Several observations point to the local state of chromatin being highly influential in the ability of the HIV provirus to overcome a major intracellular hurdle - transcription initiation. HIV-1 superinfecting already latently infected cell lines is expressed 5, implying that local factors and global cellular conditions influence gene expression independently. Cell lines harboring a single copy of a simple retroviral provirus, that of murine leukemia virus (MLV), with varying levels of expression do not differ in their capacity to support the transcription of a transiently transfected LTR-driven reporter gene 6. This suggests that factors independent of those influencing the resident provirus can affect transcription. Another finding highlighting the influence of local conditions is that silenced, methylated endogenous retro-virus (ERV) DNA becomes infectious after cloning 7.

More recently, a wealth of experiments illustrating the role of chromatin in HIV gene expression has been reported. Protein complexes involved in chromatin remodeling, including histone acetyltransferases (HATs) 8 and late SV40 transcription factor (LSF), which recruits histone deacetylase complex (HDAC-1) 9, can alter HIV-1 gene expression both in vitro and in vivo. HIV-1 expression is also affected by pharmacological modification of histone tails. Agents such as polyamides, which bind to the proviral promoter and block HDAC-1 recruitment 10, and trichostatin A, a HDAC inhibitor 11, have striking effects. Histone modifications involved in HIV gene expression have also been demonstrated by chromatin histone immunoprecipitation (ChIP) assays. HIV-1 reactivation from latently infected cell lines by either the induction of cell-cycle arrest 12 or the application of phorbol ester 13 both require the recruitment of HATs, accompanied by acetylation of histones at the HIV-1 promoter. ATP-dependent chromatin-remodeling proteins, including members of the SWI/SNF complex, are also recruited during HIV reactivation by phorbol ester application, resulting in the disruption of nucleosomes at the LTRs 14. Retinoic acid, rather specifically in HIV-1, can interfere with nucleosome remodeling at the LTRs, but not with histone acetylation, and inhibits HIV-1 transcription 15. Thus, during activation, histones associated with the HIV-1 promoter are first acetylated and chromatin-remodeling complexes are then recruited to disrupt the resident nucleosome (see 16 for a review).

Chromatin remodeling is also involved in repressing proviral gene expression. Protein factors associated with repression, such as c-Myc, occupy the HIV-1 promoter alongside HDAC-1 in a coordinated manner 17. Recruitment of HDACs, the histone methyltransferase Suv39H1, the protein HP1 (which is typical of heterochromatin) and trimethylation of H3 lysine 9 at the HIV LTR have been shown to correlate with repression of gene expression in microglial cells 18. Depleting Suv39H1 and the HP1γ subtype by siRNA increases the level of HIV gene expression 19.

Whereas the weight of evidence for the involvement of chromatin histone remodeling in the control of retroviral gene expression is compelling, the involvement of DNA methylation is more controversial. DNA methylation is associated with the recruitment of HP1, a marker of tight repression 20. In vitro, the expression levels of transfected methylated LTR-driven reporter constructs based on HIV 21, human T-cell leukemia virus type 1 (HTLV-1) 22 or ERVs 23 have been assessed. Methylation-sensitive restriction enzymes were used to detect DNA methylation in silenced transfected HIV-1 constructs 24 or in ERV LTR-driven reporter constructs 23. These studies established a clear link between DNA methylation and the absence of transcription.

Data from transiently transfected plasmids may not, however, accurately represent the behavior of integrated proviruses. For example, an integrated HTLV-1 responds differently from a transfected construct in response to extracellular stimuli 25, and in hepatocyte cell lines the promoter activity of a transfected HIV-based construct differs from that of an integrated vector 26. A further example of episomes and chromosome-associated constructs behaving differently is seen in human papillomavirus gene expression. Associating a matrix-attachment region to a human papillomavirus gene has diametrically opposite effects on gene expression depending on whether the construct is transiently transfected or stably transfected (and presumably integrated) 27.

Despite these caveats, a latent MLV provirus with a methylated genome, confirmed by methylation-sensitive restriction enzymes, can be reactivated by 5-azacytidine, an inhibitor of DNA methylation 6. In addition, methylation of the HTLV-1 proviral LTR has been assessed using methylation-sensitive restriction enzymes in cells from infected patients 28 or in transformed cell lines 29. Methylation correlated inversely with the level of viral RNA 29 and the provirus could be reactivated by 5-azacytidine 2930. Mutating the CpG sites (sites of DNA methylation) eliminates integrants that are completely silenced, strongly suggesting a role for DNA methylation in silencing 31.

In contrast, however, a methylated MLV provirus introduced into a defined site of the host-cell genome with Cre recombinase still lacks transcriptional activity after pharmacological inhibition of DNA methylation 32. More problematically, bisulfite genomic sequencing analysis on HTLV-1-infected cells from patients and transformed cell lines showed that while the 5'-LTR is hypermethylated, the 3'-LTR is hypomethylated. In neither HTLV-1 infection 30 nor in its often-used animal model, bovine leukemia virus (BLV) infection 33, does the pattern of methylation of the LTR correspond to the clinical manifestation of the infection or to disease progression.

In cell lines latently infected with HIV-1, such as ACH-2, the 5'-LTR of the provirus is hypermethylated, whereas the 3'-LTR is hypomethylated. Activating the provirus with the cytokine tumor necrosis factor-α (TNF-α) partially relieves DNA methylation of the 5'-LTR 34. However, in clones of cells derived after infection with a defective HIV-1 expressing green fluorescent protein (GFP) from the LTR, proviral expression did not correlate with DNA methylation, and bisulfite genome analysis showed most cytosine residues to be unmethylated 35. Thus, in MLV, HTLV-1 and HIV, the influence of DNA methylation on proviral behavior is controversial. Stability of gene expression may correlate more with the density of DNA methylation, as opposed to there being a binary 'methylated' or 'demethylated' state 36.

The link between chromatin remodeling and proviral activity seems incontrovertible, but how is it controlled? One possible factor is the site of integration. A study of 35 HIV-1-infected clones found heterogeneity in their individual levels of gene expression. There was no correlation between the expression level of the integrated provirus and a second transfected construct in the same clone, again implicating the local environment in controlling proviral expression 37. Analyzing the accessibility of restriction enzymes to DNA as a guide to nucleosome remodeling showed that this did correlate with the level of gene expression, despite a lack of correlation between gene expression and promoter methylation 37. In addition, latent HIV-1 proviruses are frequently found integrated near alphoid repeats, which are frequently found in heterochromatin 3839. This association with heterochromatin was further supported by a large-scale study of 971 HIV-1 integration sites that revealed that proviruses with inducible gene expression - presumably representing latent provirus - had integrated near gene deserts and centromeres, which are rich in alphoid repeats 40. Others, however, were near very highly expressed genes 40 and a study of 74 HIV-1 integration sites of latent proviruses obtained from resting CD4 T cells from 16 patients found that most of them were in actively expressed regions 41. Integration sites may, therefore, be influential, but other factors are also in operation. Consistent with the notion that viral gene expression is influenced by local factors set up at the time of integration, gene-therapy vectors containing DNA elements that can shield the provirus from the effects of adjacent chromatin, such as an MLV-based vector with a locus control region 42 or lentiviral vectors with a matrix-attachment region 43, establish high-level, position-independent gene expression. Overall, these studies strongly imply a significant role for the position of integration.

The time frame of silencing in a number of experiments is the major piece of evidence suggesting that factors other than integration site affect proviral expression. The 'site' effect on proviral chromatin configuration and gene expression would presumably be imposed soon after integration and, if it were the overriding influence, be permanent. Attenuation of gene expression has, however, been observed in longer-term culture in several experiments, mostly conducted using long-term cell clones infected with MLV or its derived vectors 32444546, sometimes with the transgene driven by a heterologous promoter rather than the viral LTR.

Even more telling, within the cell clones with provirus integrated at the same sites, variation of gene expression was observed in a number of different retroviral vectors 3144474849. Where variation exists, the level of proviral gene expression between mother and daughter cells shows some degree of correlation 4950. Silencing, where it occurred, was often linked to DNA methylation, as determined by digestion with restriction enzymes 4651 or bisulfite genomic sequencing 51. This was not universally the case, however, and variation was possible even in cells devoid of de novo methyltransferases 4748. Attempts to reactivate these proviruses using 5-azacytidine or trichostatin A after they had been silenced in long-term culture were at best only partially successful 4445464749. Variation in gene expression and the same difficulty in reversing the repression are also observed in HIV-1-infected cell clones 52. Arguably, the required strength of the reactivating stimulus may be a dose-response effect. Lorincz et al. 51 reported that more efficient reactivation could be achieved by applying trichostatin to cells pretreated with 5-azacytidine, which presumably led to the removal of the methylation mark on DNA and relieved the transgene from tight repression. Thus, the overall picture is that, whereas the chromosomal position certainly contributes to the chromatin configuration of a provirus, the level of gene expression is modulated by other factors. One possibility is that variation emerges as cell division alters existing epigenetic marks on the provirus 50. Intriguingly, there are at least two reports of upregulation of a DNA methyltransferase after HIV-1 infection 5354, suggesting the possibility of an active mechanism that silences integrated provirus.

A model for retroviral gene expression thus has to accommodate several observations. First is the strong evidence of the involvement of chromatin. Second is the correlation between DNA methylation and proviral behavior, which is not consistent in all studies: there may be a gradation of silencing from strongly repressed to unstably expressed. Thirdly, the position of integration is important. Finally, proviral shutdown can and does occur over time. Even within cell clones, variation in gene expression is common.

One possible hypothesis is that the degree of proviral gene expression reflects the permissiveness of the chromatin at the site of integration. Thus, when integration occurs in repressed chromatin, the provirus is heavily repressed, which is probably correlated with DNA methylation. Where the density of DNA methylation is less, the provirus enters a state of unstable gene expression, manifesting as variation of expression from cell to cell (variegation; Figure 1). Although this is an attractive model, it cannot completely explain all the observations listed in Box 1. Another hypothesis is that the behavior of the provirus at an early stage after infection is primarily governed by the site of integration 3755 and is only partially related to the local chromatin configuration 3538. Proviruses silenced at this point are probably more amenable to reactivation: indeed, the rate-limiting step in initiation of HIV-1 gene expression is the recruitment of the general transcription factor TFIIH 56, a relatively late step in the derepression of local chromatin. Of the proviruses that are not silenced initially, a proportion undergo shutdown with time. Proviruses silenced in this manner are tightly repressed and can be difficult to reactivate using external stimuli (Figure 2). Indeed, given the complexity of the genome, it is entirely possible that the two models are complementary: depending on the site of integration, one or other of the mechanisms depicted in Figures 1 and 2 is at work.

The additional transcriptional control mechanisms of complex retroviruses like HIV add a further level of control. Unlike simple retroviruses, where attenuation of gene expression is often observed, once transcription of an HIV-1 provirus has been established, it is extremely stable 52. This is probably due to the viral protein Tat and its response element TAR 39. Tat exerts positive feedback and enhances proviral gene expression by several mechanisms (reviewed in 5758; see also 596061). This positive-feedback axis leads to remarkably durable HIV-1 gene expression, which persists for more than 18 months once established 52. The virus-encoded transactivator Tat may be an evolutionary development by the virus to counter the cellular silencing mechanisms.

In summary, retroviral gene expression is influenced by more than one mechanism involving chromatin (Figure 2). The location of integration probably crucially affects the initial level of proviral activity. With time, the provirus may be silenced. These silencing mechanisms are likely to affect most integrated constructs, accounting for the silencing observed in simple retroviruses, HIV-1 and retroviral vectors. In HIV-1, however, silencing can be counteracted or delayed by the powerful Tat-TAR positive-feedback axis 39. The trigger event that leads to silencing is not clear. One possibility is that all proviruses are susceptible to silencing mediated by epigenetic changes through a direct mechanism, possibly via the upregulated DNA methyltransferases 5354. Such a mechanism must act early after infection.

Formation of heterochromatin is known to spread to adjacent genetic regions unless it is stopped by an insulator 626364. In the first model (Figure 1), proviral gene silencing observed in long-term cultures is one end of the spectrum of the process that relates the site of integration to proviral activity, the provirus being silenced by spreading hetero-chromatinization. Alternatively, a microRNA (miRNA)-based mechanism may be involved 65. Another possibility is that silencing is related to the cyclical expression of transcription factors, such as NFκB for HIV-1 66. In a cell infected with HIV-1, the level of NFκB dips intermittently, the production of Tat would not be maintained and the Tat-TAR positive-feedback axis would collapse. The now vulnerable provirus would be further repressed by chromatin changes that cannot be easily reversed (see Box 1). In simple retroviruses and retroviral vectors, this positive-feedback axis is absent, and therefore silencing is more frequent 324445465167.

We can begin to draw some tentative conclusions. The 'site' effect, whereby an identical provirus behaves differently according to its point of integration, argues for powerful regional control mechanisms for gene expression independent of the gene itself, providing a general defense against insertional elements. The effects of known influences on chromatin configuration clearly affect proviral gene expression, and study of individual genes in the context of proviral insertions may be illuminating to dissect out the individual contributions of methylation, acetylation and so on. Lastly, intrinsic promoter properties of the provirus have an effect that can potentially hold back the effect of regional silencing influences as long as the promoter is functioning. Ironically, the cellular silencing mechanisms actually contribute to the persistence of HIV by facilitating its evasion of drug and immunological attack.

Knowledge about the mechanism of proviral silencing and how to reverse it may lead to clinical applications in HIV infection. There have been attempts at clearing HIV from an infected patient by reactivating latent virus using interleukin-2 6869 or the histone deacetylase inhibitor valproic acid 70, thus rendering the virus susceptible to anti-retroviral therapy and immune clearance. These have been, at best, only partially successful. Understanding proviral silencing will be instrumental in devising further strategies. New insights into the control of gene expression by miRNA, which is possibly another defense against invading molecular parasites 71, will undoubtedly also have an impact 7273.

The oldest endogenous retroviruses in the human genome entered the genomes of our mammalian ancestors at around the time of the extinction of the dinosaurs 74. It might be expected that, with such a long history of coexistence between mammalian genomes and transposable elements, there would exist well developed defenses against these molecular parasites, most probably chromatin dependent. It is tempting to speculate that the silencing of retroviruses, and possibly of cellular genes, originates from such defensive mechanisms.