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.

Anomalous HIV-1 RNA, How Cap-Methylation Segregates Viral Transcripts by Form and Function.

The acquisition of m7G-cap-binding proteins is now recognized as a major variable driving the form and function of host RNAs. This manuscript compares the 5'-cap-RNA binding proteins that engage HIV-1 precursor RNAs, host mRNAs, small nuclear (sn)- and small nucleolar (sno) RNAs and sort into disparate RNA-fate pathways. Before completion of the transcription cycle, the transcription start site of nascent class II RNAs is appended to a non-templated guanosine that is methylated (m7G-cap) and bound by hetero-dimeric CBP80-CBP20 cap binding complex (CBC). The CBC is a nexus for the co-transcriptional processing of precursor RNAs to mRNAs and the snRNA and snoRNA of spliceosomal and ribosomal ribonucleoproteins (RNPs). Just as sn/sno-RNAs experience hyper-methylation of m7G-cap to trimethylguanosine (TMG)-cap, so do select HIV RNAs and an emerging cohort of mRNAs. TMG-cap is blocked from Watson:Crick base pairing and disqualified from participating in secondary structure. The HIV TMG-cap has been shown to license select viral transcripts for specialized cap-dependent translation initiation without eIF4E that is dependent upon CBP80/NCBP3. The exceptional activity of HIV precursor RNAs secures their access to maturation pathways of sn/snoRNAs, canonical and non-canonical host mRNAs in proper stoichiometry to execute the retroviral replication cycle.

Circular RNA Profiles in Viremia and ART Suppression Predict Competing circRNA-miRNA-mRNA Networks Exclusive to HIV-1 Viremic Patients.

Since the onset of the HIV-1/AIDS epidemic in 1981, 75 million people have been infected with the virus, and the disease remains a public health crisis worldwide. Circular RNAs (circRNAs) are derived from excised exons and introns during backsplicing, a form of alternative splicing. The relevance of unconventional, non-capped, and non-poly(A) transcripts to transcriptomics studies remains to be routinely investigated. Knowledge gaps to be filled are the interface between host-encoded circRNAs and viral replication in chronically progressed patients and upon treatment with antiviral drugs. We implemented a bioinformatic pipeline and repurpose publicly archived RNA sequence reads from the blood of 19 HIV-1-positive patients that previously compared transcriptomes during viremia and viremia suppression by antiretroviral therapy (ART). The in silico analysis identified viremic patients' circRNA that became undetectable after ART. The circRNAs originated from a subset of host genes enriched in the HDAC biological pathway. These circRNAs and parental mRNAs held in common a small collection of miRNA response elements (MREs), some of which were present in HIV-1 mRNAs. The function of the MRE-containing target mRNA enriched the RNA polymerase II GO pathway. To visualize the interplay between individual circRNA-miRNA-target mRNA, important for HIV-1 and potentially other diseases, an Interactive Circos tool was developed to efficiently parse the intricately competing endogenous network of circRNA-miRNA-mRNA interactions originating from seven circRNA singled out in viremic versus non-viremic patients. The combined downregulation of the identified circRNAs warrants investigation as a novel antiviral targeting strategy.

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.

Circular RNAs Are Regulators of Diverse Animal Transcriptomes: One Health Perspective.

Derived from linear (parental) precursor mRNA, circRNA are recycled exons and introns whose ends are ligated. By titrating microRNAs and RNA binding proteins, circRNA interconnect networks of competing endogenous RNAs. Without altering chromosomal DNA, circRNA regulates skeletal muscle development and proliferation, lactation, ovulation, brain development, and responses to infections and metabolic stress. This review integrates emerging knowledge of circRNA activity coming from genome-wide characterizations in many clades of animals. circRNA research addresses one of the main pillars of the One Health vision - to improve the health and productivity of food animals and generate translational knowledge in animal species.

A New Approach to 3D Modeling of Inhomogeneous Populations of Viral Regulatory RNA.

Tertiary structure (3D) is the physical context of RNA regulatory activity. Retroviruses are RNA viruses that replicate through the proviral DNA intermediate transcribed by hosts. Proviral transcripts form inhomogeneous populations due to variable structural ensembles of overlapping regulatory RNA motifs in the 5'-untranslated region (UTR), which drive RNAs to be spliced or translated, and/or dimerized and packaged into virions. Genetic studies and structural techniques have provided fundamental input constraints to begin predicting HIV 3D conformations in silico. Using SimRNA and sets of experimentally-determined input constraints of HIVNL4-3 trans-activation responsive sequence (TAR) and pairings of unique-5' (U5) with dimerization (DIS) or AUG motifs, we calculated a series of 3D models that differ in proximity of 5'-Cap and the junction of TAR and PolyA helices; configuration of primer binding site (PBS)-segment; and two host cofactors binding sites. Input constraints on U5-AUG pairings were most compatible with intramolecular folding of 5'-UTR motifs in energetic minima. Introducing theoretical constraints predicted metastable PolyA region drives orientation of 5'-Cap with TAR, U5 and PBS-segment helices. SimRNA and the workflow developed herein provides viable options to predict 3D conformations of inhomogeneous populations of large RNAs that have been intractable to conventional ensemble methods.

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.

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes. The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

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.

Isolation of Cognate Cellular and Viral Ribonucleoprotein Complexes of HIV-1 RNA Applicable to Proteomic Discovery and Molecular Investigations.

All decisions affecting the life cycle of human immunodeficiency virus (HIV-1) RNA are executed by ribonucleoprotein complexes (RNPs). HIV-1 RNA cycles through a progression of host RNPs composed of RNA-binding proteins regulating all stages of synthesis, processing, nuclear transport, translation, decay, and co-localization with assembling virions. RNA affinity chromatography is a versatile method to identify RNA-binding proteins to investigate the molecular basis of viral and cellular posttranscriptional control of gene expression. The bait is a HIV-1 RNA motif immobilized on a solid support, typically magnetic or Sepharose beads. The prey is pre-formed RNPs admixed in lysate from cells or concentrated virus particles. The methodology distinguishes high-affinity RNA-protein interactions from low-affinity complexes by increases in ionic strength during progressive elution cycles. Here, we describe RNA affinity chromatography of the 5' untranslated region of HIV-1, obtaining mixtures of high-affinity RNA binding proteins suitable for mass spectrometry and proteome identification.

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.

Cellular RNA helicases and HIV-1: insights from genome-wide, proteomic, and molecular studies.

RNA helicases are ubiquitous in plants and animals and function in many cellular processes. Retroviruses, such as human immunodeficiency virus (HIV-1), encode no RNA helicases in their genomes and utilize host cellular RNA helicases at various stages of their life cycle. Here, we briefly summarize the roles RNA helicases play in HIV-1 replication that have been identified recently, in part, through genome-wide screenings, proteomics, and molecular studies. Some of these helicases augment virus propagation while others apparently participate in antiviral defenses against viral replication.

Determination of host RNA helicases activity in viral replication.

RNA helicases are encoded by all eukaryotic and prokaryotic cells and a minority of viruses. Activity of RNA helicases is necessary for all steps in the expression of cells and viruses and the host innate response to virus infection. Their vast functional repertoire is attributable to the core ATP-dependent helicase domain in conjunction with flanking domains that are interchangeable and engage viral and cellular cofactors. Here, we address the important issue of host RNA helicases that are necessary for replication of a virus. This chapter covers approaches to identification and characterization of candidate helicases and methods to define the biochemical and biophysical parameters of specificity and functional activity of the enzymes. We discuss the context of cellular RNA helicase activity and virion-associated RNA helicases. The methodology and choice of controls fosters the assessment of the virologic scope of RNA helicases across divergent cell lineages and viral replication cycles.