Introduction

HIV-1 is the etiologic agent responsible for AIDS, a syndrome characterized by depletion of CD4+ T-lymphocytes and collapse of the immune system. People with AIDS are prone to opportunistic infections easily defended against by a normal immune system. Generally, it takes several years, post-infection, to progress to AIDS.

Since the 1990s, the development of Highly Active Anti-Retroviral Therapies (HAART) has dramatically improved the survival and life quality of HIV-1-infected individuals. Unfortunately, whereas these treatments significantly reduce the levels of viral RNA in plasma and lymphoid tissues, cessation of even prolonged highly suppressive HAART regimens results in viral load rebound to pre-therapy levels, indicating that anti-retroviral therapy of this type is unable to completely eliminate HIV-1 (Pierson et al., 2000; Persaud et al., 2003; Blankson et al., 2002; Marcello, 2006). This failure has been attributed in part to the presence of a long-lived, stable population of latently infected resting memory CD4+ T cells. Since these latently infected cells express no viral proteins, they are immuno-logically indistinguishable from uninfected cells and are insensitive to HAART (Chun et al., 1997a; Finzi et al., 1997; Wong et al., 1997). While many HIV-1-susceptible cells are fast-turnover cells, this small part of memory T cells are long-lived cells (Michie et al., 1992; Mclean and Michie, 1995). These infected cells can go dormant and stay in tissues for years despite effective HAART, thereby serving as the HIV-1 reservoirs in vivo (Wong et al., 1997). These reservoirs have such a slow rate of decay during HAART that their eradication during a human lifespan is unlikely (Finzi et al., 1999; Siliciano et al., 2003).

As with all retroviruses, HIV-1 integrates into the genome of the host cell. As a consequence, HIV-1 is confronted with a unique problem in terms of transcriptional regulation and packaging into chromatin. HIV-1 proviruses can integrate in many different sites within the host cell genome, each site with its own properties susceptible of influencing the degree of viral expression. Most cells infected by HIV-1 are productively infected, that is, they go on, within days, to complete the viral replication cycle, release progeny virus and die. However, a small fraction of incoming HIV-1 enters a latent mode of infection and constitutes a reservoir of infected cells that can produce infectious virus given appropriate stimulation. HIV-1 gene expression in these latently-infected cells can be reactivated by a wide variety of signals including cytokines such as Interleukin-2, TNFa, macrophage colony-stimulating factor (MCSF), antigens and other T-cell mitogens, glucocorticoid and thyroid hormones, bacterial infections, lipopolysaccharides,... Consequently, if HAART is ceased, viremia rapidly re-emerges, regardless of the duration of drug therapy (reviewed in Blankson et al., 2002).

At the cellular level, two major forms of HIV-1 latency have been described: pre-integration latency and post-integration latency (Bisgrove et al., 2005; Marcello, 2006). The first one which can not be taken into account for the formation of the long-term viral reservoirs, occur when virions fail to undergo integration and remain in the cytoplasm of the infected cell for days as a labile pre-integration complex. This latency form will not be further discussed in this review. Postintegration latency occurs when a provirus fails to effectively express its genome and is reversibly silenced after successful integration. This latent state is exceptionally stable and limited only by the lifespan of the infected cell and its progeny.

It has been proposed that one possible solution to the problem ofHIV-1 latency is to purge the latent reservoirs by deliberately forcing HIV-1 gene expression in these latently infected cells in presence of HAART to prevent spreading of the infection by the newly synthesized viruses (Chun et al., 1998). Such type of treatment could reduce the number of latently-infected cells by causing them to be directly killed by the cytopathic action of the virus or to be destroyed by the immune system. The definition of such strategies is clearly dependent on the knowledge of the molecular mechanisms regulating HIV-1 latency and reactivation from latency.

Much progress has recently been made to elucidate the molecular mechanisms underlying HIV-1 post-integration latency, which is intimately tied to HIV-1 transcription level. Among the possible molecular mechanisms behind HIV-1 postintegration latency are: (1) the chromatin status at the integration site; (2) the presence of the repressive nucleosome nuc-1; (3) epigenetic modifications such as acetylation (reviewed in Quivy and Van Lint, 2002); (4) the lack of activation-dependent host transcription factors such as NF-kB in resting cells; and (5) the viral trans-activator Tat, which promotes transcription via the recruitment to the HIV-1 promoter of chromatin-modifying complexes.

1.1. Integration Site

The eukaryotic genome is compacted with histones and other proteins to form chromatin, which allows for efficient storage of genetic information. However, this packaging also prevents the transcription machinery from gaining access to the DNA template (reviewed in Workman and Kingston, 1998; Felsenfeld and Groudine, 2003). The repeating unit of chromatin is the nucleosome core, composed of about 146 bp of DNA tightly wrapped, in 1.65 turns, in a left-handed superhelix around a central histone octamer which contains two molecules of each of the four core histones : H2A, H2B, H3 and H4. Two adjacent nucleosome cores are separated by a region of linker DNA (10-60 bp) that is associated with a single molecule of histone H1. Histones have a common structure, the "histone fold", consisting of two short a-helices and a long central helix separated by ^-bridges, which are required for histone-DNA and histone-histone interactions (reviewed in Hansen, 2002).

The packaging of genes into chromatin is increasingly recognized as an important component in the regulation of transcription initiation and elongation (Wolffe, 1999). Chromatin is heterogeneous in the nucleus: transcriptionally active genes are characterized by a more diffuse chromatin structure (active chromatin or euchromatin), whereas inactive genes are packaged in a highly condensed chromatin configuration that impairs access to the underlying DNA (inactive chromatin or heterochromatin) (Craig, 2005).

The histone N-terminal tails are highly basic functional domains, which are subject to multiple post-translational modifications, including acetylation, phosphorylation, methylation, poly-ADP-ribosylation, ubiquitinylation and sumoylation (reviewed in Peterson and Laniel, 2004; Margueron et al., 2005). The post-translational modifications of the histone tails can potentially alter the interaction between DNA and the histone octamers, leading to decondensation of the chromatin fiber. The "histone code" hypothesis postulates that the modifications of the histone tails are interdependent and that various combination serve to categorize regions of chromatin as transcribed, heterochromatic or centromeric (Fischle et al., 2003). For example, methylation of lysine 4 of histone H3 has been associated with active gene expression, whereas methylation of lysine 9 of histone H3 has been associated with transcriptional silencing.

The molecular mechanism underlying the specific integration of a provirus into the host cell chromatin is still poorly understood. But many studies have shown that the integration site and its corresponding chromatin environment affect HIV-1 gene expression (Nahreini and Mathews, 1997; Jordan et al., 2003). Jordan et al. developed a system to select for latently infected cellular clones (Jordan et al., 2003). The integration site of 8 latently-infected HIV cell lines, which have no basal HIV transcription, was sequenced. Half of the clones were integrated within or near alphoid repeat elements in heterochromatin. In this experiment, the latently-infected clones represented less than 0.06% of the original population. This study suggests integration into heterochromatin as a mechanism leading to HIV latency.

It has been demonstrated, however, that HIV integrate preferentially within actively transcribed genes (Schroder et al., 2002; Wu et al., 2003). An analysis of integration sites in purified resting CD4+ T cells from patients on HAART found the majority (93%) of silent provirus located within the coding region of host genes (Han et al., 2004). In this case, transcriptional interference provides another potential explanation for HIV-1 latency. Transcriptional interference can occur through several different mechanisms including enhancer trapping, promoter occlusion, or steric hindrance (reviewed in Lassen et al., 2004; Bisgrove et al., 2005). However, the replication competence of the proviruses was not analyzed in this study. It has been estimated that there are 100 defective proviruses for each latent provirus, making it likely that many of the viral integration sites represent replication-incompetent defective provirus (Chun et al., 1997a,b).

Lewinsky et al. have compared the integration sites of stably expressed proviruses with those of latent but TNFa-inducible proviruses (Lewinsky et al., 2005). Three chromosomal features corresponded with inducible expression: centromeric heterochromatin, gene deserts, and highly active host transcription units. This study is consistent with both heterochromatic silencing and transcriptional interference contributing to latency. It must be noted, however, that these two mechanisms appear to account for only a portion of latent proviruses. Other chromosomal environments unfavorable for expression may yet be found (Lewinsky et al., 2005).

It has been suggested that viral latency might also be a consequence of natural antiviral defenses in mammalian cells (Williams and Greene, 2005). The genome of HIV-1 contains numerous dsRNA regions which might silence proviral transcription via RNA interference (RNAi). HIV-1 does encode a virus specific small interfering RNA precursor which elicits antiviral restriction in human cells (Bennasser et al., 2005). It was found, however, that HIV-1 evades elicited RNAi through a suppressor of RNA silencing (SRS) function encoded in its Tat protein.

1.2. Nucleosomal Organisation of the 5' LTR

Given that HIV-1 retroviral DNA is integrated into the human genome and that cellular gene expression is controlled by local chromatin structure, viral transcription is likewise modulated by local chromatin structure.

DNase I digestion of chromatin shows that different regions of chromatin are differently susceptible to endonuclease cleavage. Small regions of the genome are exquisitely sensitive to digestion by nucleases and are called nuclease hypersensitive sites. Such sites are thought to represent nucleosome-free or -disrupted regions of chromatin which are bound by trans-acting factors, and they are generally found associated with regions of the genome that are important for the regulation of gene expression. Our laboratory has studied the chromatin organization of HIV-1 proviruses integrated in five different latently-infected cell lines by a nuclease digestion method (Verdin et al., 1993). Independently of the site of integration, two major hypersensitive sites (HS) are present in the promoter region (or Long Terminal Repeat (5' LTR)): HS2+3 and HS4 (Fig. 1b); and one major hypersensitive site in the pol gene: HS7 (Fig. 1a) (see Sections 1.4 and 1.7). Identification of these hypersensitive sites allowed our laboratory to establish the nucleosomal organisation of the 5' LTR. Two nucleosomes (called nuc-0 and nuc-1) are positioned at the viral promoter DNA at precise locations with respect to regulatory elements (Verdin et al., 1993). Nuc-0 is positioned immediately upstream of the modulatory region and nuc-1 immediately downstream of the viral transcription start site (Fig. 1b). These nucleosomes define two open regions of chromatin corresponding, respectively, to the modulatory region plus the enhancer/core promoter region (nt 200-465) (HS2+3) and to a regulatory domain in the leader region downstream of the transcription start site (nt 610-720) (HS4), where transcription factors have been found to bind in vitro and in vivo (Fig. 1b and 1c) (reviewed in al Harthi and Roebuck, 1998; Rohr et al., 2003). The location of the nucleosomes in the promoter region was also verified in vitro using the HIV-1 promoter reconstituted into chromatin and analyzed by DNase I footprinting analysis (reviewed in Van Lint, 2000). Importantly, activation of the integrated HIV-1 promoter by cytokine Tumor Necrosis Factor a (TNFa) or phorbol ester TPA is accompanied by the loss or rearrangement of the nucleosome nuc-1 near the transcription start site (Verdin et al., 1993). Therefore, chromatin modifications might result in HIV-1 promoter activation.

Chromatin-modifying complexes are classified into two major groups: (1) enzymes that control covalent modifications of the amino-terminal tails of histones (acetylation, methylation, phosphorylation, ubiquitinylation) (see Sections 1.3 and

mRNA

mRNA

5'LTR

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