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The Human Immunodeficiency Virus type 1 (HIV-1), a member of the lentivirus subfamily, infects both dividing and nondividing cells and, following reverse transcription of the viral RNA genome, integrates into the host chromatin where it enters into a latent state. Many of the factors governing viral latency remain unresolved and current antiviral treatment regimens are largely ineffective at eliminating cellular reservoirs of latent virus. The recent identification of microRNA (miRNA) encoding sequences embedded in the HIV-1 genome, and the discovery of functional virus-derived miRNAs, suggests a role for RNA Interference (RNAi) in the regulation of HIV-1 gene expression. Recently, the mammalian RNAi machinery was shown to regulate gene expression epigenetically by transcriptional modulation, providing a direct link between RNAi and a mechanism for inducing latency. Interestingly, both HIV-1 Tat, and the host TAR RNA-binding protein (TRBP), bind to the transactivating response (TAR) RNA of HIV-1 and affect the function of RNAi in human cells. Specifically, TRBP, a cofactor in Tat-TAR interactions, is a vital component of Dicer-mediated dsRNA processing. These novel observations support a central role for HIV-1 and associated host factors in regulating cellular RNAi and viral gene expression through RNA directed processes. Thus, HIV-1 may have evolved mechanisms to exploit the RNAi pathway at both the transcriptional and posttranscriptional level to affect and/or maintain a latent infection.
Lentiviruses (lentus = slow, Latin) such as the Human Immunodeficiency Viruses type 1 (HIV-1) are nononcogenic retroviruses that cause persistent infections, leading ultimately to the demise of immune regulatory cells. In the case of HIV-1, infection is inexorably followed by diseases attributable to acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi et al., 1983; Gallo et al., 1984). Clearance of lentiviral infections by the immune system is inefficient, consequently allowing for integration of proviral DNA into the genome of host cells. This feature represents a necessary prelude for the orchestration of transcriptional shutdown and a latent state in resting CD4+ cells (reviewed in Persaud et al., 2003). Since inactive, intact viral sequences remain hidden within the human genome, conventional therapeutic approaches, which are targeted to actively replicating virus, appear to be severely limited. The underlying cellular mechanisms responsible for establishing and maintaining latency are still unclear. An understanding of these mechanisms remains an important determinant for future prospects of successful long-term treatment. Recently, a large body of evidence has inferred a central role for RNA in controlling viral expression and mediating chromatin remodeling and/or maintenance (Group et al., 2005; Lu et al., 2005). In mammalian biology, as with the lower eukaryotes, the endogenous RNA interference (RNAi) pathway is likely to play an integral role in regulating viral gene expression (Seitz et al., 2003; Soifer et al., 2005). Conversely, viruses such as HIV-1 have developed appropriate counter measures, which include viral-encoded factors that inhibit the antiviral properties of RNAi (Omoto et al., 2004; Bennasser et al., 2005; Couturier and Root-Bernstein, 2005; Omoto and Fujii, 2005). However, viruses such as HIV-1 may have adopted a more subtle approach, in which the host RNAi machinery is usurped to ensure a survival advantage. In this review we hypothesize that the establishment and maintenance of HIV-1 latency may be mediated by viral expressed microRNAs (miRNAs) that operate at the transcriptional level by remodeling promoter associated chromatin, specifically nucleosomes (0), (+1), and (+2), to inactivate processive transcription from the viral long terminal repeat (LTR) promoter.
All lentiviruses utilize reverse transcriptase to convert their RNA genome into a DNA provirus (Fig. 1). Following integration and establishment of infection, the provirus exploits the cellular transcription machinery, through interactions with the 5′-LTR, to express viral RNAs. This includes the genomic RNA for incorporation into nascent virions as well as messenger RNAs for production of viral proteins (reviewed in Coffin, 1998).
The 5′-LTR contains three structural sequence elements: the U3 (−453 to −1), R (+1 to +98), and U5 (+99 to +180) regions. The basal promoter (−78 to −1) includes the TATAA box and binding sites for the transcription factor Sp1. The enhancer (−105 to −79) and modulatory regions (−454 to −104) contain NF-κB and NF-AT binding sites, respectively. The latter includes a negative regulatory element (NRE), where cellular factors such as Ap1, USF, IL-2, or c-Myb bind to and affect functions in the viral modulatory region (Fig. 1), where they have been shown to be essential for optimal promoter function (Pereira et al., 2000). Within the R region, a structured RNA element, namely, the TAR RNA (transactivation-response region), present at the 5′ end of the pregenomic mRNA sequence (+1 to +59), binds to the viral encoded transactivator protein, Tat, to induce transcriptional activation of the provirus and concomitant modification of chromatin at the integration site (Barboric and Peterlin, 2005).
Regardless of the site of viral integration, the relative regulatory domains and 5′ LTR are always packaged by two nucleosomes (Nuc), Nuc-(0) (−413 to −253) and Nuc-(+1) (+1 to +155). Nuc-(+1) is located close to the transcriptional start site, and the region between Nuc-(0) and Nuc-(+1) is bound by many of the cellular factors involved in regulating viral expression (Steger and Workman, 1997) (Fig. 2). The binding of cellular transcription factors upstream of Nuc-(+1) induces remodeling of Nuc-(+1) and subsequent transcription (Sheridan et al., 1995; Pazin et al., 1996; Steger and Workman, 1997). The importance of Nuc-(+1) for viral expression and latency is underscored by a dynamic regulation of chromatin structure surrounding the LTR promoter, which exerts an additional layer of controlled expression (Knezetic and Luse, 1986).
Upon successful infection in CD4+ T-cells, HIV-1 enters into a state of latency, a process which has been argued to be multifactorial, but which may be due to the natural progression of a CD4+ T-cell subset (whether infected or not) to shift to memory T cells (reviewed in Lassen et al., 2004). Of particular interest, is the role of the viral LTRs in establishing, maintaining, and overriding the latent state. In the absence of activation-dependent nuclear transcription factors NF-κB and NF-AT, RNA polymerase II (RNA Pol-II) associates with the 5′-LTR and transcribes relatively inefficiently. Without Tat, transcription is stalled by the presence of unphosphorylated serines 2 and 5 on the C-terminal domain (CTD) of RNA Pol-II (Zhou et al., 2004) (Fig. 2A). Basal transcription is largely non-processive when Tat is absent, resulting in very small amounts of full-length transcripts and mostly in the release of short RNAs which include the ~59 nucleotide TAR region (Fig. 2) (Kao et al., 1987).
Following Tat expression and its subsequent acetylation by p300/CBP (cAMP response element [CREB] binding protein), p300/CBP-associating factor (PCAF) and hGCN5 (Kiernan et al., 1999; Ott et al., 1999) the stalled RNA Pol-II can be activated by CTD hyperphosphorylation, which is mediated by a complex containing Tat and the positive transcription elongation factor-b (P-TEFb). P-TEFb is composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1 (Zhou et al., 2004). Once activated, RNA Pol-II–mediated transcriptional elongation can commence, resulting in full-length viral mRNAs (Fig. 2B). Provirus-infected CD4+ T cells require activation in order to express suitable quantities of Tat, which is usually mediated by the expression of cellular transcription factors such as NF-κB and NF-AT (reviewed in Lassen et al., 2004). Cytoplasmic NF-κB and NF-AT localize to the nucleus following T-cell receptor (TCR) activation of resting CD4+ T cells by antigen and/or cytokines (IL-2 or TNF-alpha; reviewed in Lassen et al., 2004).
Activated and nuclear localized NF-AT and NF-κB interact with sites in the proviral LTR promoter to induce transcription (Fig. 1). Of significance is the observation that NF-κB can also directly interact with and activate brahma-related gene (BRG)-1 (Holloway et al., 2003). In mammals the Swi/SNF complex (Fig. 2A versus 2B) functions to remodel chromatin by utilizing the hydrolysis of ATP to recruit transcription factors to target promoter regulatory sequences (reviewed in Muchardt and Yaniv 1999). The human Swi/SNF is a large (~2 MDa) multisubunit complex that is directly involved in binding to acetylated nucleosomes in vitro and in the recruitment of gene specific activators (Henderson et al., 2004). All Swi/SNF complexes have a subunit with homology to known DNA helicases (Eisen and Lucchesi 1998), one of which has been identified in mammalian cells as BRG-1 (Filetici et al., 2001). The DNA-dependent ATPase activity associated with BRG-1 is most likely the main mechanism involved in nucleosomal perturbation leading to gene expression (Tsukiyama and Wu, 1995; Varga-Weisz et al., 1995; Zhang et al., 1998) (Fig. 2). In addition, Tat has also been shown to function in the derepression of chromatin via interactions with p300/CBP, PCAF, and hGCN5, followed by subsequent enhanced histone acetyl-transferase (HAT) activity (Benkirane et al., 1998; Hottiger and Nabel, 1998; Marzio et al., 1998). Activation of HATs result in modulating chromatin to an “active” state, which correlates with enhanced transcriptional activity and methylation of lysine positions 4 and 36 on histone 3 (H3K4ME+ and H3K36ME+, respectively) (Kawasaki et al., 2005).
It is clear that the integrated provirus controls viral transcription by repressing or activating the 5′-LTR, specifically at Nuc-(+1), via the expression and access of soluble Tat to the LTR (Verdin et al., 1993). Nuc-(+1) is consistently positioned downstream of Nuc-(0) and Nuc-(−1) at a preset equilibrated distance, regardless of where the virus integrates (Verdin et al., 1993; He et al., 2002). This positional effect, along with data on HIV-1 transcriptional activation (Verdin et al., 1993; He et al. 2002), suggests that Nuc-(+1) is critical in viral replication and control. The activation of Nuc-(+1) depends not only on viral factors such as Tat but also on the acetylation of Nuc-(+1)-specific histones and other cellular transcription factors (He and Margolis, 2002). This has been recently expounded in a pilot clinical study where Margolis and colleagues used valproic acid (VPA), a histone deacetylase (HDAC) -1 inhibitor, to activate HIV-1 transcription in resting CD4+ T cells, allowing the HIV-1 fusion inhibitor enfuvirtide to inhibit the spread of activated HIV-1 from latent pools (Lehrman et al., 2005). However, there remains considerabe skepticism regarding the potential of VPA/antiretroviral treatment for the readication of viral reservoirs, since a relatively small sample size was used in this study (Smith, 2005). The order of events in the transactivation of HIV-1 from the latent/integrated isoform is depicted in Figure 2A and B. However, a mechanistic explanation for the establishment and maintenance of latency remains enigmatic. The possibility of a role for the endogenous RNAi machinery in affecting latent viral infection in CD4+ T cells is explored below.
The recent discovery and characterization of a vast array of small (21–26 nt) noncoding RNAs is dramatically changing the classical understanding of gene regulation (Zamore and Haley, 2005). In particular, short interfering RNAs (siRNAs) and microRNAs (miRNAs), which are effector sequences of the RNAi pathway, are estimated to regulate up to a third of all human genes, many of which are implicated in higher order regulatory activities. These include (but are not limited to): cellular differentiation, apoptosis, developmental timing, and regulation of transposable elements (reviewed by Kim, 2005). MicroRNAs derived from viruses have been recently identified for SV40 (Sullivan et al., 2005), Herpesvirus family members (Pfeffer et al., 2004; Cai et al., 2005; Samols et al., 2005) and HIV-1 (Omoto et al., 2004; Bennasser et al., 2005). Host-encoded miRNAs are also known to affect retrovirus primate foamy virus type 1 (PFV-1) (Lecellier et al., 2005) and Hepatitis C Virus (Jopling et al., 2005). The ever-increasing number of viral-associated miRNAs being characterized indicates an important function for small noncoding RNAs in viral regulation, adding to the complexity of host–viruses interactions.
RNAi functions largely through posttranscriptional gene silencing (PTGS) which is defined by the homology-dependent targeted degradation and/or translational suppression of mRNA (Montgomery et al., 1998; Nishikura, 2001; Sharp, 2001). siRNAs and miRNAs are short ~22-nt duplexes with 2-nt 3′-OH overhangs that are processed from precursor, longer, dsRNA triggers by the endonuclease activity of Dicer (Zamore et al., 2000; Bernstein et al., 2001; Elbashir et al., 2001). Although the pathways for generation of mature siRNAs and miRNAs are largely interchangeable, in the case of miRNAs, longer hairpin precursors of ~70 nt, or pre-miRNAs, are derived by RNA Pol II primary (pri-) miRNA transcripts that are processed by the endonuclease Drosha/DGCR8 (Lee et al., 2003) and subsequently exported from the nucleus to the cytoplasm by Exportin 5 (Lund et al., 2004) (Fig. 3). In mammalian cells, functional Dicer activity in vivo has recently been shown to require the TAR RNA binding protein (TRBP) (Chendrimada et al., 2005; Haase et al., 2005). TRBP is a host cell protein with three dsRNA binding domains, and was originally shown to bind specificlly to the HIV-1 TAR stem-loop and complement Tat-TAR binding interactions (Gatignol et al., 1991; Dorin et al., 2003). TRBP may stabilize Dicer, and appears to provide a functional link between Dicer and the endonuclease Argonaut 2 (Ago2), an important component of the RNA-induced silencing complex (RISC) (Chendrimada et al., 2005; Gregory et al., 2005). Additionally, TRBP directly inhibits protein kinase R (PKR) and the dsRNA-mediated interferon (IFN) pathway (Daher et al., 2001), further strengthening the delicate interplay between IFN and RNAi-regulated interactions.
Within RISC, the siRNA guide strand targets mRNA for degradation mediated by Ago2 cleavage (Tuschl et al., 1999; Hammond et al., 2000, 2001; Zeng and Cullen 2002; Song et al., 2004, Liu et al., 2004) (Fig. 3). Similarly, the Dicer/TRBP complex may shuttle miRNA into the RISC-like ribonucleoprotein complex (miRNP), which includes eIF2C2 and fragile X mental retardation protein (FMRP) (Mourelatos et al., 2002; Jin et al., 2004). Since miRNAs possess only partially complementary sequences with the target mRNA, silencing is generally at the level of translation and usually by targeting multiple sites located in the 3(untranslated region (UTR) (Ambros, 2004; Bartel, 2004; Kawasaki et al., 2004). MicroRNAs are found to be conserved across the eukaryotes; however, in plants miRNAs function mainly as siRNAs, guiding the cleavage of targeted mRNAs (Llave et al., 2002; Tang et al., 2003; Mansfield et al., 2004; Yekta et al., 2004).
Effector sequences of the RNAi pathway may additionally induce transcriptional gene silencing (TGS), which is characterized by the targeting of RNAi effector sequences to direct epigenetic modifications of DNA, resulting in transcriptional inhibition (Sijen et al., 2001; Pal-Bhadra et al., 2002). Small interfering RNA-mediated transcriptional suppression was first observed in transformed plants (Matzke et al., 1989), where careful analysis indicated that methylation of the targeted gene was involved in silencing (reviewed in Sijen et al., 2001; Matzke et al., 2004). RNA-dependent methylation of DNA (RdDM) within promoter regions was shown to require short RNAs which are processed by the RNAi machinery from longer dsRNA precursors in viroid-infected plants (Wassenegger et al., 1994; Mette et al., 2000).
In S. pombe RNAi-mediated TGS has been implicated in regulating heterochromatic silencing through actions of the RNA induced initiation of transciptional silencing (RITS) complex, which ultimately results in histone 3 lysine 9 methylation (H3K9ME+; Volpe et al., 2002; Noma et al., 2004). TGS has only recently been reported in human cells (Morris et al., 2004; Buhler et al., 2005; Castanotto et al., 2005; Janowski et al., 2005; Ting et al., 2005; Zhang et al., 2005) and reviewed in Morris, 2005). However, the underlying mechanism of TGS in mammalian cells is not yet completely clear but appears to involve DNA methylation and histone deacetylation/methylation (Morris et al., 2004; Buhler et al., 2005; Castanotto et al., 2005; Janowski et al., 2005; Morris, 2005; Suzuki et al., 2005; Ting et al., 2005). In human cells, TGS may also involve RITS associated factors (Noma et al., 2004); silencing has been associated with the presence of a H3K9 methylation mark at the targeted site (Buhler et al., 2005; Ting et al., 2005; Weinberg et al., 2005).
Recent experimental evidence has indicated that HIV-1 has evolved mechanism(s) to both suppress and/or usurp the RNAi pathway to maintain a latent state in infected host CD4+ cells. TRBP has the dual role of interacting with HIV-1 TAR RNA and in facilitating dsRNA processing. These processes represent an important and, perhaps, profound link between the multifactorial functions of the endogenous RNAi pathway and HIV-1 transcriptional activation. It has been previously noted that the TAR loop resembles a miRNA precursor, implying a functional role for a putative TAR-derived miRNA (Bennasser et al., 2004). However, it remains to be determined experimentally whether the processing of a putative TAR-derived pre-miRNA would be augmented by the recruitment of Dicer to the TAR loop by the Dicer/TRBP complex. Although one cannot rule out the possibility of a host gene target (see Couturier and Root Bernotein, 2005), it is possible that a TAR-derived miRNA would functionally target the complementary sequence of the TAR region itself, resulting in posttranscriptional or possibly even transcriptional inhibition of HIV-1. MicroRNAs derived from HIV-1 that correspond to sequences in Env and Nef have been detected (Omoto et al., 2004; Bennasser et al., 2005; Omoto and Fujii, 2005). Interestingly, the 3′ (end of Nef, which corresponds to the putative HIV encoded Nef microRNA, mIR-N367, also overlaps the U3 region of the HIV-1 LTR (Figs. 1 and and4).4). It is possible that during basal transcription, viral-encoded microRNAs may feedback to regulate the integrated LTR promoter by TGS (histone deacetylation/methylation and/or possibly DNA methylation) (Morris et al., 2004; Castanotto et al., 2005; Morris, 2005; Ting et al., 2005) (Fig. 4). Bennasser et al. (2005) have recently added a twist to the story by showing that high intracellular concentrations of HIV-1 Tat inhibits the activity or processivity of Dicer (Bennasser et al., 2005). Although the transactivation function (through the dsRNA binding motif) of Tat does not appear to be responsible for anti-Dicer properties, Tat may function via competitive interactions with TRBP for binding to Dicer (Fig. 4D) when present in high enough concentrations (e.g., following T-cell activation). Moreover, PKR has been shown to phosphorylate Tat (Endo-Munoz et al., 2005), which in turn, is known to inhibit PKR (Cai et al., 2000). Thus, excess Tat may supplement and/or replace the inhibitory effects on PKR by TRBP during viral replication, since any free TRBP may be utilized for viral replication. Competitive interactions between Dicer and TRBP could also be a mechanism utilized by the virus to relieve the antiviral pressures mediated by RNAi (possibly by viral-derived microRNAs/siRNAs such as TAR) when in a latent state. Interestingly, TAR may function as (1) a decoy to sequester TRBP away from Dicer and as such relieve the RNAi pressures placed on the provirus, and (2) as a microRNA to direct TGS of the provirus and function to assist in the maintance of viral latency. Thus, following TCR activation, NF-κB, NF-AT along with other transcriptional activators and chromatin remodeling factors such as BRG-1, are translocated to the nucleus to induce transcription of the previously inactive viral LTR promoter (Fig. 4D). The resulting increase in viral transcription (Nabel and Baltimore, 1987; Tong-Starksen, et al., 1987; Duh et al., 1989) would theoretically provide for enough Tat to drive transcriptional elongation (discussed above) and induce a ~100-fold increase in full-length viral transcripts (Adams et al., 1994) (Figs. 2B and and4E).4E). This would be followed by the production of infectious particles from what was previously a latent virus.
Undoubtedly, understanding the mechanism involved in establishing and maintaining viral latency remains an important objective for the successful eradication of HIV-1 infection. A number of recent findings have been juxtaposed in this review to reveal a possible role for viral encoded microRNAs in affecting latency of HIV-1. Hopefully, future experiments delving into the parameters of dsRNA-induced transcriptional gene silencing will provide further clues to the mechanism of RNA-mediated regulation of transcription, and provide new insight for future therapeutic interventions aimed at targeting latent HIV-1 infection.
We would like to thank John J. Rossi for critical evaluation of the manuscript and for helpful comments. K.V.M. is a recipient of NIH HLB R01 HL83473.