Mechanisms employed by retroviruses to exploit host factors for translational control of a complicated proteome
© Bolinger and Boris-Lawrie; licensee BioMed Central Ltd. 2009
Received: 08 August 2008
Accepted: 24 January 2009
Published: 24 January 2009
Retroviruses have evolved multiple strategies to direct the synthesis of a complex proteome from a single primary transcript. Their mechanisms are modulated by a breadth of virus-host interactions, which are of significant fundamental interest because they ultimately affect the efficiency of virus replication and disease pathogenesis. Motifs located within the untranslated region (UTR) of the retroviral RNA have established roles in transcriptional trans-activation, RNA packaging, and genome reverse transcription; and a growing literature has revealed a necessary role of the UTR in modulating the efficiency of viral protein synthesis. Examples include a 5' UTR post-transcriptional control element (PCE), present in at least eight retroviruses, that interacts with cellular RNA helicase A to facilitate cap-dependent polyribosome association; and 3' UTR constitutive transport element (CTE) of Mason-Pfizer monkey virus that interacts with Tap/NXF1 and SR protein 9G8 to facilitate RNA export and translational utilization. By contrast, nuclear protein hnRNP E1 negatively modulates HIV-1 Gag, Env, and Rev protein synthesis. Alternative initiation strategies by ribosomal frameshifting and leaky scanning enable polycistronic translation of the cap-dependent viral transcript. Other studies posit cap-independent translation initiation by internal ribosome entry at structural features of the 5' UTR of selected retroviruses. The retroviral armamentarium also commands mechanisms to counter cellular post-transcriptional innate defenses, including protein kinase R, 2',5'-oligoadenylate synthetase and the small RNA pathway. This review will discuss recent and historically-recognized insights into retrovirus translational control. The expanding knowledge of retroviral post-transcriptional control is vital to understanding the biology of the retroviral proteome. In a broad perspective, each new insight offers a prospective target for antiviral therapy and strategic improvement of gene transfer vectors.
A dual fate for unspliced retroviral mRNA: translation, encapsidation, or both?
In the cytoplasm, the retroviral primary transcript (pre-mRNA) plays a dual role as unspliced mRNA template for translation and as genomic RNA that is encapsidated into assembling virions . The RNA packaging signal in the 5' UTR of retroviral mRNA represents a pendulum that balances these possible fates of the genome-length RNA .
Retrovirus mechanisms to modulate protein synthesis
Examples of viruses reported to utilize mechanisma
Effect on translation
Internal Ribosome Entry Site
HIV-1, HIV-2, SIV, HMSV, MLV, RSV
Cap-independent translation enhancer. Ribosomes plus a subset of initiation factors internally initiate translation independently of a 5' 7-methylguanosine cap.
Post-transcriptional control element (PCE)
SNV, REV-A, HTLV-1, BLV, MPMV, FeLV,
Novel 5' terminal cap-dependent translation enhancer. Specific interaction with RNA helicase A facilitates polysome loading and efficient viral protein production. PCE is not an IRES.
Readthrough of upstream AUG codons allows translation initiation of a downstream gene (i.e. vpu and env).
Short upstream open reading frames present in 5' leader RNA attenuate translation initiation at the authentic gag-pol AUG. Effect is dependent on distance from AUG.
Stimulatory signal and slippery sequence present in mRNA induce ribosome pausing and a -1 reading frame change. Results in translation of gag-pol open reading frame to produce reverse transcriptase and other enzymatic proteins.
Termination codon readthrough
Termination codon of gag open reading frame is read as glutamate. Results in translation of gag-pol open reading frame to produce reverse transcriptase and other enzymatic proteins.
Scanning ribosome bypasses mRNA structural motif to reach AUG.
Gag-gag mRNA interaction
Gag protein binds to the 5' UTR of gag mRNA and attenuates translation efficiency.
Cis -acting repressive sequences/inhibitory sequences
AU-rich sequences present in gag, pol and env mRNA bind cellular proteins involved in mRNA metabolism and translation. This association represses cytoplasmic expression of the mRNA.
Viral regulatory protein recognizes intronic cis-acting Rev response element (RRE) and counteracts repression by INS/CRS. Trans-activates nuclear export, with coincide increases in mRNA stability and polysome loading that result in robust viral protein production. HTLV-1 Rex/RxRE and MMTV Rem/RmRE activity activate nuclear export and may likewise enhance translational output.
A long-standing issue in retrovirus biology is whether or not the processes of gag mRNA translation and virion precursor RNA encapsidation are mutually exclusive . The take-home message differs between retroviruses. For example, HIV-2 has been shown to encapsidate RNA co-translationally , while murine leukemia virus (MLV) produces two functionally distinct pools of mRNA to be used for either translation or virion assembly [8, 9]. In the case of HIV-1, unspliced RNA can be used interchangeably for translation and virion assembly [9, 10]. In distinction from HIV-2, translation is not a prerequisite to qualify unspliced HIV-1 RNA for packaging into virions .
LeBlanc and Beemon used translation-dependent nonsense mediated decay (NMD) as an innovative approach to evaluate this issue for Rous sarcoma virus (RSV) . Their study evaluated RSV molecular clones that contain artificial pre-mature termination codons (PTC). The experiments determined that unspliced PTC-containing RSV RNA, which is a substrate for translation-dependent NMD, could be packaged into virions. A follow-up study in the context of the authentic provirus determined that RSV utilizes a 3' UTR RNA stability element to evade NMD and ensure appropriate levels of gag mRNA for virion protein synthesis . The finding that unspliced RSV transcript can be a substrate for both translation and packaging into virions indicated that these processes are not mutually exclusive in this alpharetrovirus. A comprehensive review of the relationship between gag translation and virion precursor RNA packaging is presented elsewhere .
Potential for alternative translation initiation
The 5' UTR of retroviral gag pre-mRNA contains a collection of highly conserved cis-acting sequences required for several steps in virus replication. For instance, the HIV-1 5' UTR contains the Tat trans-activation response element (TAR), primer binding site, genome dimerization signal, 5' splice site and a packaging signal . Because some of these motifs are upstream of the 5' splice site, they are maintained within the 5' UTR of the ~30 alternatively spliced HIV-1 transcripts [14, 15]. This proximal section of the 5' UTR has been shown to inhibit ribosome scanning and translation initiation of a reporter RNA [14–18]. In the context of the virus, ligation of the 5' exon to various distal exons produces additional species of complex 5' UTRs that are ~350 to 775 nucleotides in length (Figure 1). These long UTRs often contain AUG or CUG sequences upstream of the authentic initiator codon [19, 20] (Figure 1), which interfere with translation initiation at the appropriate AUG . Another complicating feature is that authentic initiator codons often are located within poor Kozak consensus sequences, which may provide another regulatory feature that modulates expression of the viral proteome (reviewed in ). For example, a weak Kozak sequence surrounding the HIV-1 vpu AUG promotes translation of the downstream env gene, a process referred to as leaky scanning [14, 23]. The inhibitory features found in the HIV-1 5' UTR are also represented in all other retroviruses, as summarized for human T cell leukemia virus type 1 (HTLV-1), mouse mammary tumor virus (MMTV), and spleen necrosis virus (SNV) in Figure 1[15, 24–26]. In spite of the multiple challenges to efficient cap-dependent translation initiation, sufficient retrovirus protein production prevails.
The potential dichotomy of mechanisms governing translation initiation of retroviruses is a topic of some controversy. The use of cap-independent initiation at an internal ribosome entry sequence (IRES) has been proposed to circumvent inhibition of scanning ribosomes by the complex 5' UTR. Originally identified in the Picornaviridae, which includes poliovirus and encephalomyocarditis virus (EMCV), the IRES promotes recruitment of the 43S ribosome independently of cap-binding [27–29]. Transcripts of the Picornaviridae lack a 5' cap and provide cytoplasmic viral enzymes to inactivate factors including eIF4E, eIF4G and poly(A) binding protein (PABP) [30–32]. Consequently, picornavirus transcripts are reliant on an IRES to initiate viral protein synthesis [27, 28, 33].
By contrast, transcripts of the Retroviridae are considered to bear a 5' cap and therefore IRES-dependent initiation is not necessarily critical. In support of this idea, translation of the avian spleen necrosis virus is reduced when cap-dependent translation is inhibited by infection with EMCV . Nevertheless, the search for IRES activity in the Retroviridae has been extensive with IRES-like activity proposed for at least six retroviruses, including HIV-1 and HIV-2 [35–37], simian immunodeficiency (SIV) , RSV , and murine leukemia viruses (Friend and Moloney strains, F-MLV and MoMLV, respectively) [40–42], and Harvey murine sarcoma virus (HMSV)  (Table 1 and reviewed in ). Studies to identify internal initiation in isolated viral UTR segments have primarily utilized the transfection of bicistronic reporter plasmids. A caveat to this approach is false-positive IRES activity attributable to cryptic promoter activity or splicing of the test sequence. A case study of bicistronic reporter plasmids that employed extensive RNA analysis determined that 5' UTR sequences of HTLV-1, REV-A, or SNV produced multiple transcripts that correlated with false-positive IRES activity . The false-positive activity was validated by the observation that transfection of homologous in vitro transcribed RNA did not recapitulate IRES activity. A possible caveat is that the transfected RNAs may fail to interact with necessary IRES-transacting factors (ITAFs) in the nucleus. An alternative approach to measure HIV-1 IRES employed poliovirus infection to inhibit cap-dependent translation initiation. The results determined that HIV-1 Gag protein synthesis is sustained from a heterologous reporter plasmid during poliovirus infection . Unexpectedly, the putative IRES activity was conferred by sequences downstream of the gag translation initiation codon, rather than the 5' UTR. In summary, utilization of internal ribosome entry at retroviral IRES remains a controversial subject, and conditional IRES activity is an intriguing possible explanation for the disparate results. An alternative scenario is that features of the complex 5' UTR direct mechanistically uncharacterized virus-host interactions to modulate cap-dependent initiation. This scenario and its perspective into the translation of complex cellular mRNAs are discussed in the next section.
Cap-dependent retrovirus translation enhancers
Retrovirus:host interactions involved in retroviral translation control
Examples of interacting virusa
Effect on translation
Interacts with retrovirus protein & RNA
Protein kinase R (PKR)
HIV-1 Tat reduces PKR autophosphorylation. Tat and eIF2α compete as substrates of PKR. High levels of HIV-1 TAR RNA or HTLV-1 RxRE inhibit PKR autophosphorylation.
Small RNA pathway components
(Dicer & Drosha)
PFV Tas and HIV-1 Tat act as RNA silencing suppressors that combat the antiviral effect of small RNA pathway. Also miRNAs may be encoded by retroviruses that downregulate host antiviral defense.
Interacts with retrovirus RNA
TAR RNA binding protein (TRBP)
Binding of TRBP to HIV-1 TAR RNA results in increased HIV-1 transcription and translation.
HIV-1 5' UTR RNA binds 2-5OAS resulting in RNAseL activity in vitro. HIV-1 infection is associated with reduced interferon production and reduced 2-5A:RNAseL binding, allowing HIV-1 mRNA to evade cleavage by RNaseL. HTLV-1 RxRE activates 2-5OAS in vitro.
RNA helicase A
(RHA or DHX9)
SNV, REV-A, HTLV-1, MPMV, HFV, FeLV, BLV, HIV-1
RHA binds PCE mRNA leading to increased polysome association and efficient protein synthesis.
In overexpression experiments, hyper-phosphorylated 9G8 binds constitutive transport element-containing reporter mRNA resulting in increased polysome accumulation and protein synthesis.
Sam68, SLM-1, SLM-2
HIV-1, HTLV-1, EIAV, MPMV
Sam68, SLM-1 and SLM-2 act synergistically with HIV-1 Rev, HTLV-1 Rex and EIAV ERev to facilitate expression and proper cytoplasmic localization of RRE-containing mRNA. Sam68 also enhances translation of mRNA containing the MPMV constitutive transport element.
hnRNPE1 binds HIV-1 mRNA at the exon splicing silencer in Rev exon (ESSE) and reduces Gag, Env, and Rev protein production.
MLV reverse transcriptase binds eRF1 promoting readthrough of the gag termination codon to produce proteins encoded by gag-pol.
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 .
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  (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 . 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) . 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 . 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 . 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% . 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 . 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 . 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 . Overexpression of 9G8 in 293T cells produced a 10-fold increase in Gag protein production from the reporter RNA . 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" ). 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 . 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 . 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) . 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 . Other feasible explanations are the disruption of another step in the translation process or the reduced stability of the nascent polypeptide.
Initiation and beyond: Retrovirus protein production is reliant on ribosome frameshifting and leaky scanning
Retroviruses employ economical strategies of post-transcriptional control that culminate in expression of multiple viral open reading frames from a relatively small (~10 kb) genome. The strategies include alternative translation initiation, modulation of elongation and polypeptide termination.
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 ). An economic strategy of leaky scanning provides HIV-1 Vpu and Env protein synthesis from a single bicistronic vpu-env mRNA . 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 . When the context of the vpu AUG is mutated to a match a strong Kozak consensus and reduce readthrough, env translation is abrogated . By contrast, a higher level of env translation is achieved upon mutation of the vpu AUG . 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 .
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  These results reinforced the important role of the upsteam AUG in tempering env translation initiation . 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 . 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 . 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 . 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 . 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 . 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) . 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 . 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 . 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 .
Retroviral regulatory export proteins Rev, Rex, and Rem may moonlight as translation stimulation factors
The HIV-1 post-transcriptional regulatory protein, Rev, is a 116 amino acid nuclear-cytoplasmic shuttling RNA binding protein [105–107] that is required for delivery of genome-length, unspliced RNA to the cytoplasm for subsequent translation and/or packaging into virions . Synonymous loci have been identified in other complex retroviruses, including HTLV-1 and HTLV-2 Rex/RxRE and mouse mammary tumor virus (MMTV) Rem/RmRE [108–111].
Extensive experimentation with reporter plasmids and HIV-1 provirus has characterized Rev/RRE as a potent molecular switch that significantly trans-activates cytoplasmic accumulation of intron-containing RNAs. In the absence of Rev, cis-acting inhibitory sequences cause nuclear retention and low steady-state accumulation of these RNAs [112–116]. In the presence of Rev, the stability and nuclear export of RRE-containing transcripts are activated . In addition to trans-activation of HIV-1 mRNA export, Rev has been identified to promote translation of RRE-containing mRNA [118, 119]. D'Agostino et al. demonstrated that co-transfection of Rev with gag-RRE reporter plasmids in HeLa-Tat cells yielded a discordant relationship between the increase in cytoplasmic RNA and Gag protein levels. Addition of Rev caused a 4 to 16-fold increase in cytoplasmic accumulation of reporter RNA while Gag protein level increased by a discordant 850-fold. Ribosomal profile analysis indicated an increase in gag polysome association from 4% to 20% upon addition of Rev. The results indicated that Rev/RRE activity increases the cytoplasmic utilization of the Rev-dependent mRNA. A similar conclusion was reached by Arrigo et al., who transfected lymphoid cells with HIV-1 provirus that either lacked or contained the Rev open reading frame [118, 120]. The presence of Rev increased polysome association of gag, vif, vpr, and vpu/env mRNA, indicating that Rev increases the translational efficiency of Rev-dependent transcripts. The observation by Cochrane and colleagues that Rev/RRE activity requires interaction with newly synthesized RNA  is consistent with the theme that nuclear interactions facilitate cytoplasmic utilization of retroviral RNA, as discussed above for RHA/PCE and 9GA/CTE.
A translational role for Rev-like proteins encoded by other retroviruses remains to be investigated in detail. Notably, study of HTLV-2 Rex-2/RxRE activity measured a 7-to-9 fold increase in cytoplasmic accumulation of gag RNA that was accompanied by a discordant 130-fold increase in steady state Gag protein, which is reminiscent of HIV-1 Rev/RRE activity [119, 122]. Given the similarity of domain structure between HIV-1 Rev, HTLV-1 Rex and MMTV Rem, the conservation of functional activity in both nuclear export and cytoplasmic translation is an expectation (Table 2).
Study of the cellular protein Sam68 has produced additional insights into post-transcriptional control of retroviral gene expression. Sam68 and Sam68-like proteins SLM-1 and SLM-2 act synergistically with Rev to increase expression of RRE-containing mRNAs [73, 123]. Synergistic activity is also observed by co-expression of Sam68 and HTLV-1 Rex or EIAV ERev . Furthermore, C-terminal truncation of Sam68, which deletes the nuclear localization signal, generates an isoform that inhibits Rev activity and negated the effects of wild type Sam68, SLM-1 or SLM-2 on HIV-1 gene expression [73, 123]. The cytoplasmically-restricted Sam68 mutant did not inhibit Rev shuttling ability, but changed the cytoplasmic distribution of RRE-containing unspliced HIV-1 env reporter RNA from a dispersed pattern to sequestration at the nuclear periphery. Addition of the SV40 large T antigen nuclear localization signal to the Sam68 mutant restored proper distribution of cytoplasmic RRE-RNA. These observations posited that interaction of Sam68 and RRE in the nucleus is critical for the target RNA to associate with translation machinery in the cytoplasm . In sum, results with Rev, Sam68, RHA, 9G8 and hnRNPE1 echo the theme that nuclear interactions are important for productive retrovirus translation in the cytoplasm.
Rev/RRE regulation of viral protein production involves derepression of cis-acting repressive sequences (CRS, also referred to as instability sequences [INS]) present in HIV-1 gag, gag-pol, and env mRNA [112, 115, 125]. When placed 3' to chloroamphenicol acetyltransferase reporter gene, AU-rich segments of gag, pol, or env coding regions substantially reduced reporter gene activity [112, 115, 125]. Addition of the Rev responsive element (RRE) in cis and Rev protein in trans alleviated gene repression. In this reporter system, cytoplasmic accumulation and steady-state RNA levels were relatively unaffected, although the addition of CRS to a different reporter caused nuclear retention in the absence of Rev . RNA affinity assays indicated that CRS interacted with cellular splicing factor hnRNPC1 and potentially other undefined factor(s). The nuclear retention of viral mRNA was attributed to sequestration in RNA-protein complexes that are inaccessible for nuclear export .
Further study of HIV-1 cis-acting repressive sequences by Schwartz et al (referred to as INS elements) showed that AU-rich regions of p17gag transcript decreased mRNA stability and reduced the steady-state abundance of viral RNA . Similar to experiments described above, co-transfection of Rev expression plasmid relieved the inhibitory effect of INS on HIV-1 tat-RRE-gag reporter RNA. Mutations in the INS lead to Rev-independent gene expression and eliminated the instability phenotype. Multiple proteins have been identified to bind INS elements, including hnRNP A1 [127, 128], polypyrimidine tract binding protein (PTB) [127, 128], polypyrimidine tract-binding protein-associated splicing factor (PSF)  and poly(A)-binding protein (PABP) . The binding partners have been postulated to coordinate interrelated steps in HIV-1 RNA post-transcriptional control. For example, hnRNP A1 was found to stimulate Rev-dependent mRNA export ; PSF modulates nuclear retention ; and PABP modulates transcript stability . Overexpression of PSF decreases the abundance of all HIV-1 transcripts, with the most substantial effect on unspliced and singly spliced species . Binding of PABP to INS RNA correlated with reduced mRNA stability, which indicated that PABP association at regions other than the polyA tail could disrupt or compete the formation of RNP necessary for efficient translation initiation.
In addition to affecting HIV-1 RNA export and stability, codon bias that results from the unusually high AU content of HIV-1 transcripts may attenuate translation. The presence of rare codons has been demonstrated to induce ribosome pausing that culminates in RNA turnover (Reviewed by Hentze and Kulozik ). Codon optimization of HIV-1 env and gag-pol sequences has been shown to increase viral protein production in human cells [133, 134]. Furthermore, codon optimization of gag-pol increased RNA abundance and eliminated the requirement for Rev/RRE in HIV-1 based vector systems . The observations that codon optimization eliminates Rev/RRE dependence support a positive role for Rev/RRE in stability and translation of HIV-1 transcripts.
Although studies have observed CNS/INS to modulate HIV-1 RNA by different post-transcriptional mechanisms, the convergent conclusion is that these viral sequences interact with cellular proteins involved in splicing and other steps in mRNA metabolism to balance virus protein production. Furthermore, these studies emphasize that Rev/RRE is a multifunctional protein/RNA interaction which can trans-activate the stability of RRE-containing viral mRNAs, their export from the nucleus, and their translation in the cytoplasm. The combination of these roles makes Rev/RRE a strategic regulatory axis in HIV-1 replication.
A balancing act: The ability of retroviruses to elicit or block innate host defense pathways
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 . 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 . 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 ). 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 , productive HIV-1 infection causes a decrease in PKR protein levels . A separate study indicated that a high concentration of TAR-containing RNA can inhibit PKR activation, possibly by sequestration of the protein . 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 .
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 . 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 . 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 . 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 . 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) . TRBP (also designated TRBP1) was discovered to bind HIV-1 TAR in a cDNA screen in HeLa cells . An isoform designated TRBP2 has been identified that includes a 21 amino acid extension at the N-terminus . Because both TRBP isoforms have been reported to activate the HIV-1 LTR in human and murine cells, they appear to be functionally equivalent .
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 ; and ii) binding to HIV-1 TAR and Rev response element (RRE) RNA resulting in enhanced virus expression . 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 . 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 . 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 .
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 . 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 . 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 . Homan and colleagues utilized a stable 2-5A agonist called 2-5AN6B, which is taken up into the cytoplasm of cultured T cells and subsequently activates RNaseL . They found that treatment of PBMCs with 2-5AN6B 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 2-5AN6B 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 . 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 . 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 . 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 . Interestingly, Tas RSS activity is recapitulated across the plant and animal kingdoms .
A role for RNA silencing in HIV-1 replication kinetics has been documented in both peripheral blood mononuclear cells  and in resting primary CD4+ T cells . Mechanistically, host RNA silencing inhibits translation of HIV-1 gag-pol mRNA and possibly all HIV-1 transcripts . 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 . 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 . Suppression of miRNA-directed activity against HIV-1 was first documented by Bennasser et al. , but remains controversial . Tat RSS activity is modest and not global, and is demonstrated in several [176, 178] but not all assays . Tat RSS activity is dependent on the double-stranded RNA binding domain , which is a feature conserved in many plant virus RSS . Like PFV-1 Tas, HIV-1 Tat displays cross-kingdom RSS activity .
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 . Second, plant virus RSS  or Ebola V35  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 .
Additional evidence for HIV-1 modulation of the host small RNA pathway has been presented by Jeang and colleagues . 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 . 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 .
In conclusion, investigations continue to unveil retrovirus-host interplay in post-transcriptional control. A recurrent theme is employment of viral strategies that leverage antiviral defenses to achieve balanced viral gene expression. The fundamental knowledge of retrovirus translational control continues to expand. Each new finding has potential utility to devising new strategies for antiviral therapy and improving retroviral vector transduction. A common theme of retrovirus translational control is that an RNA structural motif interacts with viral or cellular protein to modulate balanced levels of structural and enzymatic proteins necessary for viral replication (summarized in Tables 1 and 2). These RNA-protein interactions have already been employed to improve gene transfer vectors [184–186]. Further understanding of these RNA-protein interactions may ultimately generate specialized vectors or helper virus cassettes that modulate interactions with translation modulatory proteins, such as RNA helicase A, 9G8, or hnRNP E1, or other yet-to-be identified interaction partners.
Another significant arena is the understanding of viral proteins that alter cellular post-transcriptional innate defenses (summarized in Figure 2 and Table 2). A protein of obvious importance is HIV-1 Tat, which in addition to its essential role in virus-specific transcription, has the ability to block the antiviral PKR response and act as an RNA silencing suppressor. Given its multi-faceted activity, Tat could be a sophisticated target for antiviral therapy. The other HIV-1 regulatory protein Rev has already been targeted in clinical trial , but this approach to influencing viral translational control remains to be realized. Decoding the molecular mechanisms of retrovirus translational control is valuable to fundamental knowledge and to the development of targeted antiviral strategies and gene transfer tools.
We thank Mr. Shuiming Qian and Dr. Kathleen Hayes-Ozello for comments on the text and Mr. Tim Vojt for figure preparation. This work was supported by grants from the National Institutes of Health National Cancer Institute RO1CA108882; P01CA16058; and P30CA100730.
- Kozak M: The scanning model for translation: an update. J Cell Biol. 1989, 108: 229-241.PubMedGoogle Scholar
- Merrick WC: Cap-dependent and cap-independent translation in eukaryotic systems. Gene. 2004, 332: 1-11.PubMedGoogle Scholar
- Kozak M: Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984, 12: 857-872.PubMed CentralPubMedGoogle Scholar
- Pelletier J, Sonenberg N: Insertion mutagenesis to increase secondary structure within the 5' noncoding region of a eukaryotic mRNA reduces translational efficiency. Cell. 1985, 40: 515-526.PubMedGoogle Scholar
- Butsch M, Boris-Lawrie K: Destiny of unspliced retroviral RNA: ribosome and/or virion?. J Virol. 2002, 76: 3089-3094.PubMed CentralPubMedGoogle Scholar
- Anderson EC, Lever AM: Human immunodeficiency virus type 1 Gag polyprotein modulates its own translation. J Virol. 2006, 80: 10478-10486.PubMed CentralPubMedGoogle Scholar
- Kaye JF, Lever AM: Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation. J Virol. 1999, 73: 3023-3031.PubMed CentralPubMedGoogle Scholar
- Levin JG, Grimley PM, Ramseur JM, Berezesky IK: Deficiency of 60 to 70S RNA in murine leukemia virus particles assembled in cells treated with actinomycin D. J Virol. 1974, 14: 152-161.PubMed CentralPubMedGoogle Scholar
- Levin JG, Rosenak MJ: Synthesis of murine leukemia virus proteins associated with virions assembled in actinomycin D-treated cells: evidence for persistence of viral messenger RNA. Proc Natl Acad Sci USA. 1976, 73: 1154-1158.PubMed CentralPubMedGoogle Scholar
- Butsch M, Boris-Lawrie K: Translation is not required to generate virion precursor RNA in human immunodeficiency virus type 1-infected T cells. J Virol. 2000, 74: 11531-11537.PubMed CentralPubMedGoogle Scholar
- LeBlanc JJ, Beemon KL: Unspliced Rous sarcoma virus genomic RNAs are translated and subjected to nonsense-mediated mRNA decay before packaging. J Virol. 2004, 78: 5139-5146.PubMed CentralPubMedGoogle Scholar
- Weil JE, Beemon KL: A 3' UTR sequence stabilizes termination codons in the unspliced RNA of Rous sarcoma virus. RNA. 2006, 12: 102-110.PubMed CentralPubMedGoogle Scholar
- Berkhout B: Structure and function of the human immunodeficiency virus leader RNA. Prog Nucleic Acid Res Mol Biol. 1996, 54: 1-34.PubMedGoogle Scholar
- Schwartz S, Felber BK, Benko DM, Fenyo EM, Pavlakis GN: Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1. J Virol. 1990, 64: 2519-2529.PubMed CentralPubMedGoogle Scholar
- Purcell DF, Martin MA: Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity. J Virol. 1993, 67: 6365-6378.PubMed CentralPubMedGoogle Scholar
- Parkin NT, Cohen EA, Darveau A, Rosen C, Haseltine W, Sonenberg N: Mutational analysis of the 5' non-coding region of human immunodeficiency virus type 1: effects of secondary structure on translation. EMBO J. 1988, 7: 2831-2837.PubMed CentralPubMedGoogle Scholar
- Miele G, Mouland A, Harrison GP, Cohen E, Lever AM: The human immunodeficiency virus type 1 5' packaging signal structure affects translation but does not function as an internal ribosome entry site structure. J Virol. 1996, 70: 944-951.PubMed CentralPubMedGoogle Scholar
- Geballe AP, Gray MK: Variable inhibition of cell-free translation by HIV-1 transcript leader sequences. Nucleic Acids Res. 1992, 20: 4291-4297.PubMed CentralPubMedGoogle Scholar
- Dasso MC, Jackson RJ: Efficient initiation of mammalian mRNA translation at a CUG codon. Nucleic Acids Res. 1989, 17: 6485-6497.PubMed CentralPubMedGoogle Scholar
- Hann SR: Regulation and function of non-AUG-initiated proto-oncogenes. Biochimie. 1994, 76: 880-886.PubMedGoogle Scholar
- Das AT, van Dam AP, Klaver B, Berkhout B: Improved envelope function selected by long-term cultivation of a translation-impaired HIV-1 mutant. Virology. 1998, 244: 552-562.PubMedGoogle Scholar
- Yilmaz A, Bolinger C, Boris-Lawrie K: Retrovirus translation initiation: Issues and hypotheses derived from study of HIV-1. Curr HIV Res. 2006, 4: 131-139.PubMedGoogle Scholar
- Schwartz S, Felber BK, Pavlakis GN: Mechanism of translation of monocistronic and multicistronic human immunodeficiency virus type 1 mRNAs. Mol Cell Biol. 1992, 12: 207-219.PubMed CentralPubMedGoogle Scholar
- Nicot C, Harrod RL, Ciminale V, Franchini G: Human T-cell leukemia/lymphoma virus type 1 nonstructural genes and their functions. Oncogene. 2005, 24: 6026-6034.PubMedGoogle Scholar
- Coffin JM, Hughes SH, Varmus HE: Retroviruses. 1997, Cold Spring Harbor: Cold Spring Harbor Laboratory PressGoogle Scholar
- Bandyopadhyay PK, Temin HM: Expression from an internal AUG codon of herpes simplex thymidine kinase gene inserted in a retrovirus vector. Mol Cell Biol. 1984, 4: 743-748.PubMed CentralPubMedGoogle Scholar
- Pelletier J, Kaplan G, Racaniello VR, Sonenberg N: Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region. Mol Cell Biol. 1988, 8: 1103-1112.PubMed CentralPubMedGoogle Scholar
- Jang SK, Krausslich HG, Nicklin MJ, Duke GM, Palmenberg AC, Wimmer E: A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol. 1988, 62: 2636-2643.PubMed CentralPubMedGoogle Scholar
- Hellen CU, Sarnow P: Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 2001, 15: 1593-1612.PubMedGoogle Scholar
- Etchison D, Milburn SC, Edery I, Sonenberg N, Hershey JW: Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex. J Biol Chem. 1982, 257: 14806-14810.PubMedGoogle Scholar
- Joachims M, Van Breugel PC, Lloyd RE: Cleavage of poly(A)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro. J Virol. 1999, 73: 718-727.PubMed CentralPubMedGoogle Scholar
- Kerekatte V, Keiper BD, Badorff C, Cai A, Knowlton KU, Rhoads RE: Cleavage of Poly(A)-binding protein by coxsackievirus 2A protease in vitro and in vivo: another mechanism for host protein synthesis shutoff?. J Virol. 1999, 73: 709-717.PubMed CentralPubMedGoogle Scholar
- Chen CY, Sarnow P: Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs. Science. 1995, 268: 415-417.PubMedGoogle Scholar
- Bolinger C, Yilmaz A, Hartman TR, Kovacic MB, Fernandez S, Ye J, et al: RNA helicase A interacts with divergent lymphotropic retroviruses and promotes translation of human T-cell leukemia virus type 1. Nucleic Acids Res. 2007, 35: 2629-2642.PubMed CentralPubMedGoogle Scholar
- Brasey A, Lopez-Lastra M, Ohlmann T, Beerens N, Berkhout B, Darlix JL, et al: The leader of human immunodeficiency virus type 1 genomic RNA harbors an internal ribosome entry segment that is active during the G2/M phase of the cell cycle. J Virol. 2003, 77: 3939-3949.PubMed CentralPubMedGoogle Scholar
- Buck CB, Shen X, Egan MA, Pierson TC, Walker CM, Siliciano RF: The human immunodeficiency virus type 1 gag gene encodes an internal ribosome entry site. J Virol. 2001, 75: 181-191.PubMed CentralPubMedGoogle Scholar
- Herbreteau CH, Weill L, Decimo D, Prevot D, Darlix JL, Sargueil B, et al: HIV-2 genomic RNA contains a novel type of IRES located downstream of its initiation codon. Nat Struct Mol Biol. 2005Google Scholar
- Ohlmann T, Lopez-Lastra M, Darlix JL: An internal ribosome entry segment promotes translation of the simian immunodeficiency virus genomic RNA. J Biol Chem. 2000, 275: 11899-11906.PubMedGoogle Scholar
- Deffaud C, Darlix JL: Rous sarcoma virus translation revisited: characterization of an internal ribosome entry segment in the 5' leader of the genomic RNA. J Virol. 2000, 74: 11581-11588.PubMed CentralPubMedGoogle Scholar
- Deffaud C, Darlix JL: Characterization of an internal ribosomal entry segment in the 5' leader of murine leukemia virus env RNA. J Virol. 2000, 74: 846-850.PubMed CentralPubMedGoogle Scholar
- Berlioz C, Darlix JL: An internal ribosomal entry mechanism promotes translation of murine leukemia virus gag polyprotein precursors. J Virol. 1995, 69: 2214-2222.PubMed CentralPubMedGoogle Scholar
- Vagner S, Waysbort A, Marenda M, Gensac MC, Amalric F, Prats AC: Alternative translation initiation of the Moloney murine leukemia virus mRNA controlled by internal ribosome entry involving the p57/PTB splicing factor. J Biol Chem. 1995, 270: 20376-20383.PubMedGoogle Scholar
- Berlioz C, Torrent C, Darlix JL: An internal ribosomal entry signal in the rat VL30 region of the Harvey murine sarcoma virus leader and its use in dicistronic retroviral vectors. J Virol. 1995, 69: 6400-6407.PubMed CentralPubMedGoogle Scholar
- Butsch M, Hull S, Wang Y, Roberts TM, Boris-Lawrie K: The 5' RNA terminus of spleen necrosis virus contains a novel posttranscriptional control element that facilitates human immunodeficiency virus Rev/RRE-independent Gag production. J Virol. 1999, 73: 4847-4855.PubMed CentralPubMedGoogle Scholar
- Roberts TM, Boris-Lawrie K: The 5' RNA terminus of spleen necrosis virus stimulates translation of nonviral mRNA. J Virol. 2000, 74: 8111-8118.PubMed CentralPubMedGoogle Scholar
- Hartman TR, Qian S, Bolinger C, Fernandez S, Schoenberg DR, Boris-Lawrie K: RNA helicase A is necessary for translation of selected messenger RNAs. Nat Struct Mol Biol. 2006, 13: 509-516.PubMedGoogle Scholar
- Hull S, Boris-Lawrie K: RU5 of Mason-Pfizer monkey virus 5' long terminal repeat enhances cytoplasmic expression of human immunodeficiency virus type 1 gag-pol and nonviral reporter RNA. J Virol. 2002, 76: 10211-10218.PubMed CentralPubMedGoogle Scholar
- Roberts TM, Boris-Lawrie K: Primary sequence and secondary structure motifs in spleen necrosis virus RU5 confer translational utilization of unspliced human immunodeficiency virus type 1 reporter RNA. J Virol. 2003, 77: 11973-11984.PubMed CentralPubMedGoogle Scholar
- Russell RA, Zeng Y, Erlwein O, Cullen BR, McClure MO: The R region found in the human foamy virus long terminal repeat is critical for both Gag and Pol protein expression. J Virol. 2001, 75: 6817-6824.PubMed CentralPubMedGoogle Scholar
- Yamasaki Y, Narain S, Yoshida H, Hernandez L, Barker T, Hahn PC, et al: Autoantibodies to RNA helicase A: a new serologic marker of early lupus. Arthritis Rheum. 2007, 56: 596-604.PubMedGoogle Scholar
- Wei X, Pacyna-Gengelbach M, Schluns K, An Q, Gao Y, Cheng S, et al: Analysis of the RNA helicase A gene in human lung cancer. Oncol Rep. 2004, 11: 253-258.PubMedGoogle Scholar
- Zhong X, Safa AR: RNA helicase A in the MEF1 transcription factor complex up-regulates the MDR1 gene in multidrug-resistant cancer cells. J Biol Chem. 2004, 279: 17134-17141.PubMedGoogle Scholar
- Takeda Y, Caudell P, Grady G, Wang G, Suwa A, Sharp GC, et al: Human RNA helicase A is a lupus autoantigen that is cleaved during apoptosis. J Immunol. 1999, 163: 6269-6274.PubMedGoogle Scholar
- Chester N, Kuo F, Kozak C, O'Hara CD, Leder P: Stage-specific apoptosis, developmental delay, and embryonic lethality in mice homozygous for a targeted disruption in the murine Bloom's syndrome gene. Genes Dev. 1998, 12: 3382-3393.PubMed CentralPubMedGoogle Scholar
- Robb GB, Rana TM: RNA helicase A interacts with RISC in human cells and functions in RISC loading. Mol Cell. 2007, 26: 523-537.PubMedGoogle Scholar
- Moore MJ: From birth to death: the complex lives of eukaryotic mRNAs. Science. 2005, 309: 1514-1518.PubMedGoogle Scholar
- Ernst RK, Bray M, Rekosh D, Hammarskjold ML: A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA. Mol Cell Biol. 1997, 17: 135-144.PubMed CentralPubMedGoogle Scholar
- Pasquinelli AE, Ernst RK, Lund E, Grimm C, Zapp ML, Rekosh D, et al: The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway. EMBO J. 1997, 16: 7500-7510.PubMed CentralPubMedGoogle Scholar
- Jin L, Guzik BW, Bor YC, Rekosh D, Hammarskjold ML: Tap and NXT promote translation of unspliced mRNA. Genes Dev. 2003, 17: 3075-3086.PubMed CentralPubMedGoogle Scholar
- Swartz JE, Bor YC, Misawa Y, Rekosh D, Hammarskjold ML: The shuttling SR protein 9G8 plays a role in translation of unspliced mRNA containing a constitutive transport element. J Biol Chem. 2007, 282: 19844-19853.PubMedGoogle Scholar
- Gruter P, Tabernero C, von Kobbe C, Schmitt C, Saavedra C, Bachi A, et al: TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus. Mol Cell. 1998, 1: 649-659.PubMedGoogle Scholar
- Huang Y, Steitz JA: Splicing factors SRp20 and 9G8 promote the nucleocytoplasmic export of mRNA. Mol Cell. 2001, 7: 899-905.PubMedGoogle Scholar
- Huang Y, Gattoni R, Stevenin J, Steitz JA: SR splicing factors serve as adapter proteins for TAP-dependent mRNA export. Mol Cell. 2003, 11: 837-843.PubMedGoogle Scholar
- Huang Y, Yario TA, Steitz JA: A molecular link between SR protein dephosphorylation and mRNA export. Proc Natl Acad Sci USA. 2004, 101: 9666-9670.PubMed CentralPubMedGoogle Scholar
- Bray M, Prasad S, Dubay JW, Hunter E, Jeang KT, Rekosh D, et al: A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent. Proc Natl Acad Sci USA. 1994, 91: 1256-1260.PubMed CentralPubMedGoogle Scholar
- Hull S, Boris-Lawrie K: Analysis of synergy between divergent simple retrovirus posttranscriptional control elements. Virology. 2003, 317: 146-154.PubMedGoogle Scholar
- Thermann R, Hentze MW: Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation. Nature. 2007, 447: 875-878.PubMedGoogle Scholar
- Sanford JR, Longman D, Caceres JF: Multiple roles of the SR protein family in splicing regulation. Prog Mol Subcell Biol. 2003, 31: 33-58.PubMedGoogle Scholar
- Sanford JR, Gray NK, Beckmann K, Caceres JF: A novel role for shuttling SR proteins in mRNA translation. Genes Dev. 2004, 18: 755-768.PubMed CentralPubMedGoogle Scholar
- McLaren M, Asai K, Cochrane A: A novel function for Sam68: enhancement of HIV-1 RNA 3' end processing. RNA. 2004, 10: 1119-1129.PubMed CentralPubMedGoogle Scholar
- Reddy TR, Tang H, Xu W, Wong-Staal F: Sam68, RNA helicase A and Tap cooperate in the post-transcriptional regulation of human immunodeficiency virus and type D retroviral mRNA. Oncogene. 2000, 19: 3570-3575.PubMedGoogle Scholar
- Coyle JH, Guzik BW, Bor YC, Jin L, Eisner-Smerage L, Taylor SJ, et al: Sam68 enhances the cytoplasmic utilization of intron-containing RNA and is functionally regulated by the nuclear kinase Sik/BRK. Mol Cell Biol. 2003, 23: 92-103.PubMed CentralPubMedGoogle Scholar
- Soros VB, Carvajal HV, Richard S, Cochrane AW: Inhibition of human immunodeficiency virus type 1 Rev function by a dominant-negative mutant of Sam68 through sequestration of unspliced RNA at perinuclear bundles. J Virol. 2001, 75: 8203-8215.PubMed CentralPubMedGoogle Scholar
- Woolaway K, Asai K, Emili A, Cochrane A: hnRNP E1 and E2 have distinct roles in modulating HIV-1 gene expression. Retrovirology. 2007, 4: 28-PubMed CentralPubMedGoogle Scholar
- Ostareck DH, Ostareck-Lederer A, Wilm M, Thiele BJ, Mann M, Hentze MW: mRNA silencing in erythroid differentiation: hnRNP K and hnRNP E1 regulate 15-lipoxygenase translation from the 3' end. Cell. 1997, 89: 597-606.PubMedGoogle Scholar
- Schwartz S, Felber BK, Fenyo EM, Pavlakis GN: Env and Vpu proteins of human immunodeficiency virus type 1 are produced from multiple bicistronic mRNAs. J Virol. 1990, 64: 5448-5456.PubMed CentralPubMedGoogle Scholar
- Schubert U, Bour S, Willey RL, Strebel K: Regulation of virus release by the macrophage-tropic human immunodeficiency virus type 1 AD8 isolate is redundant and can be controlled by either Vpu or Env. J Virol. 1999, 73: 887-896.PubMed CentralPubMedGoogle Scholar
- Anderson JL, Johnson AT, Howard JL, Purcell DF: Both linear and discontinuous ribosome scanning are used for translation initiation from bicistronic human immunodeficiency virus type 1 env mRNAs. J Virol. 2007, 81: 4664-4676.PubMed CentralPubMedGoogle Scholar
- Krummheuer J, Johnson AT, Hauber I, Kammler S, Anderson JL, Hauber J, et al: A minimal uORF within the HIV-1 vpu leader allows efficient translation initiation at the downstream env AUG. Virology. 2007, 363: 261-271.PubMedGoogle Scholar
- Pooggin MM, Futterer J, Skryabin KG, Hohn T: A short open reading frame terminating in front of a stable hairpin is the conserved feature in pregenomic RNA leaders of plant pararetroviruses. J Gen Virol. 1999, 80 (Pt 8): 2217-2228.PubMedGoogle Scholar
- Pooggin MM, Hohn T, Futterer J: Role of a short open reading frame in ribosome shunt on the cauliflower mosaic virus RNA leader. J Biol Chem. 2000, 275: 17288-17296.PubMedGoogle Scholar
- Pooggin MM, Futterer J, Skryabin KG, Hohn T: Ribosome shunt is essential for infectivity of cauliflower mosaic virus. Proc Natl Acad Sci USA. 2001, 98: 886-891.PubMed CentralPubMedGoogle Scholar
- Ryabova LA, Pooggin MM, Hohn T: Viral strategies of translation initiation: ribosomal shunt and reinitiation. Prog Nucleic Acid Res Mol Biol. 2002, 72: 1-39.PubMedGoogle Scholar
- Ryabova LA, Hohn T: Ribosome shunting in the cauliflower mosaic virus 35S RNA leader is a special case of reinitiation of translation functioning in plant and animal systems. Genes Dev. 2000, 14: 817-829.PubMed CentralPubMedGoogle Scholar
- Browne GJ, Proud CG: Regulation of peptide-chain elongation in mammalian cells. Eur J Biochem. 2002, 269: 5360-5368.PubMedGoogle Scholar
- Jacks T, Varmus HE: Expression of the Rous sarcoma virus pol gene by ribosomal frameshifting. Science. 1985, 230: 1237-1242.PubMedGoogle Scholar
- Jacks T, Power MD, Masiarz FR, Luciw PA, Barr PJ, Varmus HE: Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature. 1988, 331: 280-283.PubMedGoogle Scholar
- Jacks T, Madhani HD, Masiarz FR, Varmus HE: Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region. Cell. 1988, 55: 447-458.PubMedGoogle Scholar
- Falk H, Mador N, Udi R, Panet A, Honigman A: Two cis-acting signals control ribosomal frameshift between human T-cell leukemia virus type II gag and pro genes. J Virol. 1993, 67: 6273-6277.PubMed CentralPubMedGoogle Scholar
- Mador N, Panet A, Honigman A: Translation of gag, pro, and pol gene products of human T-cell leukemia virus type 2. J Virol. 1989, 63: 2400-2404.PubMed CentralPubMedGoogle Scholar
- Dulude D, Baril M, Brakier-Gingras L: Characterization of the frameshift stimulatory signal controlling a programmed -1 ribosomal frameshift in the human immunodeficiency virus type 1. Nucleic Acids Res. 2002, 30: 5094-5102.PubMed CentralPubMedGoogle Scholar
- Staple DW, Butcher SE: Solution structure and thermodynamic investigation of the HIV-1 frameshift inducing element. J Mol Biol. 2005, 349: 1011-1023.PubMedGoogle Scholar
- Gaudin C, Mazauric MH, Traikia M, Guittet E, Yoshizawa S, Fourmy D: Structure of the RNA signal essential for translational frameshifting in HIV-1. J Mol Biol. 2005, 349: 1024-1035.PubMedGoogle Scholar
- Dulude D, Berchiche YA, Gendron K, Brakier-Gingras L, Heveker N: Decreasing the frameshift efficiency translates into an equivalent reduction of the replication of the human immunodeficiency virus type 1. Virology. 2006, 345: 127-136.PubMedGoogle Scholar
- Leger M, Dulude D, Steinberg SV, Brakier-Gingras L: The three transfer RNAs occupying the A, P and E sites on the ribosome are involved in viral programmed -1 ribosomal frameshift. Nucleic Acids Res. 2007, 35: 5581-5592.PubMed CentralPubMedGoogle Scholar
- Gendron K, Charbonneau J, Dulude D, Heveker N, Ferbeyre G, Brakier-Gingras L: The presence of the TAR RNA structure alters the programmed -1 ribosomal frameshift efficiency of the human immunodeficiency virus type 1 (HIV-1) by modifying the rate of translation initiation. Nucleic Acids Res. 2008, 36: 30-40.PubMed CentralPubMedGoogle Scholar
- Frolova L, Le GX, Zhouravleva G, Davydova E, Philippe M, Kisselev L: Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase. RNA. 1996, 2: 334-341.PubMed CentralPubMedGoogle Scholar
- Song H, Mugnier P, Das AK, Webb HM, Evans DR, Tuite MF, et al: The crystal structure of human eukaryotic release factor eRF1 – mechanism of stop codon recognition and peptidyl-tRNA hydrolysis. Cell. 2000, 100: 311-321.PubMedGoogle Scholar
- Bertram G, Bell HA, Ritchie DW, Fullerton G, Stansfield I: Terminating eukaryote translation: domain 1 of release factor eRF1 functions in stop codon recognition. RNA. 2000, 6: 1236-1247.PubMed CentralPubMedGoogle Scholar
- Hatfield DL, Levin JG, Rein A, Oroszlan S: Translational suppression in retroviral gene expression. Adv Virus Res. 1992, 41: 193-239.PubMedGoogle Scholar
- Yoshinaka Y, Katoh I, Copeland TD, Oroszlan S: Translational readthrough of an amber termination codon during synthesis of feline leukemia virus protease. J Virol. 1985, 55: 870-873.PubMed CentralPubMedGoogle Scholar
- Yoshinaka Y, Katoh I, Copeland TD, Oroszlan S: Murine leukemia virus protease is encoded by the gag-pol gene and is synthesized through suppression of an amber termination codon. Proc Natl Acad Sci USA. 1985, 82: 1618-1622.PubMed CentralPubMedGoogle Scholar
- Orlova M, Yueh A, Leung J, Goff SP: Reverse transcriptase of Moloney murine leukemia virus binds to eukaryotic release factor 1 to modulate suppression of translational termination. Cell. 2003, 115: 319-331.PubMedGoogle Scholar
- Felsenstein KM, Goff SP: Expression of the gag-pol fusion protein of Moloney murine leukemia virus without gag protein does not induce virion formation or proteolytic processing. J Virol. 1988, 62: 2179-2182.PubMed CentralPubMedGoogle Scholar
- Meyer BE, Malim MH: The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm. Genes Dev. 1994, 8: 1538-1547.PubMedGoogle Scholar
- Felber BK, Hadzopoulou-Cladaras M, Cladaras C, Copeland T, Pavlakis GN: rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc Natl Acad Sci USA. 1989, 86: 1495-1499.PubMed CentralPubMedGoogle Scholar
- Richard N, Iacampo S, Cochrane A: HIV-1 Rev is capable of shuttling between the nucleus and cytoplasm. Virology. 1994, 204: 123-131.PubMedGoogle Scholar
- Hidaka M, Inoue J, Yoshida M, Seiki M: Post-transcriptional regulator (rex) of HTLV-1 initiates expression of viral structural proteins but suppresses expression of regulatory proteins. EMBO J. 1988, 7: 519-523.PubMed CentralPubMedGoogle Scholar
- Seiki M, Inoue J, Hidaka M, Yoshida M: Two cis-acting elements responsible for posttranscriptional trans-regulation of gene expression of human T-cell leukemia virus type I. Proc Natl Acad Sci USA. 1988, 85: 7124-7128.PubMed CentralPubMedGoogle Scholar
- Indik S, Gunzburg WH, Salmons B, Rouault F: A novel, mouse mammary tumor virus encoded protein with Rev-like properties. Virology. 2005, 337: 1-6.PubMedGoogle Scholar
- Mertz JA, Simper MS, Lozano MM, Payne SM, Dudley JP: Mouse mammary tumor virus encodes a self-regulatory RNA export protein and is a complex retrovirus. J Virol. 2005, 79: 14737-14747.PubMed CentralPubMedGoogle Scholar
- Cochrane AW, Jones KS, Beidas S, Dillon PJ, Skalka AM, Rosen CA: Identification and characterization of intragenic sequences which repress human immunodeficiency virus structural gene expression. J Virol. 1991, 65: 5305-5313.PubMed CentralPubMedGoogle Scholar
- Maldarelli F, Martin MA, Strebel K: Identification of posttranscriptionally active inhibitory sequences in human immunodeficiency virus type 1 RNA: novel level of gene regulation. J Virol. 1991, 65: 5732-5743.PubMed CentralPubMedGoogle Scholar
- Schwartz S, Campbell M, Nasioulas G, Harrison J, Felber BK, Pavlakis GN: Mutational inactivation of an inhibitory sequence in human immunodeficiency virus type 1 results in Rev-independent gag expression. J Virol. 1992, 66: 7176-7182.PubMed CentralPubMedGoogle Scholar
- Schwartz S, Felber BK, Pavlakis GN: Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein. J Virol. 1992, 66: 150-159.PubMed CentralPubMedGoogle Scholar
- Schneider R, Campbell M, Nasioulas G, Felber BK, Pavlakis GN: Inactivation of the human immunodeficiency virus type 1 inhibitory elements allows Rev-independent expression of Gag and Gag/protease and particle formation. J Virol. 1997, 71: 4892-4903.PubMed CentralPubMedGoogle Scholar
- Malim MH, Cullen BR: Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes. Mol Cell Biol. 1993, 13: 6180-6189.PubMed CentralPubMedGoogle Scholar
- Arrigo SJ, Chen IS: Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs. Genes Dev. 1991, 5: 808-819.PubMedGoogle Scholar
- D'Agostino DM, Felber BK, Harrison JE, Pavlakis GN: The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs. Mol Cell Biol. 1992, 12: 1375-1386.PubMed CentralPubMedGoogle Scholar
- Arrigo SJ, Weitsman S, Rosenblatt JD, Chen IS: Analysis of rev gene function on human immunodeficiency virus type 1 replication in lymphoid cells by using a quantitative polymerase chain reaction method. J Virol. 1989, 63: 4875-4881.PubMed CentralPubMedGoogle Scholar
- Iacampo S, Cochrane A: Human immunodeficiency virus type 1 Rev function requires continued synthesis of its target mRNA. J Virol. 1996, 70: 8332-8339.PubMed CentralPubMedGoogle Scholar
- Kusuhara K, Anderson M, Pettiford SM, Green PL: Human T-cell leukemia virus type 2 Rex protein increases stability and promotes nuclear to cytoplasmic transport of gag/pol and env RNAs. J Virol. 1999, 73: 8112-8119.PubMed CentralPubMedGoogle Scholar
- Reddy TR, Xu W, Mau JK, Goodwin CD, Suhasini M, Tang H, et al: Inhibition of HIV replication by dominant negative mutants of Sam68, a functional homolog of HIV-1 Rev. Nat Med. 1999, 5: 635-642.PubMedGoogle Scholar
- Modem S, Badri KR, Holland TC, Reddy TR: Sam68 is absolutely required for Rev function and HIV-1 production. Nucleic Acids Res. 2005, 33: 873-879.PubMed CentralPubMedGoogle Scholar
- Rosen CA, Terwilliger E, Dayton A, Sodroski JG, Haseltine WA: Intragenic cis-acting art gene-responsive sequences of the human immunodeficiency virus. Proc Natl Acad Sci USA. 1988, 85: 2071-2075.PubMed CentralPubMedGoogle Scholar
- Olsen HS, Cochrane AW, Rosen C: Interaction of cellular factors with intragenic cis-acting repressive sequences within the HIV genome. Virology. 1992, 191: 709-715.PubMedGoogle Scholar
- Black AC, Luo J, Chun S, Bakker A, Fraser JK, Rosenblatt JD: Specific binding of polypyrimidine tract binding protein and hnRNP A1 to HIV-1 CRS elements. Virus Genes. 1996, 12: 275-285.PubMedGoogle Scholar
- Black AC, Luo J, Watanabe C, Chun S, Bakker A, Fraser JK, et al: Polypyrimidine tract-binding protein and heterogeneous nuclear ribonucleoprotein A1 bind to human T-cell leukemia virus type 2 RNA regulatory elements. J Virol. 1995, 69: 6852-6858.PubMed CentralPubMedGoogle Scholar
- Zolotukhin AS, Michalowski D, Bear J, Smulevitch SV, Traish AM, Peng R, et al: PSF acts through the human immunodeficiency virus type 1 mRNA instability elements to regulate virus expression. Mol Cell Biol. 2003, 23: 6618-6630.PubMed CentralPubMedGoogle Scholar
- Afonina E, Neumann M, Pavlakis GN: Preferential binding of poly(A)-binding protein 1 to an inhibitory RNA element in the human immunodeficiency virus type 1 gag mRNA. J Biol Chem. 1997, 272: 2307-2311.PubMedGoogle Scholar
- Najera I, Krieg M, Karn J: Synergistic stimulation of HIV-1 rev-dependent export of unspliced mRNA to the cytoplasm by hnRNP A1. J Mol Biol. 1999, 285: 1951-1964.PubMedGoogle Scholar
- Hentze MW, Kulozik AE: RNA surveillance and nonsense-mediated decay. Cell. 1999, 96: 307-310.PubMedGoogle Scholar
- Haas J, Park EC, Seed B: Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr Biol. 1996, 6: 315-324.PubMedGoogle Scholar
- Kotsopoulou E, Kim VN, Kingsman AJ, Kingsman SM, Mitrophanous KA: A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene. J Virol. 2000, 74: 4839-4852.PubMed CentralPubMedGoogle Scholar
- Muesing MA, Smith DH, Capon DJ: Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein. Cell. 1987, 48: 691-701.PubMedGoogle Scholar
- Roy S, Agy M, Hovanessian AG, Sonenberg N, Katze MG: The integrity of the stem structure of human immunodeficiency virus type 1 Tat-responsive sequence of RNA is required for interaction with the interferon-induced 68,000-Mr protein kinase. J Virol. 1991, 65: 632-640.PubMed CentralPubMedGoogle Scholar
- Patel RC, Stanton P, McMillan NM, Williams BR, Sen GC: The interferon-inducible double-stranded RNA-activated protein kinase self-associates in vitro and in vivo. Proc Natl Acad Sci USA. 1995, 92: 8283-8287.PubMed CentralPubMedGoogle Scholar
- Wu S, Kaufman RJ: A model for the double-stranded RNA (dsRNA)-dependent dimerization and activation of the dsRNA-activated protein kinase PKR. J Biol Chem. 1997, 272: 1291-1296.PubMedGoogle Scholar
- Thomis DC, Samuel CE: Mechanism of interferon action: evidence for intermolecular autophosphorylation and autoactivation of the interferon-induced, RNA-dependent protein kinase. J Virol. 1993, 67: 7695-7700.PubMed CentralPubMedGoogle Scholar
- Galabru J, Hovanessian A: Autophosphorylation of the protein kinase dependent on double-stranded RNA. J Biol Chem. 1987, 262: 15538-15544.PubMedGoogle Scholar
- Langland JO, Jacobs BL: Cytosolic double-stranded RNA-dependent protein kinase is likely a dimer of partially phosphorylated Mr = 66,000 subunits. J Biol Chem. 1992, 267: 10729-10736.PubMedGoogle Scholar
- Merrick WC: Mechanism and regulation of eukaryotic protein synthesis. Microbiol Rev. 1992, 56: 291-315.PubMed CentralPubMedGoogle Scholar
- Roy S, Katze MG, Parkin NT, Edery I, Hovanessian AG, Sonenberg N: Control of the interferon-induced 68-kilodalton protein kinase by the HIV-1 tat gene product. Science. 1990, 247: 1216-1219.PubMedGoogle Scholar
- Gunnery S, Rice AP, Robertson HD, Mathews MB: Tat-responsive region RNA of human immunodeficiency virus 1 can prevent activation of the double-stranded-RNA-activated protein kinase. Proc Natl Acad Sci USA. 1990, 87: 8687-8691.PubMed CentralPubMedGoogle Scholar
- Mordechai E, Kon N, Henderson EE, Suhadolnik RJ: Activation of the interferon-inducible enzymes, 2',5'-oligoadenylate synthetase and PKR by human T-cell leukemia virus type I Rex-response element. Virology. 1995, 206: 913-922.PubMedGoogle Scholar
- Brand SR, Kobayashi R, Mathews MB: The Tat protein of human immunodeficiency virus type 1 is a substrate and inhibitor of the interferon-induced, virally activated protein kinase, PKR. J Biol Chem. 1997, 272: 8388-8395.PubMedGoogle Scholar
- McMillan NA, Chun RF, Siderovski DP, Galabru J, Toone WM, Samuel CE, et al: HIV-1 Tat directly interacts with the interferon-induced, double-stranded RNA-dependent kinase, PKR. Virology. 1995, 213: 413-424.PubMedGoogle Scholar
- Endo-Munoz L, Warby T, Harrich D, McMillan NA: Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription. Virol J. 2005, 2: 17-PubMed CentralPubMedGoogle Scholar
- Gatignol A, Buckler-White A, Berkhout B, Jeang KT: Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. Science. 1991, 251: 1597-1600.PubMedGoogle Scholar
- Bannwarth S, Talakoub L, Letourneur F, Duarte M, Purcell DF, Hiscott J, et al: Organization of the human tarbp2 gene reveals two promoters that are repressed in an astrocytic cell line. J Biol Chem. 2001, 276: 48803-48813.PubMedGoogle Scholar
- Duarte M, Graham K, Daher A, Battisti PL, Bannwarth S, Segeral E, et al: Characterization of TRBP1 and TRBP2. Stable stem-loop structure at the 5' end of TRBP2 mRNA resembles HIV-1 TAR and is not found in its processed pseudogene. J Biomed Sci. 2000, 7: 494-506.PubMedGoogle Scholar
- Benkirane M, Neuveut C, Chun RF, Smith SM, Samuel CE, Gatignol A, et al: Oncogenic potential of TAR RNA binding protein TRBP and its regulatory interaction with RNA-dependent protein kinase PKR. EMBO J. 1997, 16: 611-624.PubMed CentralPubMedGoogle Scholar
- Soumelis V, Scott I, Gheyas F, Bouhour D, Cozon G, Cotte L, et al: Depletion of circulating natural type 1 interferon-producing cells in HIV-infected AIDS patients. Blood. 2001, 98: 906-912.PubMedGoogle Scholar
- Feldman S, Stein D, Amrute S, Denny T, Garcia Z, Kloser P, et al: Decreased interferon-alpha production in HIV-infected patients correlates with numerical and functional deficiencies in circulating type 2 dendritic cell precursors. Clin Immunol. 2001, 101: 201-210.PubMedGoogle Scholar
- Murray HW, Rubin BY, Masur H, Roberts RB: Impaired production of lymphokines and immune (gamma) interferon in the acquired immunodeficiency syndrome. N Engl J Med. 1984, 310: 883-889.PubMedGoogle Scholar
- Dorin D, Bonnet MC, Bannwarth S, Gatignol A, Meurs EF, Vaquero C: The TAR RNA-binding protein, TRBP, stimulates the expression of TAR-containing RNAs in vitro and in vivo independently of its ability to inhibit the dsRNA-dependent kinase PKR. J Biol Chem. 2003, 278: 4440-4448.PubMedGoogle Scholar
- Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K, et al: TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature. 2005, 436: 740-744.PubMed CentralPubMedGoogle Scholar
- Bennasser Y, Yeung ML, Jeang KT: HIV-1 TAR RNA subverts RNA interference in transfected cells through sequestration of TAR RNA-binding protein, TRBP. J Biol Chem. 2006, 281: 27674-27678.PubMedGoogle Scholar
- Gatignol A, Laine S, Clerzius G: Dual role of TRBP in HIV replication and RNA interference: viral diversion of a cellular pathway or evasion from antiviral immunity?. Retrovirology. 2005, 2: 65-PubMed CentralPubMedGoogle Scholar
- Dong B, Silverman RH: 2-5A-dependent RNase molecules dimerize during activation by 2-5A. J Biol Chem. 1995, 270: 4133-4137.PubMedGoogle Scholar
- Nilsen TW, Weissman SG, Baglioni C: Role of 2',5'-oligo(adenylic acid) polymerase in the degradation of ribonucleic acid linked to double-stranded ribonucleic acid by extracts of interferon-treated cells. Biochemistry. 1980, 19: 5574-5579.PubMedGoogle Scholar
- Min JY, Krug RM: The primary function of RNA binding by the influenza A virus NS1 protein in infected cells: Inhibiting the 2'–5' oligo (A) synthetase/RNase L pathway. Proc Natl Acad Sci USA. 2006, 103: 7100-7105.PubMed CentralPubMedGoogle Scholar
- Cayley PJ, Davies JA, McCullagh KG, Kerr IM: Activation of the ppp(A2'p)nA system in interferon-treated, herpes simplex virus-infected cells and evidence for novel inhibitors of the ppp(A2'p)nA-dependent RNase. Eur J Biochem. 1984, 143: 165-174.PubMedGoogle Scholar
- Rivas C, Gil J, Melkova Z, Esteban M, Diaz-Guerra M: Vaccinia virus E3L protein is an inhibitor of the interferon (i.f.n.)-induced 2-5A synthetase enzyme. Virology. 1998, 243: 406-414.PubMedGoogle Scholar
- SenGupta DN, Silverman RH: Activation of interferon-regulated, dsRNA-dependent enzymes by human immunodeficiency virus-1 leader RNA. Nucleic Acids Res. 1989, 17: 969-978.PubMed CentralPubMedGoogle Scholar
- Wu JM, Chiao JW, Maayan S: Diagnostic value of the determination of an interferon-induced enzyme activity: decreased 2',5'-oligoadenylate dependent binding protein activity in AIDS patient lymphocytes. AIDS Res. 1986, 2: 127-131.PubMedGoogle Scholar
- Homan JW, Steele AD, Martinand-Mari C, Rogers TJ, Henderson EE, Charubala R, et al: Inhibition of morphine-potentiated HIV-1 replication in peripheral blood mononuclear cells with the nuclease-resistant 2-5A agonist analog, 2-5A(N6B). J Acquir Immune Defic Syndr. 2002, 30: 9-20.PubMedGoogle Scholar
- Dimitrova DI, Reichenbach NL, Yang X, Pfleiderer W, Charubala R, Gaughan JP, et al: Inhibition of HIV type 1 replication in CD4+ and CD14+ cells purified from HIV type 1-infected individuals by the 2-5A agonist immunomodulator, 2-5A(N6B). AIDS Res Hum Retroviruses. 2007, 23: 123-134.PubMed CentralPubMedGoogle Scholar
- Kumar A: RNA interference: a multifaceted innate antiviral defense. Retrovirology. 2008, 5: 17-PubMed CentralPubMedGoogle Scholar
- Lecellier CH, Dunoyer P, Arar K, Lehmann-Che J, Eyquem S, Himber C, et al: A cellular microRNA mediates antiviral defense in human cells. Science. 2005, 308: 557-560.PubMedGoogle Scholar
- Cullen BR: Is RNA interference involved in intrinsic antiviral immunity in mammals?. Nat Immunol. 2006, 7: 563-567.PubMedGoogle Scholar
- Berkhout B, Jeang KT: RISCy business: MicroRNAs, pathogenesis, and viruses. J Biol Chem. 2007, 282: 26641-26645.PubMedGoogle Scholar
- Triboulet R, Mari B, Lin YL, Chable-Bessia C, Bennasser Y, Lebrigand K, et al: Suppression of microRNA-silencing pathway by HIV-1 during virus replication. Science. 2007, 315: 1579-1582.PubMedGoogle Scholar
- Huang J, Wang F, Argyris E, Chen K, Liang Z, Tian H, et al: Cellular microRNAs contribute to HIV-1 latency in resting primary CD4+ T lymphocytes. Nat Med. 2007, 13: 1241-1247.PubMedGoogle Scholar
- Qu F, Morris TJ: Suppressors of RNA silencing encoded by plant viruses and their role in viral infections. FEBS Lett. 2005, 579: 5958-5964.PubMedGoogle Scholar
- Qian S, Zhong X, Yu L, Ding B, Haan PD, Boris-Lawrie K: HIV-1 Tat RNA silencing suppressor activity is conserved across kingdoms and counteracts translational repression of HIV-1. Proc Natl Acad Sci U S A. 2009, 106 (2): 605-610.PubMed CentralPubMedGoogle Scholar
- Yeung ML, Benkirane M, Jeang KT: Small non-coding RNAs, mammalian cells, and viruses: regulatory interactions?. Retrovirology. 2007, 4: 74-PubMed CentralPubMedGoogle Scholar
- Bennasser Y, LE SY, Benkirane M, Jeang KT: Evidence that HIV-1 encodes an siRNA and a suppressor of RNA silencing. Immunity. 2005, 22: 607-619.PubMedGoogle Scholar
- Lin J, Cullen BR: Analysis of the interaction of primate retroviruses with the human RNA interference machinery. J Virol. 2007, 81: 12218-12226.PubMed CentralPubMedGoogle Scholar
- Haasnoot J, de Vries W, Geutjes EJ, Prins M, de Haan P, Berkhout B: The Ebola virus VP35 protein is a suppressor of RNA silencing. PLoS Pathog. 2007, 3: e86-PubMed CentralPubMedGoogle Scholar
- Yeung ML, Bennasser Y, Myers TG, Jiang G, Benkirane M, Jeang KT: Changes in microRNA expression profiles in HIV-1-transfected human cells. Retrovirology. 2005, 2: 81-PubMed CentralPubMedGoogle Scholar
- Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K, et al: TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature. 2005, 436: 740-744.PubMed CentralPubMedGoogle Scholar
- Cullen BR: Viruses and microRNAs. Nat Genet. 2006, 38 (Suppl): S25-S30.PubMedGoogle Scholar
- Rizvi TA, Schmidt RD, Lew KA, Keeling ME: Rev/RRE-independent Mason-Pfizer monkey virus constitutive transport element-dependent propagation of SIVmac239 vectors using a single round of replication assay. Virology. 1996, 222: 457-463.PubMedGoogle Scholar
- Zufferey R, Donello JE, Trono D, Hope TJ: Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol. 1999, 73: 2886-2892.PubMed CentralPubMedGoogle Scholar
- Yilmaz A, Fernandez S, Lairmore MD, Boris-Lawrie K: Coordinate enhancement of transgene transcription and translation in a lentiviral vector. Retrovirology. 2006, 3: 13-PubMed CentralPubMedGoogle Scholar
- Bahner I, Sumiyoshi T, Kagoda M, Swartout R, Peterson D, Pepper K, et al: Lentiviral vector transduction of a dominant-negative Rev gene into human CD34+ hematopoietic progenitor cells potently inhibits human immunodeficiency virus-1 replication. Mol Ther. 2007, 15: 76-85.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.