Mechanisms employed by retroviruses to exploit host factors for translational control of a complicated proteome

Many retroviruses utilize a 5' terminal post-transcriptional control element responsive to cellular RNA helicase A

While cap-independent initiation at an IRES is one approach for viral mRNAs to overcome barriers to ribosome scanning, another is represented by the post-transcriptional control element (PCE) (Table 1). Similar to the IRES, the PCE initially was identified in viral mRNA and subsequently in cellular transcripts [34, 44–47]. Accordingly, study of retroviral PCEs provides a window into translation control of complex cellular mRNAs [46].

PCE is a redundant stem-loop RNA structure that was initially identified in the 5' UTR of avian spleen necrosis virus (SNV) and subsequently in a growing collection of retroviruses across the metazoa including Mason-Pfizer monkey virus (MPMV), human foamy virus (HFV), reticuloendotheliosis virus strain A (REV-A), human T-cell leukemia virus type 1 (HTLV-1), feline leukemia virus (FeLV), bovine leukemia virus (BLV) [34, 44, 45, 47–49] and HIV-1 (Bolinger and Boris-Lawrie, unpublished data). A significant step toward understanding the control mechanism enforced by PCE was the discovery that cellular RNA helicase A (RHA or DHX9) specifically associates with PCE and is critical for robust translation of PCE-containing mRNAs [46] (Table 2). RHA is a multifunctional DEIH box helicase and RNA binding protein, and deregulation of RHA has been associated with various cancers and autoimmune disease [50–53]. In addition, RHA knockout in mice is embryonic lethal [54]. Roles for RHA in transcription and pre-mRNA splicing have been characterized, and new evidence indicates a role for RHA in loading of guide-strand siRNA onto the RNA interference silencing complex (RISC) [55]. Hartman et al. determined that RHA interacts with PCE-containing mRNAs in the nucleus and cytoplasm, and postulated that RHA contributes to RNA/RNP remodeling that facilitates polyribosome association [46]. Upon RHA downregulation, PCE-containing mRNAs still accumulate in the cytoplasm, however they are translationally-silent and possibly sequestered in RNA storage granules. Likewise, non-functional PCE mutant RNAs accumulate in the cytoplasm; these transcripts lack efficient interaction with RHA and are poorly translated [44, 46–48]. Experiments utilizing SNV PCE-HIV gag reporter RNA determined that RHA downregulation specifically decreased the rate of Rev/RRE-independent Gag protein production, independently of a change in global protein or RNA synthesis [46]. The effect of RHA on Gag production occurs at the post-transcriptional level because quantitative RNA analyses detected no significant change in steady-state gag mRNA levels or nuclear/cytoplasmic distribution.

The PCE/RHA RNA switch is also operative in human retroviruses. The R and U5 sequences of the HIV-1 and HTLV-1 5' LTR function coordinately to confer RHA-dependent PCE activity (Bolinger, Sharma, Singh, Boris-Lawrie, unpublished data). Experiments with HTLV-1 provirus indicated that downregulation of endogenous RHA significantly reduced polysome accumulation of HTLV-1 gag mRNA from 75% to ~10% [34]. Control experiments determined that RHA downregulation was specific to HTLV-1 gag and did not affect gapdh RNA or global cellular translation [34, 46]. Experiments with HIV-1 provirus indicated that RHA downregulation reduces HIV-1 gag translation (Bolinger, Sharma, Singh, Boris-Lawrie, unpublished data). In summary, RHA/PCE operates a 5' proximal RNA switch that is critical for translation of many retroviruses (Tables 1 and 2).

Nuclear interaction with host proteins facilitates retrovirus translation

The post-transcriptional processes of mRNA splicing, export, and translation are mechanistically linked and unspliced host pre-mRNA is typically a poor substrate for nuclear export or cytoplasmic translation [56]. However, retroviruses utilize the unspliced pre-mRNA as template for synthesis of essential structural and enzymatic proteins. Retroviruses have therefore evolved specialized mechanisms to ensure efficient export and translation independently of cellular default controls. Cis-acting RNA elements and interactive partners, such as HIV-1 Rev responsive element (RRE) and Rev, HTLV-1 Rex responsive element (RxRE) and Rex, or Mason-Pfizer monkey (MPMV) virus constitutive transport element (CTE) are necessary for efficient nuclear export of unspliced viral pre-mRNA. While HIV-1 RRE or HTLV-1 RxRE interact with Rev or Rex to connect to the CRM1 export receptor, CTE of the genetically simpler MPMV directly interacts with the Tap/NXF1 nuclear export receptor [57–60]. Another nuclear protein, 9G8, is recruited to the MPMV CTE during transcription; subsequent dephosphorylation triggers recruitment of Tap/NXF1 [58, 61–65]. The association of Tap/NXF1 with CTE is critical for the export of intron-containing MPMV mRNA into the cytoplasm, where it remains associated with 9G8 [59, 60].

In a particular cellular environment CTE was shown to enhance the translational efficiency of HIV-1 gag-pol mRNA in conjunction with the PCE located in the MPMV 5' LTR [66]. The synergistic effect of PCE and CTE on protein production was observed in monkey COS cells, but not human 293 cells. Translation enhancement was also dependent on the presence of a retroviral promoter, which posited co-transcriptional recruitment of a cellular factor that is more available in Cos cells than 293 cells. Consistent with this idea, overexpression of Tap/NXF1 increased production of the Gag reporter protein by 30-fold in 293 cells independently of an increase in total gag mRNA abundance or cytoplasmic accumulation. The increase in translational utilization of the RNA was conferred specifically by CTE, since reporters containing PCE but not CTE, were not affected by Tap/NXF1 overexpression. These results provided an example of nuclear virus-host interaction that modulates translation.

In a separate study, splicing regulatory protein 9G8 was identified as a cellular protein that increases cytoplasmic utilization of the HIV-1 gag-pol-CTE reporter mRNA [66]. Overexpression of 9G8 in 293T cells produced a 10-fold increase in Gag protein production from the reporter RNA [60]. The overexpression of 9G8 did not alter cytoplasmic accumulation of the reporter RNA but increased polyribosome association by 10-fold. Ribosomal profiles and immunoblots indicated that hyperphosphorylated 9G8 was associated with high molecular weight complexes that were sensitive to EDTA treatment, indicating that 9G8 likely associated with polyribosomes and not other heavy complexes (referred to as "pseudo-polysomes" [67]). These results bolstered the recent realization that splicing regulatory proteins provide functional linkage between multiple steps of post-transcriptional gene regulation, and that the linkage is co-opted during retrovirus replication [68, 69].

Sam68 (Src-associated in mitosis 68) is another host protein that increases cytoplasmic utilization of CTE-containing mRNA [70]. Sam68 has been shown to functionally synergize with Rev-like proteins of complex retroviruses to bolster viral post-transcriptional gene expression [71, 72]. Sam68:RNA interaction in the nucleus has been shown to facilitate the association of viral RNA with translation machinery in the cytoplasm, resulting in enhanced protein production [73]. Sam68 activity is addressed in more detail below.

hnRNP E1 negatively influences HIV-1 protein production

RHA, 9G8, and Sam68 are examples of nucleocytoplasmic shuttling proteins that promote efficient viral protein production. By contrast, hnRNP E1 provides an antagonistic effect on HIV-1 protein synthesis. hnRNP E1 is a nuclear protein that interacts with the HIV-1 exon splicing silencer in Rev exon (ESSE3) [74]. Contrary to the name, this interaction is not associated with significant change in viral RNA splicing. Instead, overexpression of hnRNP E1 caused a substantial decrease in Gag (p55 and p24), Env (gp160/gp120), and Rev production. Fractionation assays indicated that the decreased level of Rev protein remained sufficient for export of HIV-1 Rev-dependent env RNA. A complementary experiment utilizing siRNA downregulation of endogenous hnRNP E1 recapitulated a significant change in Gag and Env protein without observable effect on RNA abundance or splicing. These results indicated that hnRNP E1 negatively affects HIV-1 translation. A possible mechanism is that hnRNP E1 blocks the association of the 60S ribosome with the 43S initiation complex once the initiation codon is reached, as has been observed for hnRNP E1 and r15-LOX mRNA [75]. Other feasible explanations are the disruption of another step in the translation process or the reduced stability of the nascent polypeptide.

Control of translation by RNA localization and leaky scanning

For genetically simple retroviruses, inefficient splicing produces Gag, Gag-Pol and Env open reading frames on separate transcripts. Genetically complex retroviruses, such as HIV-1, employ alternative splicing to produce open reading frames for regulatory and accessory proteins on additional transcripts (Figure 1) [14, 15]. As summarized above, the 5' UTR of both unspliced and spliced retroviral transcripts contain features that impede ribosomes scan and alternative mechanisms are expected to choreograph viral protein production (reviewed in [22]). An economic strategy of leaky scanning provides HIV-1 Vpu and Env protein synthesis from a single bicistronic vpu-env mRNA [76]. Schwartz and colleagues demonstrated that translation of env is reliant on a weak Kozak consensus surrounding the vpu AUG. As a result, ribosomes scan past the vpu AUG to reach the env AUG and initiate translation of env, which is a process referred to as leaky scanning [23]. When the context of the vpu AUG is mutated to a match a strong Kozak consensus and reduce readthrough, env translation is abrogated [23]. By contrast, a higher level of env translation is achieved upon mutation of the vpu AUG [77]. Thus, leaky scanning through the vpu AUG is an important mechanism to achieve balanced expression of these HIV-1 gene products and is important for efficient virus replication [76].

A recent study of the 5' UTR of 16 env RNA isoforms produced by alternative splicing identified that production of Vpu is largely dependent on inclusion of exons that exclude the upstream rev AUG [78] These results reinforced the important role of the upsteam AUG in tempering env translation initiation [78]. Anderson and colleagues found that the four env mRNA isoforms containing exon 5E (isoforms 1,5,8, and 13) produced approximately four-fold more Vpu accessory protein in comparison to the rev AUG-containing isoforms. Mutation of the rev AUG in env2 increased Vpu production to a level similar to env1, indicating that initiation at the upstream rev AUG significantly depletes scanning ribosomes to initiate at the vpu initiation codon, consistent with the leaky scanning mechanism. However, in contrast to previous studies by Schwartz and colleagues, the presence of upstream AUGs had little effect on Env synthesis, suggesting the possibility of translation via a discontinuous scanning mechanism (IRES or ribosome shunt). The authors showed that the changes in protein expression were not attributable to aberrant transcripts from cryptic splicing, promoter activity or differential mRNA stability. A companion study by Krummheuer and colleagues suggested that a minimal five nucleotide open reading frame upstream of the vpu AUG acts as a ribosome pausing site, which is a feature of the ribosomal shunt characterized in cauliflower mosaic virus [79–84]. The cap-dependent ribosomal shunt is an intriguing alternative initiation mechanism that deserves further analysis in retroviruses.

Ribosomal frameshifting during translation elongation

Translation initiation has been studied intensively to understand mechanisms controlling cellular and viral protein synthesis. The translation elongation cycle, which is a closely regulated and high energy consuming process, also plays a profound role in the regulation of protein synthesis [85]. After initiation, phosphorylated elongation factors 1A and 1B (eEF1A and eEF1B) mediate amino acyl-tRNA recruitment to the ribosome A site and GDP/GTP exchange, respectively. Ribosome translocation occurs when elongation factor 2 (eEF2) associates with the ribosome and binds GTP to move the tRNA into the ribosome P site. This translocation results in a one-codon shift of the ribosome relative to the mRNA. mRNA containing a combination of repetitive sequences, referred to as slippery sequence, and stable secondary structure can pause ribosome locomotion leading to a mRNA reading frame change, referred to as programmed frameshift [86–88]. Essential retrovirus enzymatic proteins protease, integrase, and reverse transcriptase are encoded by the pro and pol genes that are distal to the Gag open reading frame in the viral unspliced mRNA. For lentiviruses and deltaviruses, the Pro and Pol coding regions are in a different reading frame than Gag. A -1 ribosome frameshift is necessary for synthesis of the Gag-Pro-Pol polyprotein, which is self-cleaved by the viral protease during maturation [87]. HIV-1 requires a single frameshift to produce Gag-Pol, while other retroviruses, such as HTLV-2, employ two separate frameshifts to produce Gag-Pro and Gag-Pro-Pol [89, 90]. Frameshifting occurs at an approximate rate of 1 Gag-Pol for every 20 Gag molecules synthesized [25, 87]. Disruption of either an upstream heptanucleotide slippery sequence (UUUUUUA in HIV-1) or an RNA stem-loop pseudoknot structure downstream of the frameshift site (referred to as the stimulatory signal) [91–93] alters frameshift efficiency and is deleterious to virus replication [94]. Recent work has indicated that both the seven nucleotides of the slippery sequence and the three preceding nucleotides are essential to maintain proper Gag-Pol ratio [95]. Brakier-Gingras and colleagues proposed an elegant model in which -1 frameshifting involves tRNA interaction with the ribosome at not only the P and A sites, but also with the E site [95]. Bicistronic reporters were constructed containing the HIV-1 seven nucleotide slippery sequence directly preceded by three nucleotides derived from the full 10 nucleotide slippery sequence of multiple viruses, including HTLV-1 and equine infectious anemia virus (EIAV). The upstream coding region contained renilla luciferase as a transfection efficiency control and firefly luciferase reporter downstream of the viral slippery sequence. Firefly Luciferase protein is observable in the event of a -1 ribosome frameshift. Results from transiently transfected 293T cells indicated that maintenance of the frameshift ratios required the authentic decanucleotide sequences from each virus. These results implicate that specific tRNA occupancy at the ribosome E site is important for -1 frameshift efficiency.

A similar reporter system was used to study the relationship between frameshifting and the activity of protein kinase R (PKR) [96]. This study found that inhibition of PKR activity by transfection of high levels of TAR RNA into Jurkat T cells or 293T cells resulted in decreased frameshifting efficiency. This effect occurred whether TAR was present in the reporter mRNA or expressed in trans from a separate plasmid. By contrast, activation of PKR by transfection of low amounts of TAR RNA increased frameshifting efficiency by 140%. TAR RNA had no effect on frameshifting after downregulation of PKR by siRNAs in 293T cells. Furthermore, the introduction of a TAR mutant deficient in PKR binding had no effect on frameshifting. The results indicated that TAR-PKR interaction contributes to efficient viral frameshifting. The proposed model is that translation initiation efficiency and frameshifting are inversely correlated. When the rate of translation initiation is slow (due to activation of PKR by TAR), frameshifting occurs at a higher rate because spacing between ribosomes increases and each ribosome encounters the frameshifting signal. Conversely, frameshifting decreases when the rate of initiation is increased because the stimulatory signal does not have time to refold and ribosomes continue to translate without pausing.

Modulation of translation termination by MLV reverse transcriptase

Translation termination occurs when the cellular release factor eRF1 recognizes a stop codon and GTP is hydrolyzed by eRF3 [97]. eRF1 has a structure similar to tRNA and is thought to bind the ribosome A site in a similar fashion to tRNA; it is proposed that stop codon recognition occurs through anticodon mimicry [98, 99]. The hallmark enzyme of the retrovirus family, reverse transcriptase, is encoded by the pol gene and is absolutely essential for viral replication. For synthesis of infectious retrovirus, pol RNA is translated by a ribosomal frameshift at a slippery sequence (as in HIV-1) or by readthrough of the gag termination codon (as in FeLV and MLV) [87, 100]. In the case of readthrough, a UAG stop codon is read as glutamine and translation proceeds to generate the Gag-Pol polyprotein [101, 102]. A combination of yeast two-hybrid, in vitro, and in vivo studies by Orlova and colleagues demonstrated that MLV reverse transcriptase binds to eRF1 through a direct protein-protein interaction that enhances readthrough of the gag stop codon [103]. This interaction appears to be specific to MLV, as HIV-1 reverse transcriptase, which does not require termination codon readthrough for Pol synthesis, did not bind eRF1. The interaction of MLV RT with eRF1 allows the RT to self-regulate translation termination, thereby maintaining an appropriate ratio of Gag:Gag-Pol protein, which is critical to generate infectious virus [104].

HIV-1 vs. the PKR pathway

HIV-1 TAR is a highly-conserved stable RNA stem loop that interacts with Tat protein to regulate viral transcription [135]. TAR has been shown to negatively regulate translation by at least two mechanisms: i) inhibition of scanning ribosomes on HIV-1 mRNA [16–18], and ii) activation of the interferon-induced serine/threonine protein kinase R (PKR) when at a low concentration [136]. Active PKR is composed of two N-terminal double-stranded RNA binding motifs (dsRBM) and a C-terminal kinase domain. The intra-molecular interaction between the dsRBM and kinase domain form an inactive "closed" conformation (Figure 2) [137, 138]. A change to an "open" conformation is triggered by association of PKR with double-stranded RNA (including HIV-1 TAR), leading to PKR dimerization and autophosphorylation [139–141]. In its active form, PKR phosphorylates Ser-51 on the α subunit of eukaryotic initiation factor 2 (eIF2α), which blocks eIF2α activation of the guanine exchange factor eIF2B (for a general review, see [142]). The disruption of eIF2α function by PKR ultimately reduces global cellular translation.

HIV-1 is among a variety of eukaryotic viruses that have evolved to circumvent this cellular defense mechanism. Roy and colleagues found that although low levels of TAR RNA bind and activate PKR [136], productive HIV-1 infection causes a decrease in PKR protein levels [143]. A separate study indicated that a high concentration of TAR-containing RNA can inhibit PKR activation, possibly by sequestration of the protein [144]. The double-stranded nature of the HTLV-1 Rex response element (RxRE) inflicted a similar fate for PKR, affecting PKR autophosphorylation that diminished with increasing concentration of RxRE-containing RNA [145].

The reduction in PKR activity observed by Roy and colleagues occurred with both HIV-1 infection and expression of HIV-1 Tat alone, implicating Tat as a major contributor to the downregulation [143]. The action of Tat was specific to PKR, since another interferon-response pathway, 2',5'-oligoadenylate synthesis (2-5OAS), was not affected by Tat alone. Later in vitro work identified the basic region of Tat as a substrate of PKR, and found that Tat can compete with eIF2α for phosphorylation [146]. Pre-incubation of PKR with increasing concentrations of Tat protein in in vitro kinase assays showed that Tat effectively reduced autophosphorylation of PKR in response to dsRNA. This study indicated that both the one exon (Tat 72) and two exon (Tat 86) variants of Tat were phosphorylation substrates of PKR (72 and 86 refer to predicted Tat proteins in their amino acid lengths). McMillan and colleagues found that only Tat 86 could be phosphorylated by pre-activated PKR although both Tat variants interacted with PKR [147]. While Tat 72 was not a phosphorylation substrate for PKR, in vitro pulldown and kinase assays indicated that Tat 72 binds to PKR and acutely inhibits its autophosphorylation and its ability to phosphorylate Tat 86. In vivo interaction between PKR and Tat 72 was verified in both a HeLa cell line and an in HIV-1 infected T cells. Overexposure of immunoblots indicated a low, yet detectable interaction between PKR and Tat 86, consistent with Tat 86 being a substrate of PKR. Although not demonstrated, it seems likely that the interactions between PKR and Tat variants occur in the context of TAR RNA due to their shared ability to bind TAR. Separate work has determined that phosphorylation of Tat 86 by PKR increases its interaction with TAR RNA and triggers more robust transcription of viral RNA [148]. An intriguing possibility is that the Tat variants are temporally controlled during the HIV-1 lifecycle to strategically modulate viral protein production. Overall, the analyses of HIV-1/PKR interplay indicate that although the HIV-1 TAR structure should elicit activation of the PKR antiviral defense (which it does at low concentration), Tat uses two strategies to combat this action: blocking the autophosphorylation of PKR; or acting as a substrate competitor to reduce the level of phosphorylated eIF2α thereby altering global translation. An overview of HIV-1/PKR interplay is depicted in Figure 2.

An additional strategy to counter innate defense is the HIV-1 interaction with a cellular double-stranded RNA binding protein, TAR RNA binding protein (TRBP) (Table 2) [149]. TRBP (also designated TRBP1) was discovered to bind HIV-1 TAR in a cDNA screen in HeLa cells [149]. An isoform designated TRBP2 has been identified that includes a 21 amino acid extension at the N-terminus [150]. Because both TRBP isoforms have been reported to activate the HIV-1 LTR in human and murine cells, they appear to be functionally equivalent [151].

TRBP proteins facilitate HIV-1 production through at least two mechanisms: i) potently inhibiting interferon-α induced PKR autophosphorylation through a direct protein:protein interaction [152]; and ii) binding to HIV-1 TAR and Rev response element (RRE) RNA resulting in enhanced virus expression [149]. A protective activity of TRBP on HIV-1 was demonstrated by Benkirane et al., who found that CEM T cells infected with HIV-1 expressing TRBP1 were resistant to the antiviral effect of interferon-α treatment [152]. Interferon-α repression of HIV-1 replication is due largely to PKR activation; therefore, a role for TRBP proteins in the PKR pathway was investigated. Stable expression of TRBP1 in HeLa cells reduced phosphorylation of PKR in response to interferon-α. Co-immunoprecipitation assays indicated an RNA-independent interaction between TRBP1 and PKR; however, treatment with interferon-α reduced this interaction in a dose-dependent manner. Immunoblotting revealed that INF-α inversely affected steady-state levels of PKR (increased) and TRBP1 (decreased) proteins, which could explain the apparent reduction in protein-protein association. Separate studies have reported that HIV-1 infection reduces the production of interferon-α, -β, and -γ in both T cells and monocytes, which could be a cause for the correlated reduction of PKR activation in response to HIV-1 [153–155]. These strategic observations illuminate the vast web of interactions that HIV-1 can balance to sustain productive virus replication (Figure 2).

TRBP proteins directly modulate retrovirus translation

Structural features of TAR RNA reduce the translation efficiency of heterologous reporter mRNAs, and the introduction of point mutations that reduce the free energy of TAR restores translation of a TAR-CAT reporter RNA in vitro [17, 156]. Addition of TRBP1 purified from E. coli caused a significant increase in translation of wild-type TAR-CAT RNA. Interestingly, both CAT and TAR-CAT RNAs elicited activation of PKR, as evidenced by the phosphorylation of eIF2α. This result suggested that the reduced translation of TAR-CAT mRNA and the subsequent rescue by TRBP1 was at least in part independent of a PKR-specific effect. Consistent with this idea, low concentrations of TRBP2 facilitated translation of TAR-Luciferase mRNA in transiently transfected PKR-deficient mouse embryonic fibroblasts [156]. Importantly, this experiment demonstrated that both TRBP1 and TRBP2 isoforms can impact the translation of TAR-containing RNAs. Experiments with truncated domains of TRBP2 showed that the double-stranded RNA binding domains singly or in combination were responsible for the stimulatory effect on reporter protein production. A comparison of reporter protein and mRNA levels showed that the effect induced by the TRBP proteins was attributable to increased translation and not increased steady-state mRNA levels. Taken together, these results provide an example of how HIV-1 RNA has evolved to utilize cellular proteins to boost translation (Table 2). A growing list of roles have been identified for TRBP proteins, including a key role in the small RNA pathway in a ribonucleoprotein complex with siRNA, Dicer, and Ago2 [157, 158]. This and additional implications of TRBP activities on HIV-1 replication are discussed below and reviewed elsewhere [159].

HIV-1 versus the 2'–5' oligoadenylate pathway

Studies of infected patients have indicated that HIV-1 affects 2'–5' oligoadenylate synthetase (2-5OAS) (Figure 2), which is a key enzyme in the regulation of an additional interferon-induced antiviral defense. 2-5OAS binds dsRNA longer than 60 bases through its amino-terminal residues. This triggers 2-5OAS to synthesize 2',5'-oligoadenylate (2-5A), a small molecule that binds to ankyrin repeats in the enzyme RNaseL and causes its dimerization and activation [160, 161]. Once activated, RNaseL cleaves viral dsRNA with the mission of blocking viral protein production. Multiple viruses, including HIV-1, have devised a strategy to cripple this host defense to promote virus survival [162–164]. Association of HIV-1 RNA with 2-5OAS was investigated by SenGupta and Silverman, who incubated affinity resins bound to either HIV-1 5' UTR RNA or poly r(I):r(C) with extracts of interferon-treated HeLa cells prior to activation of 2-5A synthesis by magnesium and ATP. Both RNAs bound 2-5OAS; however, 2-5A synthesis was lower by a factor of five in response to the HIV-1 RNA substrate [165]. A similar study found that in vitro transcribed HTLV-1 RxRE RNA stimulated 2-5A synthesis from both interferon-treated HeLa cells and assays that used purified 2-5OAS [145]. The level of 2-5A production in response to RxRE RNA was similar to that induced by TAR RNA, although poly r(I):r(C) RNA induced 2-5A synthesis more potently than either RxRE or TAR. The authors suggested that the increased ability of poly r(I):r(C) to induce 2-5A production relates to the vast size difference of the molecule compared to RxRE or TAR RNA; this size difference increases the interaction between 2-5OAS and dsRNA.

Although structures in the retroviral genome apparently trigger activation of the 2-5OAS pathway, the virus may stop RNA degradation at a later stage in the signaling cascade. In support of this idea, a 65% reduction in the binding affinity of 2-5A to RNase L was observed in lymphocytes of HIV-1-infected patients [153–155, 166]. Conversely, recent work has found that activation of 2-5A activity can combat HIV-1 replication [167]. Homan and colleagues utilized a stable 2-5A agonist called 2-5A^N6B, which is taken up into the cytoplasm of cultured T cells and subsequently activates RNaseL [167]. They found that treatment of PBMCs with 2-5A^N6B inhibited the generation of infectious HIV-1, increased the expression of interferon-α and -γ, and increased the expression of chemokines. Later work showed that 2-5A^N6B caused a significant decrease in HIV-1 Gag protein production at 96 hours post-infection of CD4+ lymphocytes cultured from healthy donors. This decrease also was observed in the supernatant of CD4+ and CD14+ cells isolated from HIV-1 seropositive patients [168]. Taken together, these results indicate that HIV-1 targets both the PKR and 2-5OAS antiviral pathways to protect viral RNA from degradation, which ultimately protects translation and the production of virus particles.

Retrovirus interplay with the small RNA pathway

The small RNA pathway operates a versatile innate antiviral defense, and retroviruses are likely to exploit some aspect of this antiviral defense [169]. MicroRNAs (miRNAs) are short non-coding regulatory RNAs (~21 to 25 nucleotides) expressed by viral genomes and their eukaryotic host [170, 171]. The interaction of endogenously expressed miRNAs with distinct targeted mRNA potently down-regulates gene expression by triggering specific degradation or translational inhibition of the transcript. miRNAs have the potential to abrogate the deleterious effects of human retrovirus infection or at least slow disease progression [172–174]. While details of interplay between plant viruses and host RNA silencing are robust, details of retrovirus interplay with host RNA silencing are just beginning to develop.

Early evidence of host miRNA activity in retrovirus infection emerged from the finding that miR-32 downregulated primate foamy virus type 1 in human cells [170]. This study also identified that the PFV-1 Tas protein can act as an RNA silencing suppressor (RSS). RSSs play a well-described role in the pathogenesis of plant viruses, but remain a controversial phenomenon for animal viruses [175]. Interestingly, Tas RSS activity is recapitulated across the plant and animal kingdoms [170].

A role for RNA silencing in HIV-1 replication kinetics has been documented in both peripheral blood mononuclear cells [173] and in resting primary CD4+ T cells [174]. Mechanistically, host RNA silencing inhibits translation of HIV-1 gag-pol mRNA and possibly all HIV-1 transcripts [176]. This activity occurs independently of a change in steady state gag-pol mRNA and reflects an anti-viral activity of the host cell miRNAs. An examination of the 3' UTR of HIV-1 transcripts from several strains has identified binding sites for a cluster of cellular miRNAs [174]. The identified miRNAs are enriched in resting CD4+ T cells compared to activated CD4+ T cells. Inhibition of these miRNAs correlated with increased viral protein production and virus particle production in resting CD4+ T cells without altering the amount of spliced or unspliced HIV-1 mRNA. These results unveiled an important role for cellular miRNAs in HIV-1 latency.

HIV-1 has been shown to utilize several strategies to counter host RNA silencing: suppression, protection of viral RNA in viral nucleocapsid, evasion, modulation and adaptation to RNA silencing [177]. Suppression of miRNA-directed activity against HIV-1 was first documented by Bennasser et al. [178], but remains controversial [179]. Tat RSS activity is modest and not global, and is demonstrated in several [176, 178] but not all assays [179]. Tat RSS activity is dependent on the double-stranded RNA binding domain [178], which is a feature conserved in many plant virus RSS [175]. Like PFV-1 Tas, HIV-1 Tat displays cross-kingdom RSS activity [176].

Multiple lines of evidence indicate a physiological role for RSS activity in productive expression of the HIV-1 provirus. First, downregulation of Dicer and/or Drosha, which suppressed the small RNA pathway, enhanced HIV-1 replication kinetics in chronically infected human PBMC and Jurkat T cells [173]. Second, plant virus RSS [176] or Ebola V35 [180] can replace the Tat RSS activity. Third, the attenuation in gag mRNA translation by RNA silencing is exacerbated by the K51A substitution in the Tat double-stranded RNA-binding domain [176, 178]. Finally, when Dicer was down-regulated, this change rescued robust gag translation and bolstered HIV-1 virion production in a continuous cell line [176].

Additional evidence for HIV-1 modulation of the host small RNA pathway has been presented by Jeang and colleagues [158]. This study found that TAR:TRBP interaction effectively sequestered TRBP from Dicer. The outcome was a reduction in the processing of reporter shRNA (luciferase) and three endogenous pre-miRNAs (miR-16, miR-93, and miR-221) that are downregulated by HIV-1 [181]. The implicit consequence of TRBP sequestration by TAR is less TRBP is available to interact with Dicer and assist in loading of guide-strand miRNA into the RISC complex [158, 182]. Taken together with the identification of Tat RSS activity, the results indicate redundant strategies operated by TAR RNA and Tat RSS protein temper host RNA silencing of HIV-1 gene expression.

In sum, the small RNA pathway represents yet another host post-transcriptional mechanism that the retrovirus leverages to its benefit. An interesting notion is that tissue-specific miRNAs may influence retrovirus evolution [183].