Publications by authors named "Kathleen Boris-Lawrie"

37 Publications

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

Front Genet 2020 18;11:999. Epub 2020 Sep 18.

Department of Veterinary and Biomedical Sciences, Veterinary Medicine Graduate Program, University of Minnesota Twin Cities, Saint Paul, MN, United States.

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.
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http://dx.doi.org/10.3389/fgene.2020.00999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531264PMC
September 2020

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

Viruses 2020 09 29;12(10). Epub 2020 Sep 29.

Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, MN 55108, USA.

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 HIV 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.
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http://dx.doi.org/10.3390/v12101108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650772PMC
September 2020

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

J Biol Chem 2020 05 20;295(22):7763-7773. Epub 2020 Apr 20.

Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, Minnesota 55108

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 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 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 mRNA is a component of a previously unknown ribonucleoprotein complex. Results from the tandem IP indicated that another component of the -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 -containing polysomes. Mutational analysis uncovered two dsRNA-binding domains of RHA that are necessary to tether -NCBP1/NCBP3 to polysomes. We also found that 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.
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http://dx.doi.org/10.1074/jbc.RA119.012005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261793PMC
May 2020

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

J Biol Chem 2019 07 7;294(30):11473-11485. Epub 2019 Jun 7.

Department of Biochemistry, University of Missouri, Columbia, Missouri 65211

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 conditions that mimic the early reverse transcription steps prior to capsid core uncoating. We did not observe RHA-mediated structural remodeling of the tRNA-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.
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http://dx.doi.org/10.1074/jbc.RA119.007679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6663884PMC
July 2019

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

Nucleic Acids Res 2018 08;46(14):7366-7378

Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, MN 55108, USA.

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.
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http://dx.doi.org/10.1093/nar/gky369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101577PMC
August 2018

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

J Vis Exp 2017 01 16(119). Epub 2017 Jan 16.

Department of Veterinary & Biomedical Sciences, University of Minnesota; Department of Veterinary Biosciences, Ohio State University;

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.
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http://dx.doi.org/10.3791/54391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352254PMC
January 2017

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

J Mol Biol 2016 06 21;428(11):2418-2429. Epub 2016 Apr 21.

Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jmb.2016.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4884555PMC
June 2016

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

Methods Mol Biol 2016 ;1354:133-46

Department of Veterinary Biosciences, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.

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.
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http://dx.doi.org/10.1007/978-1-4939-3046-3_9DOI Listing
October 2016

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

Virology 2015 Dec 13;486:307-20. Epub 2015 Nov 13.

Department Veterinary & Biomedical Sciences, University of Minnesota, 205 VSB, 1971 Commonwealth Avenue, Saint Paul, MN 55108. Electronic address:

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.
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http://dx.doi.org/10.1016/j.virol.2015.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877169PMC
December 2015

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

Neoplasia 2013 Sep;15(9):1049-63

Center for Childhood Cancer, Research Institute at Nationwide Children's Hospital, Columbus, OH.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3769884PMC
http://dx.doi.org/10.1593/neo.13286DOI Listing
September 2013

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

Virus Res 2013 Feb 16;171(2):357-65. Epub 2012 Jul 16.

Molecular Virology Section 1, Laboratory of Molecular, Microbiology, The National Institute of Allergy and Infectious Diseases, The National Institutes of Health, Bethesda, MD 20892, USA.

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.
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http://dx.doi.org/10.1016/j.virusres.2012.06.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493675PMC
February 2013

Determination of host RNA helicases activity in viral replication.

Methods Enzymol 2012 ;511:405-35

Department of Veterinary Biosciences, Ohio State University, Columbus, Ohio, USA.

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.
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http://dx.doi.org/10.1016/B978-0-12-396546-2.00019-XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862593PMC
October 2012

Thriving under stress: selective translation of HIV-1 structural protein mRNA during Vpr-mediated impairment of eIF4E translation activity.

PLoS Pathog 2012 22;8(3):e1002612. Epub 2012 Mar 22.

Department of Veterinary Biosciences, Ohio State University, Columbus, Ohio, United States of America.

Translation is a regulated process and is pivotal to proper cell growth and homeostasis. All retroviruses rely on the host translational machinery for viral protein synthesis and thus may be susceptible to its perturbation in response to stress, co-infection, and/or cell cycle arrest. HIV-1 infection arrests the cell cycle in the G2/M phase, potentially disrupting the regulation of host cell translation. In this study, we present evidence that HIV-1 infection downregulates translation in lymphocytes, attributable to the cell cycle arrest induced by the HIV-1 accessory protein Vpr. The molecular basis of the translation suppression is reduced accumulation of the active form of the translation initiation factor 4E (eIF4E). However, synthesis of viral structural proteins is sustained despite the general suppression of protein production. HIV-1 mRNA translation is sustained due to the distinct composition of the HIV-1 ribonucleoprotein complexes. RNA-coimmunoprecipitation assays determined that the HIV-1 unspliced and singly spliced transcripts are predominantly associated with nuclear cap binding protein 80 (CBP80) in contrast to completely-spliced viral and cellular mRNAs that are associated with eIF4E. The active translation of the nuclear cap binding complex (CBC)-bound viral mRNAs is demonstrated by ribosomal RNA profile analyses. Thus, our findings have uncovered that the maintenance of CBC association is a novel mechanism used by HIV-1 to bypass downregulation of eIF4E activity and sustain viral protein synthesis. We speculate that a subset of CBP80-bound cellular mRNAs contribute to recovery from significant cellular stress, including human retrovirus infection.
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http://dx.doi.org/10.1371/journal.ppat.1002612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3310836PMC
July 2012

Tat RNA silencing suppressor activity contributes to perturbation of lymphocyte miRNA by HIV-1.

Retrovirology 2011 May 13;8:36. Epub 2011 May 13.

Department of Veterinary Biosciences, Center for Retrovirus Research, Ohio State University, Columbus OH, USA.

Background: MicroRNA (miRNA)-mediated RNA silencing is integral to virtually every cellular process including cell cycle progression and response to virus infection. The interplay between RNA silencing and HIV-1 is multifaceted, and accumulating evidence posits a strike-counterstrike interface that alters the cellular environment to favor virus replication. For instance, miRNA-mediated RNA silencing of HIV-1 translation is antagonized by HIV-1 Tat RNA silencing suppressor activity. The activity of HIV-1 accessory proteins Vpr/Vif delays cell cycle progression, which is a process prominently modulated by miRNA. The expression profile of cellular miRNA is altered by HIV-1 infection in both cultured cells and clinical samples. The open question stands of what, if any, is the contribution of Tat RNA silencing suppressor activity or Vpr/Vif activity to the perturbation of cellular miRNA by HIV-1.

Results: Herein, we compared the perturbation of miRNA expression profiles of lymphocytes infected with HIV-1(NL4-3) or derivative strains that are deficient in Tat RNA silencing suppressor activity (Tat K51A substitution) or ablated of the vpr/vif open reading frames. Microarrays recapitulated the perturbation of the cellular miRNA profile by HIV-1 infection. The miRNA expression trends overlapped ~50% with published microarray results on clinical samples from HIV-1 infected patients. Moreover, the number of miRNA perturbed by HIV-1 was largely similar despite ablation of Tat RSS activity and Vpr/Vif; however, the Tat RSS mutation lessened HIV-1 downregulation of twenty-two miRNAs.

Conclusions: Our study identified miRNA expression changes attributable to Tat RSS activity in HIV-1(NL4-3). The results accomplish a necessary step in the process to understand the interface of HIV-1 with host RNA silencing activity. The overlap in miRNA expression trends observed between HIV-1 infected CEMx174 lymphocytes and primary cells supports the utility of cultured lymphocytes as a tractable model to investigate interplay between HIV-1 and host RNA silencing. The subset of miRNA determined to be perturbed by Tat RSS in HIV-1 infection provides a focal point to define the gene networks that shape the cellular environment for HIV-1 replication.
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http://dx.doi.org/10.1186/1742-4690-8-36DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3120759PMC
May 2011

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

Nucleic Acids Res 2011 May 18;39(9):3724-34. Epub 2011 Jan 18.

Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT 06510, USA.

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.
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http://dx.doi.org/10.1093/nar/gkq1350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3089476PMC
May 2011

RNA helicases: emerging roles in viral replication and the host innate response.

RNA Biol 2010 Nov-Dec;7(6):775-87. Epub 2010 Nov 1.

Department of Veterinary Biosciences, Ohio State University, Columbus, OH, USA.

RNA helicases serve multiple roles at the virus-host interface. In some situations, RNA helicases are essential host factors to promote viral replication; however, in other cases they serve as a cellular sensor to trigger the antiviral state in response to viral infection. All family members share the conserved ATP-dependent catalytic core linked to different substrate recognition and protein-protein interaction domains. These flanking domains can be shuffled between different helicases to achieve functional diversity. This review summarizes recent studies, which have revealed two types of activity by RNA helicases. First, RNA helicases are catalysts of progressive RNA-protein rearrangements that begin at gene transcription and culminate in mRNA translation. Second, RNA helicases can act as a scaffold for alternative protein-protein interactions that can defeat the antiviral state. The mounting fundamental understanding of RNA helicases is being used to develop selective and efficacious drugs against human and animal pathogens. The analysis of RNA helicases in virus model systems continues to provide insights into virology, cell biology and immunology, and has provided fresh perspective to continue unraveling the complexity of virus-host interactions.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3073335PMC
http://dx.doi.org/10.4161/rna.7.6.14249DOI Listing
July 2011

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

J Biol Chem 2011 Feb 1;286(7):5328-37. Epub 2010 Dec 1.

Center for Retrovirus Research, Department of Veterinary Biosciences, Ohio State University, Columbus, Ohio 43210, USA.

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-β-β-β-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.
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http://dx.doi.org/10.1074/jbc.M110.176339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037645PMC
February 2011

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

Nucleic Acids Res 2010 Mar 9;38(5):1686-96. Epub 2009 Dec 9.

Department of Veterinary Biosciences, Center for Retrovirus Research and Center for RNA Biology, The Ohio State University, Columbus, OH 43210-1093, USA.

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.
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http://dx.doi.org/10.1093/nar/gkp1075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2836548PMC
March 2010

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

Retrovirology 2009 Jan 24;6. Epub 2009 Jan 24.

Center for Retrovirus Research, Department of Veterinary Biosciences, Molecular, Cellular, and Developmental Biology graduate program, The Ohio State University, Columbus, OH, USA.

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.
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http://dx.doi.org/10.1186/1742-4690-6-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2657110PMC
January 2009

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 Jan 2;106(2):605-10. Epub 2009 Jan 2.

Center for Retrovirus Research and Department of Veterinary Biosciences, Molecular, Cellular and Developmental Biology Graduate Program, Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA.

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.
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http://dx.doi.org/10.1073/pnas.0806822106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2626750PMC
January 2009

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

Retrovirology 2007 Dec 10;4:88. Epub 2007 Dec 10.

The 2007 M. Jeang Retrovirology Prize has been awarded to Dr. Karen L. Beemon.
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http://dx.doi.org/10.1186/1742-4690-4-88DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2211504PMC
December 2007

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

Nucleic Acids Res 2007 10;35(8):2629-42. Epub 2007 Apr 10.

Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210-1093, USA.

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.
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http://dx.doi.org/10.1093/nar/gkm124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1885656PMC
June 2007

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

J Virol 2006 Sep;80(17):8566-81

Department of Plant Cellular and Molecular Biology, Ohio State University, Columbus, 43210, USA.

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.
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http://dx.doi.org/10.1128/JVI.00837-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1563885PMC
September 2006

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

Nat Struct Mol Biol 2006 Jun 7;13(6):509-16. Epub 2006 May 7.

Center for Retrovirus Research and Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210 USA.

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.
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http://dx.doi.org/10.1038/nsmb1092DOI Listing
June 2006

Retrovirus translation initiation: Issues and hypotheses derived from study of HIV-1.

Curr HIV Res 2006 Apr;4(2):131-9

Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210, USA.

Human immunodeficiency virus type 1 (HIV-1) has a small, multifunctional genome that encodes a relatively large and complex proteome. The virus has adopted specialized post-transcriptional control mechanisms to maximize its coding capacity while economically maintaining the information stored in cis-acting replication sequences. The conserved features of the 5' untranslated region of all viral transcripts suggest they are poor substrates for cap-dependent ribosome scanning and provide a compelling rationale for internal initiation of translation. This article summarizes key experimental results of studies that have evaluated HIV-1 translation initiation. A model is discussed in which cap-dependent and cap-independent initiation mechanisms of HIV-1 co-exist to ensure viral protein production in the context of 1) structured replication motifs that inhibit ribosome scanning, and 2) alterations in host translation machinery in response to HIV-1 infection or other cellular stresses. We discuss key issues that remain to be understood and suggest parameters to validate internal initiation activity in HIV-1 and other retroviruses.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863997PMC
http://dx.doi.org/10.2174/157016206776055039DOI Listing
April 2006

Coordinate enhancement of transgene transcription and translation in a lentiviral vector.

Retrovirology 2006 Feb 15;3:13. Epub 2006 Feb 15.

Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA.

Background: Coordinate enhancement of transgene transcription and translation would be a potent approach to significantly improve protein output in a broad array of viral vectors and nonviral expression systems. Many vector transgenes are complementary DNA (cDNA). The lack of splicing can significantly reduce the efficiency of their translation. Some retroviruses contain a 5' terminal post-transcriptional control element (PCE) that facilitates translation of unspliced mRNA. Here we evaluated the potential for spleen necrosis virus PCE to stimulate protein production from HIV-1 based lentiviral vector by: 1) improving translation of the internal transgene transcript; and 2) functionally synergizing with a transcriptional enhancer to achieve coordinate increases in RNA synthesis and translation.

Results: Derivatives of HIV-1 SIN self-inactivating lentiviral vector were created that contain PCE and cytomegalovirus immediate early enhancer (CMV IE). Results from transfected cells and four different transduced cell types indicate that: 1) PCE enhanced transgene protein synthesis; 2) transcription from the internal promoter is enhanced by CMV IE; 3) PCE and CMV IE functioned synergistically to significantly increase transgene protein yield; 4) the magnitude of translation enhancement by PCE was similar in transfected and transduced cells; 5) differences were observed in steady state level of PCE vector RNA in transfected and transduced cells; 6) the lower steady state was not attributable to reduced RNA stability, but to lower cytoplasmic accumulation in transduced cells.

Conclusion: PCE is a useful tool to improve post-transcriptional expression of lentiviral vector transgene. Coordinate enhancement of transcription and translation is conferred by the combination of PCE with CMV IE transcriptional enhancer and increased protein yield up to 11 to 17-fold in transfected cells. The incorporation of the vector provirus into chromatin correlated with reduced cytoplasmic accumulation of PCE transgene RNA. We speculate that epigenetic modulation of promoter activity altered cotranscriptional recruitment of RNA processing factors and reduced the availability of fully processed transcript or the efficiency of export from the nucleus. Our results provide an example of the dynamic interplay between the transcription and post-transcription steps of gene expression and document that introduction of heterologous gene expression signals can yield disparate effects in transfected versus transduced cells.
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http://dx.doi.org/10.1186/1742-4690-3-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1388234PMC
February 2006

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

J Virol 2006 Jan;80(1):181-91

Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210, USA.

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.
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http://dx.doi.org/10.1128/JVI.80.1.181-191.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1317543PMC
January 2006

Human T lymphotropic virus type 1 accessory protein p12I modulates calcium-mediated cellular gene expression and enhances p300 expression in T lymphocytes.

AIDS Res Hum Retroviruses 2005 Apr;21(4):273-84

Center for Retrovirus Research and Department of Veterinary Biosciences, Ohio State University, Columbus, Ohio 43210, USA.

Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T cell leukemia/lymphoma (ATLL), an aggressive CD4+ T lymphocyte malignancy. Activation of T lymphocytes is required for effective retroviral integration into the host cell genome and subsequent viral replication, but the molecular mechanisms involved in HTLV-1-mediated T cell activation remain unclear. HTLV-1 encodes various accessory proteins such as p12I, which has been demonstrated to be critical for HTLV-1 infectivity in vivo in rabbits and in vitro in quiescent primary human T lymphocytes. This hydrophobic protein localizes in the endoplasmic reticulum, increases intracellular calcium, and activates nuclear factor of activated T cell-mediated transcription. To further elucidate the role of p12I in regulation of cellular gene expression, we performed gene array analysis on stable p12I-expressing Jurkat T cells, using Affymetrix U133A arrays. Our data indicate that p12I altered the expression of genes associated with a network of interrelated pathways including T cell signaling, cell proliferation, and apoptosis. Expression of several calcium-regulated genes was found to be altered by p12I, consistent with known properties of the viral protein. Gene array findings were confirmed by semiquantitative RT-PCR in Jurkat T cells and primary CD4+ T lymphocytes. Furthermore, dose-dependent expression of p12I in Jurkat T cells resulted in significant increases in p300 and p300-dependent transcription. This is the first report of a viral protein influencing the transcription of p300, a rate-limiting coadapter critical in HTLV-1-mediated T cell activation. Collectively, our data strongly indicate that HTLV-1 p12I modulates cellular gene expression patterns to hasten the activation of T lymphocytes and thereby promote efficient viral infection.
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http://dx.doi.org/10.1089/aid.2005.21.273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2668121PMC
April 2005

Human T lymphotropic virus type-1 p30II alters cellular gene expression to selectively enhance signaling pathways that activate T lymphocytes.

Retrovirology 2004 Nov 23;1:39. Epub 2004 Nov 23.

Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210, USA.

Background: Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T-cell leukemia/lymphoma and is implicated in a variety of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13II and p30II, which are incompletely defined in the virus life cycle or HTLV-1 pathogenesis. Proviral clones of the virus with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. Exogenous expression of p30II differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and represses tax/rex RNA nuclear export.

Results: Herein, we further characterized the role of p30II in regulation of cellular gene expression, using stable p30II expression system employing lentiviral vectors to test cellular gene expression with Affymetrix U133A arrays, representing approximately 33,000 human genes. Reporter assays in Jurkat T cells and RT-PCR in Jurkat and primary CD4+ T-lymphocytes were used to confirm selected gene expression patterns. Our data reveals alterations of interrelated pathways of cell proliferation, T-cell signaling, apoptosis and cell cycle in p30II expressing Jurkat T cells. In all categories, p30II appeared to be an overall repressor of cellular gene expression, while selectively increasing the expression of certain key regulatory genes.

Conclusions: We are the first to demonstrate that p30II, while repressing the expression of many genes, selectively activates key gene pathways involved in T-cell signaling/activation. Collectively, our data suggests that this complex retrovirus, associated with lymphoproliferative diseases, relies upon accessory gene products to modify cellular environment to promote clonal expansion of the virus genome and thus maintain proviral loads in vivo.
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http://dx.doi.org/10.1186/1742-4690-1-39DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC538277PMC
November 2004