Retroviral PBS-segment sequence and structure: Orchestrating early and late replication events.

An essential regulatory hub for retroviral replication events, the 5' untranslated region (UTR) encodes an ensemble of cis-acting replication elements that overlap in a logical manner to carry out divergent RNA activities in cells and in virions. The primer binding site (PBS) and primer activation sequence initiate the reverse transcription process in virions, yet overlap with structural elements that regulate expression of the complex viral proteome. PBS-segment also encompasses the attachment site for Integrase to cut and paste the 3' long terminal repeat into the host chromosome to form the provirus and purine residues necessary to execute the precise stoichiometry of genome-length transcripts and spliced viral RNAs. Recent genetic mapping, cofactor affinity experiments, NMR and SAXS have elucidated that the HIV-1 PBS-segment folds into a three-way junction structure. The three-way junction structure is recognized by the host's nuclear RNA helicase A/DHX9 (RHA). RHA tethers host trimethyl guanosine synthase 1 to the Rev/Rev responsive element (RRE)-containing RNAs for m7-guanosine Cap hyper methylation that bolsters virion infectivity significantly. The HIV-1 trimethylated (TMG) Cap licenses specialized translation of virion proteins under conditions that repress translation of the regulatory proteins. Clearly host-adaption and RNA shapeshifting comprise the fundamental basis for PBS-segment orchestrating both reverse transcription of virion RNA and the nuclear modification of m7G-Cap for biphasic translation of the complex viral proteome. These recent observations, which have exposed even greater complexity of retroviral RNA biology than previously established, are the impetus for this article. Basic research to fully comprehend the marriage of PBS-segment structures and host RNA binding proteins that carry out retroviral early and late replication events is likely to expose an immutable virus-specific therapeutic target to attenuate retrovirus proliferation.

Blocking tri-methylguanosine synthase 1 (TGS1) stops anchorage-independent growth of canine sarcomas.

Tri methylguanosine synthase 1 (TGS1) is the enzyme that hyper methylates the hallmark 7-methyl-guanosine cap (m7G-cap) appended to the transcription start site of RNAs. The m7G-cap and the eIF4E-cap binding protein guide canonical cap-dependent translation of mRNAs, whereas hyper methylated cap, m2,2,7G-cap (TMG) lacks adequate eIF4E affinity and licenses entry into a different translation initiation pathway. The potential role for TGS1 and TMG-capped mRNA in neoplastic growth is unknown. Canine sarcoma has high translational value to the human disease. Cumulative downregulation of protein synthesis in osteosarcoma OSCA-40 was achieved cooperatively by siTGS1 and Torin-1. Torin-1 inhibited the proliferation of three canine sarcoma explants in a reversible manner that was eliminated by siRNA-downregulation of TGS1. TGS1 failure prevented the anchorage-independent growth of osteo- and hemangio-sarcomas and curtailed sarcoma recovery from mTOR inhibition. RNA immunoprecipitation studies identified TMG-capped mRNAs encoding TGS1, DHX9 and JUND. TMG-tgs1 transcripts were downregulated by leptomycin B and TGS1 failure was compensated by eIF4E mRNP-dependent tgs1 mRNA translation affected by mTOR. The evidence documents TMG-capped mRNAs are hallmarks of the investigated neoplasms and synergy between TGS1 specialized translation and canonical translation is involved in sarcoma recovery from mTOR inhibition. Therapeutic targeting of TGS1 activity in cancer is ripe for future exploration.

HIV-1 hypermethylated guanosine cap licenses specialized translation unaffected by mTOR.

Appended to the 5' end of nascent RNA polymerase II transcripts is 7-methyl guanosine (m7G-cap) that engages nuclear cap-binding complex (CBC) to facilitate messenger RNA (mRNA) maturation. Mature mRNAs exchange CBC for eIF4E, the rate-limiting translation factor that is controlled through mTOR. Experiments in immune cells have now documented HIV-1 incompletely processed transcripts exhibited hypermethylated m7G-cap and that the down-regulation of the trimethylguanosine synthetase-1-reduced HIV-1 infectivity and virion protein synthesis by several orders of magnitude. HIV-1 cap hypermethylation required nuclear RNA helicase A (RHA)/DHX9 interaction with the shape of the 5' untranslated region (UTR) primer binding site (PBS) segment. Down-regulation of RHA or the anomalous shape of the PBS segment abrogated hypermethylated caps and derepressed eIF4E binding for virion protein translation during global down-regulation of host translation. mTOR inhibition was detrimental to HIV-1 proliferation and attenuated Tat, Rev, and Nef synthesis. This study identified mutually exclusive translation pathways and the calibration of virion structural/accessory protein synthesis with de novo synthesis of the viral regulatory proteins. The hypermethylation of select, viral mRNA resulted in CBC exchange to heterodimeric CBP80/NCBP3 that expanded the functional capacity of HIV-1 in immune cells.

The three-way junction structure of the HIV-1 PBS-segment binds host enzyme important for viral infectivity.

HIV-1 reverse transcription initiates at the primer binding site (PBS) in the viral genomic RNA (gRNA). Although the structure of the PBS-segment undergoes substantial rearrangement upon tRNALys3 annealing, the proper folding of the PBS-segment during gRNA packaging is important as it ensures loading of beneficial host factors. DHX9/RNA helicase A (RHA) is recruited to gRNA to enhance the processivity of reverse transcriptase. Because the molecular details of the interactions have yet to be defined, we solved the solution structure of the PBS-segment preferentially bound by RHA. Evidence is provided that PBS-segment adopts a previously undefined adenosine-rich three-way junction structure encompassing the primer activation stem (PAS), tRNA-like element (TLE) and tRNA annealing arm. Disruption of the PBS-segment three-way junction structure diminished reverse transcription products and led to reduced viral infectivity. Because of the existence of the tRNA annealing arm, the TLE and PAS form a bent helical structure that undergoes shape-dependent recognition by RHA double-stranded RNA binding domain 1 (dsRBD1). Mutagenesis and phylogenetic analyses provide evidence for conservation of the PBS-segment three-way junction structure that is preferentially bound by RHA in support of efficient reverse transcription, the hallmark step of HIV-1 replication.

The mRNA encoding the JUND tumor suppressor detains nuclear RNA-binding proteins to assemble polysomes that are unaffected by mTOR.

One long-standing knowledge gap is the role of nuclear proteins in mRNA translation. Nuclear RNA helicase A (DHX9/RHA) is necessary for the translation of the mRNAs of JUND (JunD proto-oncogene AP-1 transcription factor subunit) and HIV-1 genes, and nuclear cap-binding protein 1 (NCBP1)/CBP80 is a component of HIV-1 polysomes. The protein kinase mTOR activates canonical messenger ribonucleoproteins by post-translationally down-regulating the eIF4E inhibitory protein 4E-BP1. We posited here that NCBP1 and DHX9/RHA (RHA) support a translation pathway of JUND RNA that is independent of mTOR. We present evidence from reciprocal immunoprecipitation experiments indicating that NCBP1 and RHA both are components of messenger ribonucleoproteins in several cell types. Moreover, tandem affinity and RT-quantitative PCR results revealed that JUND mRNA is a component of a previously unknown ribonucleoprotein complex. Results from the tandem IP indicated that another component of the JUND-containing ribonucleoprotein complex is NCBP3, a recently identified ortholog of NCBP2/CBP20. We also found that NCBP1, NCBP3, and RHA, but not NCBP2, are components of JUND-containing polysomes. Mutational analysis uncovered two dsRNA-binding domains of RHA that are necessary to tether JUND-NCBP1/NCBP3 to polysomes. We also found that JUND translation is unaffected by inhibition of mTOR, unless RHA was down-regulated by siRNA. These findings uncover a noncanonical cap-binding complex consisting of NCBP1/NCBP3 and RHA substitutes for the eukaryotic translation initiation factors 4E and 4G and activates mTOR-independent translation of the mRNA encoding the tumor suppressor JUND.

Virion-associated, host-derived DHX9/RNA helicase A enhances the processivity of HIV-1 reverse transcriptase on genomic RNA.

DHX9/RNA helicase A (RHA) is a host RNA helicase that participates in many critical steps of the HIV-1 life cycle. It co-assembles with the viral RNA genome into the capsid core. Virions deficient in RHA are less infectious as a result of reduced reverse transcription efficiency, demonstrating that the virion-associated RHA promotes reverse transcription before the virion gains access to the new host's RHA. Here, we quantified reverse-transcription intermediates in HIV-1-infected T cells to clarify the mechanism by which RHA enhances HIV-1 reverse transcription efficiency. Consistently, purified recombinant human RHA promoted reverse transcription efficiency under in vitro conditions that mimic the early reverse transcription steps prior to capsid core uncoating. We did not observe RHA-mediated structural remodeling of the tRNALys3-viral RNA-annealed complex. RHA did not enhance the DNA synthesis rate until incorporation of the first few nucleotides, suggesting that RHA participates primarily in the elongation phase of reverse transcription. Pre-steady-state and steady-state kinetic studies revealed that RHA has little impact on the kinetics of single-nucleotide incorporation. Primer extension assays performed in the presence of trap dsDNA disclosed that RHA enhances the processivity of HIV-1 reverse transcriptase (RT). The biochemical assays used here effectively reflected and explained the low RT activity in HIV-1 virions produced from RHA-depleted cells. Moreover, RT activity in our assays indicated that RHA in HIV-1 virions is required for the efficient catalysis of (-)cDNA synthesis during viral infection before capsid uncoating. Our study identifies RHA as a processivity factor of HIV-1 RT.

Identification of conserved, primary sequence motifs that direct retrovirus RNA fate.

Precise stoichiometry of genome-length transcripts and alternatively spliced mRNAs is a hallmark of retroviruses. We discovered short, guanosine and adenosine sequence motifs in the 5'untranslated region of several retroviruses and ascertained the reasons for their conservation using a representative lentivirus and genetically simpler retrovirus. We conducted site-directed mutagenesis of the GA-motifs in HIV molecular clones and observed steep replication delays in T-cells. Quantitative RNA analyses demonstrate the GA-motifs are necessary to retain unspliced viral transcripts from alternative splicing. Mutagenesis of the GA-motifs in a C-type retrovirus validate the similar downregulation of unspliced transcripts and virion structural protein. The evidence from cell-based co-precipitation studies shows the GA-motifs in the 5'untranslated region confer binding by SFPQ/PSF, a protein co-regulated with T-cell activation. Diminished SFPQ/PSF or mutation of either GA-motif attenuates the replication of HIV. The interaction of SFPQ/PSF with both GA-motifs is crucial for maintaining the stoichiometry of the viral transcripts and does not affect packaging of HIV RNA. Our results demonstrate the conserved GA-motifs direct the fate of retrovirus RNA. These findings have exposed an RNA-based molecular target to attenuate retrovirus replication.

DHX9/RHA Binding to the PBS-Segment of the Genomic RNA during HIV-1 Assembly Bolsters Virion Infectivity.

Cellular RNA-binding proteins incorporated into virions during human immunodeficiency virus type 1 (HIV-1) assembly promote the replication efficiency of progeny virions. Despite its critical role in bolstering virion infectivity, the molecular basis for the incorporation of DHX9/RNA helicase A (RHA) to virions remains unclear. Here, cell-based experiments demonstrate that the truncation of segments of the HIV-1 5'-untranslated region (5'-UTR) distinct from the core encapsidation sequence eliminated virion incorporation of RHA, indicating that RHA recruitment is mediated by specific interactions with the HIV-1 5'-UTR. In agreement with biological data, isothermal titration calorimetry determined that the dimer conformation of the 5'-UTR binds one RHA molecule per RNA strand, and the interaction is independent of nucleocapsid protein binding. NMR spectra employing a deuterium-labeling approach enabled resolution of the dimeric 5'-UTR in complex with the RHA N-terminal domain. The structure of the large molecular mass complex was dependent on RHA binding to a double-stranded region of the primer binding site (PBS)-segment of the 5'-UTR. A single A-to-C substitution was sufficient to disrupt biophysical conformation and attenuate virion infectivity in cell-based assays. Taken together, our studies demonstrate the structural basis for HIV-1 genomic RNA to recruit beneficial cellular cofactor to virions. The support of progeny virion infectivity by RHA is attributable to structure-dependent binding at the PBS-segment of the HIV-1 5'-UTR during virus assembly.

HIV-1 and two avian retroviral 5' untranslated regions bind orthologous human and chicken RNA binding proteins.

Essential host cofactors in retrovirus replication bind cis-acting sequences in the 5'untranslated region (UTR). Although host RBPs are crucial to all aspects of virus biology, elucidating their roles in replication remains a challenge to the field. Here RNA affinity-coupled-proteomics generated a comprehensive, unbiased inventory of human and avian RNA binding proteins (RBPs) co-isolating with 5'UTRs of HIV-1, spleen necrosis virus and Rous sarcoma virus. Applying stringent biochemical and statistical criteria, we identified 185 RBP; 122 were previously implicated in retrovirus biology and 63 are new to the 5'UTR proteome. RNA electrophoretic mobility assays investigated paralogs present in the common ancestor of vertebrates and one hnRNP was identified as a central node to the biological process-anchored networks of HIV-1, SNV, and RSV 5' UTR-proteomes. This comprehensive view of the host constituents of retroviral RNPs is broadly applicable to investigation of viral replication and antiviral response in both human and avian cell lineages.

Stress-induced isoforms of MDM2 and MDM4 correlate with high-grade disease and an altered splicing network in pediatric rhabdomyosarcoma.

Pediatric rhabdomyosarcoma (RMS) is a morphologically and genetically heterogeneous malignancy commonly classified into three histologic subtypes, namely, alveolar, embryonal, and anaplastic. An issue that continues to challenge effective RMS patient prognosis is the dearth of molecular markers predictive of disease stage irrespective of tumor subtype. Our study involving a panel of 70 RMS tumors has identified specific alternative splice variants of the oncogenes Murine Double Minute 2 (MDM2) and MDM4 as potential biomarkers for RMS. Our results have demonstrated the strong association of genotoxic-stress inducible splice forms MDM2-ALT1 (91.6% Intergroup Rhabdomyosarcoma Study Group stage 4 tumors) and MDM4-ALT2 (90.9% MDM4-ALT2-positive T2 stage tumors) with high-risk metastatic RMS. Moreover, MDM2-ALT1-positive metastatic tumors belonged to both the alveolar (50%) and embryonal (41.6%) subtypes, making this the first known molecular marker for high-grade metastatic disease across the most common RMS subtypes. Furthermore, our results show that MDM2-ALT1 expression can function by directly contribute to metastatic behavior and promote the invasion of RMS cells through a matrigel-coated membrane. Additionally, expression of both MDM2-ALT1 and MDM4-ALT2 increased anchorage-independent cell-growth in soft agar assays. Intriguingly, we observed a unique coordination in the splicing of MDM2-ALT1 and MDM4-ALT2 in approximately 24% of tumor samples in a manner similar to genotoxic stress response in cell lines. To further explore splicing network alterations with possible relevance to RMS disease, we used an exon microarray approach to examine stress-inducible splicing in an RMS cell line (Rh30) and observed striking parallels between stress-responsive alternative splicing and constitutive splicing in RMS tumors.

Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes.

The stem cell protein Lin28 functions to inhibit the biogenesis of a group of miRNAs but also stimulates the expression of a subset of mRNAs at the post-transcriptional level, the underlying mechanism of which is not yet understood. Here we report the characterization of the molecular interplay between Lin28 and RNA helicase A (RHA) known to play an important role in remodeling ribonucleoprotein particles during translation. We show that reducing Lin28 expression results in decreased RHA association with polysomes while increasing Lin28 expression leads to elevated RHA association. Further, the carboxyl terminus of Lin28 is necessary for interaction with both the amino and carboxyl termini of RHA. Importantly, a carboxyl terminal deletion mutant of Lin28 that retains RNA-binding activity fails to interact with RHA and exhibits dominant-negative effects on Lin28-dependent stimulation of translation. Taken together, these results lead us to suggest that Lin28 may stimulate translation by actively recruiting RHA to polysomes.

Features of double-stranded RNA-binding domains of RNA helicase A are necessary for selective recognition and translation of complex mRNAs.

The DExH protein RNA helicase A (RHA) plays numerous roles in cell physiology, and post-transcriptional activation of gene expression is a major role among them. RHA selectively activates translation of complex cellular and retroviral mRNAs. Although RHA requires interaction with structural features of the 5'-UTR of these target mRNAs, the molecular basis of their translation activation by RHA is poorly understood. RHA contains a conserved ATPase-dependent helicase core that is flanked by two α-ß-ß-ß-α double-stranded RNA-binding domains at the N terminus and repeated arginine-glycine residues at the C terminus. The individual recombinant N-terminal, central helicase, and C-terminal domains were evaluated for their ability to specifically interact with cognate RNAs by in vitro biochemical measurements and mRNA translation assays in cells. The results demonstrate that N-terminal residues confer selective interaction with retroviral and junD target RNAs. Conserved lysine residues in the distal α-helix of the double-stranded RNA-binding domains are necessary to engage structural features of retroviral and junD 5'-UTRs. Exogenous expression of the N terminus coprecipitates junD mRNA and inhibits the translation activity of endogenous RHA. The results indicate that the molecular basis for the activation of translation by RHA is recognition of target mRNA by the N-terminal domain that tethers the ATP-dependent helicase for rearrangement of the complex 5'-UTR.

RNA helicase A modulates translation of HIV-1 and infectivity of progeny virions.

Retroviruses rely on host RNA-binding proteins to modulate various steps in their replication. Previously several animal retroviruses were determined to mediate Dhx9/RNA helicase A (RHA) interaction with a 5' terminal post-transcriptional control element (PCE) for efficient translation. Herein PCE reporter assays determined HTLV-1 and HIV-1 RU5 confer orientation-dependent PCE activity. The effect of Dhx9/RHA down-regulation and rescue with siRNA-resistant RHA on expression of HIV-1(NL4-3) provirus determined that RHA is necessary for efficient HIV-1 RNA translation and requires ATPase-dependent helicase function. Quantitative analysis determined HIV-1 RNA steady-state and cytoplasmic accumulation were not reduced; rather the translational activity of viral RNA was reduced. Western blotting determined that RHA-deficient virions assemble with Lys-tRNA synthetase, exhibit processed reverse transcriptase and contain similar level of viral RNA, but they are poorly infectious on primary lymphocytes and HeLa cells. The results demonstrate RHA is an important host factor within the virus-producer cell and within the viral particle. The identification of RHA-dependent PCE activity in cellular junD RNA and in six of seven genera of Retroviridae suggests conservation of this translational control mechanism among vertebrates, and convergent evolution of Retroviridae to utilize this host mechanism.

HIV-1 Tat RNA silencing suppressor activity is conserved across kingdoms and counteracts translational repression of HIV-1.

The RNA silencing pathway is an intracellular innate response to virus infections and retro-transposons. Many plant viruses counter this host restriction by RNA silencing suppressor (RSS) activity of a double-stranded RNA-binding protein, e.g., tomato bushy stunt virus P19. Here, we demonstrate P19 and HIV-1 Tat function across the plant and animal kingdoms and suppress a common step in RNA silencing that is downstream of small RNA maturation. Our experiments reveal that RNA silencing in HIV-1 infected human cells severely attenuates the translational output of the unspliced HIV-1 gag mRNA, and possibly all HIV-1 transcripts. The attenuation in gag mRNA translation is exacerbated by K51A substitution in the Tat double-stranded RNA-binding domain. Tat, plant virus RSS, or Dicer downregulation rescues robust gag translation and bolsters HIV-1 virion production. The reversal of HIV-1 translation repression by plant RSS supports the recent finding in Arabidopsis that plant miRNAs operate by translational inhibition. Our results identify common features between RNA silencing suppression of plant and animal viruses. We suggest that RNA silencing-mediated translation repression plays a strategic role in determining the viral set-point in a newly HIV-1-infected patient.

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

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.

Bridging fundamental RNA biology, retroviral replication, and oncogenesis: Karen Beemon wins the 2007 Retrovirology Prize.

The 2007 M. Jeang Retrovirology Prize has been awarded to Dr. Karen L. Beemon.

RNA helicase A interacts with divergent lymphotropic retroviruses and promotes translation of human T-cell leukemia virus type 1.

The 5' untranslated region (UTR) of retroviruses contain structured replication motifs that impose barriers to efficient ribosome scanning. Two RNA structural motifs that facilitate efficient translation initiation despite a complex 5' UTR are internal ribosome entry site (IRES) and 5' proximal post-transcriptional control element (PCE). Here, stringent RNA and protein analyses determined the 5' UTR of spleen necrosis virus (SNV), reticuloendotheliosis virus A (REV-A) and human T-cell leukemia virus type 1 (HTLV-1) exhibit PCE activity, but not IRES activity. Assessment of SNV translation initiation in the natural context of the provirus determined that SNV is reliant on a cap-dependent initiation mechanism. Experiments with siRNAs identified that REV-A and HTLV-1 PCE modulate post-transcriptional gene expression through interaction with host RNA helicase A (RHA). Analysis of hybrid SNV/HTLV-1 proviruses determined SNV PCE facilitates Rex/Rex responsive element-independent Gag production and interaction with RHA is necessary. Ribosomal profile analyses determined that RHA is necessary for polysome association of HTLV-1 gag and provide direct evidence that RHA is necessary for efficient HTLV-1 replication. We conclude that PCE/RHA is an important translation regulatory axis of multiple lymphotropic retroviruses. We speculate divergent retroviruses have evolved a convergent RNA-protein interaction to modulate translation of their highly structured mRNA.

Tertiary structural and functional analyses of a viroid RNA motif by isostericity matrix and mutagenesis reveal its essential role in replication.

RNA-templated RNA replication is essential for viral or viroid infection, as well as for regulation of cellular gene expression. Specific RNA motifs likely regulate various aspects of this replication. Viroids of the Pospiviroidae family, as represented by the Potato spindle tuber viroid (PSTVd), replicate in the nucleus by utilizing DNA-dependent RNA polymerase II. We investigated the role of the loop E (sarcin/ricin) motif of the PSTVd genomic RNA in replication. A tertiary-structural model of this motif, inferred by comparative sequence analysis and comparison with nuclear magnetic resonance and X-ray crystal structures of loop E motifs in other RNAs, is presented in which core non-Watson-Crick base pairs are precisely specified. Isostericity matrix analysis of these base pairs showed that the model accounts for the reported natural sequence variations and viable experimental mutations in loop E motifs of PSTVd and other viroids. Furthermore, isostericity matrix analysis allowed us to design disruptive, as well as compensatory, mutations of PSTVd loop E. Functional analyses of such mutants by in vitro and in vivo experiments demonstrated that loop E structural integrity is crucial for replication, specifically during transcription. Our results suggest that the PSTVd loop E motif exists and functions in vivo and provide loss-of-function genetic evidence for the essential role of a viroid RNA three-dimensional motif in rolling-circle replication. The use of isostericity matrix analysis of non-Watson-Crick base pairing to rationalize mutagenesis of tertiary motifs and systematic in vitro and in vivo functional assays of mutants offers a novel, comprehensive approach to elucidate the tertiary-structure-function relationships for RNA motifs of general biological significance.

RNA helicase A is necessary for translation of selected messenger RNAs.

RNA helicase A (RHA) is a highly conserved DEAD-box protein that activates transcription, modulates RNA splicing and binds the nuclear pore complex. The life cycle of typical mRNA involves RNA processing and translation after ribosome scanning of a relatively unstructured 5' untranslated region (UTR). The precursor RNAs of retroviruses and selected cellular genes harbor a complex 5' UTR and use a yet-to-be-identified host post-transcriptional effector to stimulate efficient translation. Here we show that RHA recognizes a structured 5'-terminal post-transcriptional control element (PCE) of a retrovirus and the JUND growth-control gene. RHA interacts with PCE RNA in the nucleus and cytoplasm, facilitates polyribosome association and is necessary for its efficient translation. Our results reveal a previously unidentified role for RHA in translation and implicate RHA as an integrative effector in the continuum of gene expression from transcription to translation.

Human T-cell leukemia virus open reading frame II encodes a posttranscriptional repressor that is recruited at the level of transcription.

Human T-cell leukemia virus (HTLV) infection is a chronic, lifelong infection that is associated with the development of leukemia and neurological disease after a long latency period, and the mechanism by which the virus is able to evade host immune surveillance is elusive. Besides the structural and enzymatic proteins, HTLV encodes regulatory (Tax and Rex) and accessory (open reading frame I [ORF I] and ORF II) proteins. Tax activates viral and cellular transcription and promotes T-cell growth and malignant transformation. Rex acts posttranscriptionally to facilitate cytoplasmic expression of incompletely spliced viral mRNAs. Recently, we reported that the accessory gene products of HTLV-1 and HTLV-2 ORF II (p30II and p28II, respectively) are able to restrict viral replication. These proteins act as negative regulators of both Tax and Rex by binding to and retaining their mRNA in the nucleus, leading to reduced protein expression and virion production. Here, we show that p28II is recruited to the viral promoter in a Tax-dependent manner. After recruitment to the promoter, p28II or p30II then travels with the transcription elongation machinery until its target mRNA is synthesized. Experiments artificially directing these proteins to the promoter indicate that p28II, unlike HTLV-1 p30II, displays no transcriptional activity. Furthermore, the tethering of p28II directly to tax/rex mRNA resulted in repression of Tax function, which could be attributed to the ability of p28II to block TAP/p15-mediated enhancement of Tax expression. p28II-mediated reduction of viral replication in infected cells may permit survival of the cells by allowing escape from immune recognition, which is consistent with the critical role of HTLV accessory proteins in viral persistence in vivo.