip down into a kinetic well of repressed expression, where counterregulatory cellular influences enforce repression and silence the provirus.32
Posttranslational histone modifications, previously referred to as the “histone code,” are distinct modifications occurring at particular sites on the histone tail that can direct protein complexes to interact with the histone–DNA multimer, directing gene activity.33
These modifications may in general make chromatin more or less accessible to transcription factors, but also form biophysical marks on genes that signal the ordered recruitment of complexes of regulatory factors that up- or down-regulate gene expression. Such epigenetic histone markings appear to play a key contributory role in regulating HIV expression, and in particular in establishing LTR quiescence and latency. Furthermore, to add another layer of complexity, many of the host enzymes that modify histones also modify other cellular proteins, and so the regulatory network that may down-modulate HIV gene expression may be linked to other biochemical cellular events that enforce HIV latency.34,35
Initial studies by Verdin demonstrated that a strictly positioned nucleosome (Nuc-1) was found at the viral RNA start site (+10 to +155), and increased accessibility of chromatin near Nuc-1 associated with transcriptional activation. Furthermore, histone deacetylase inhibitors were shown to up-regulate LTR expression.36,37
Later, recruitment and occupancy of histone deacetylase 1 (HDAC1) at the HIV LTR were shown to directly mediate transcriptional silencing.38–40
Further study of the role of HDACs in LTR regulation revealed multiple cellular DNA-binding protein complexes that could recruit HDACs to the integrated provirus.41–45
Recently it has been found that the class I HDACs 1, 2, and 3 predominate at the HIV LTR in CD4+
T cell models, and that viral outgrowth could be induced from the resting CD4+
T cells of HIV-infected, aviremic, ART-treated patients by selective HDAC inhibitors (HDACis) targeting these same class I HDACs.46–52
Other epigenetic modifications, such as methylation of histones or of DNA itself, contribute to the regulation of proviral latency, and may be targets for therapy. CpG methylation of HIV promoter DNA has been shown to contribute to establish a durable, “locked” state that is difficult to reactivate. Histone methyltransferases such as EZH2 and SUV39H1 can regulate HIV-1 transcription by inducing histone H3 at lysine 9 (H3K9) methylation, and other repressive proteins can accumulate on transcriptionally inactive proviruses.53–56
Pearson and colleagues further corroborated those findings and showed that progressive iterative histone modifications drive a proviral promoter into latency in primary CD4+
Further restrictions to proviral expression and the escape from latency may exist beyond the step of the production of the initial viral mRNA transcripts. Host enzymes may inhibit the activity Tat protein that is produced by any initial burst of viral expression, raising another obstacle to the escape from latency.58,59
HIV mRNA export may be impaired in resting T cells, posing another barrier to production of provirus.28
Host miRNAs may also impede HIV mRNA expression or translation.26,27
Overall, the view of the establishment and maintenance of latent proviral infection is one of a dynamic process in which latency is established by a series of or accumulation of infrequent events. However, once established, expression of the proviral promoter is then restricted on numerous levels. The first translational challenge is to develop approaches that are capable of safely and effectively overcoming the obstacles to proviral production. This in and of itself may allow purging of the persistent proviral reservoir, or an alterative additional step may need to be taken to ensure the clearance of productively infected cells.