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Abstract
Recent advances in the study of virus-cell interactions have improved our understanding of how viruses that replicate their genomes in the nucleus (e.g., retroviruses, hepadnaviruses, herpesviruses, and a subset of RNA viruses) hijack cellular pathways to export these genomes to the cytoplasm where they access virion egress pathways. These findings shed light on novel aspects of viral life cycles relevant to the development of new antiviral strategies and can yield new tractable, virus-based tools for exposing additional secrets of the cell. The goal of this review is to summarize defined and emerging modes of virus-host interactions that drive the transit of viral genomes out of the nucleus across the nuclear envelope barrier, with an emphasis on retroviruses that are most extensively studied. In this context, we prioritize discussion of recent progress in understanding the trafficking and function of the human immunodeficiency virus type 1 Rev protein, exemplifying a relatively refined example of stepwise, cooperativity-driven viral subversion of multi-subunit host transport receptor complexes.
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Affiliation(s)
- Ryan T. Behrens
- Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin, USA
| | - Nathan M. Sherer
- McArdle Laboratory for Cancer Research and Carbone Cancer Center, University of Wisconsin, Madison, Wisconsin, USA
- Institute for Molecular Virology, University of Wisconsin, Madison, Wisconsin, USA
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2
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Guo J, Zhu Y, Ma X, Shang G, Liu B, Zhang K. Virus Infection and mRNA Nuclear Export. Int J Mol Sci 2023; 24:12593. [PMID: 37628773 PMCID: PMC10454920 DOI: 10.3390/ijms241612593] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2023] [Revised: 07/29/2023] [Accepted: 08/03/2023] [Indexed: 08/27/2023] Open
Abstract
Gene expression in eukaryotes begins with transcription in the nucleus, followed by the synthesis of messenger RNA (mRNA), which is then exported to the cytoplasm for its translation into proteins. Along with transcription and translation, mRNA export through the nuclear pore complex (NPC) is an essential regulatory step in eukaryotic gene expression. Multiple factors regulate mRNA export and hence gene expression. Interestingly, proteins from certain types of viruses interact with these factors in infected cells, and such an interaction interferes with the mRNA export of the host cell in favor of viral RNA export. Thus, these viruses hijack the host mRNA nuclear export mechanism, leading to a reduction in host gene expression and the downregulation of immune/antiviral responses. On the other hand, the viral mRNAs successfully evade the host surveillance system and are efficiently exported from the nucleus to the cytoplasm for translation, which enables the continuation of the virus life cycle. Here, we present this review to summarize the mechanisms by which viruses suppress host mRNA nuclear export during infection, as well as the key strategies that viruses use to facilitate their mRNA nuclear export. These studies have revealed new potential antivirals that may be used to inhibit viral mRNA transport and enhance host mRNA nuclear export, thereby promoting host gene expression and immune responses.
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Affiliation(s)
- Jiayin Guo
- University of Chinese Academy of Sciences, Beijing 100049, China; (J.G.); (Y.Z.); (X.M.)
| | - Yaru Zhu
- University of Chinese Academy of Sciences, Beijing 100049, China; (J.G.); (Y.Z.); (X.M.)
| | - Xiaoya Ma
- University of Chinese Academy of Sciences, Beijing 100049, China; (J.G.); (Y.Z.); (X.M.)
| | - Guijun Shang
- Shanxi Provincial Key Laboratory of Protein Structure Determination, Shanxi Academy of Advanced Research and Innovation, Taiyuan 030012, China;
| | - Bo Liu
- Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai 200031, China
- Shanghai Huashen Institute of Microbes and Infections, Shanghai 200052, China
| | - Ke Zhang
- Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai 200031, China
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3
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Khan M, Hou S, Chen M, Lei H. Mechanisms of RNA export and nuclear retention. WILEY INTERDISCIPLINARY REVIEWS. RNA 2023; 14:e1755. [PMID: 35978483 DOI: 10.1002/wrna.1755] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/10/2022] [Revised: 06/21/2022] [Accepted: 07/06/2022] [Indexed: 05/13/2023]
Abstract
With the identification of huge amount of noncoding RNAs in recent years, the concept of RNA localization has extended from traditional mRNA export to RNA export of mRNA and ncRNA as well as nuclear retention of ncRNA. This review aims to summarize the recent findings from studies on the mechanisms of export of different RNAs and nuclear retention of some lncRNAs in higher eukaryotes, with a focus on splicing-dependent TREX recruitment for the export of spliced mRNA and the sequence-dependent mechanism of mRNA export in the absence of splicing. In addition, evidence to support the involvement of m6 A modification in RNA export with the coordination between the methylase complex and TREX complex as well as sequence-dependent nuclear retention of lncRNA is recapitulated. Finally, a model of sequence-dependent RNA localization is proposed along with the many questions that remain to be answered. This article is categorized under: RNA Export and Localization > RNA Localization RNA Export and Localization > Nuclear Export/Import.
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Affiliation(s)
- Misbah Khan
- Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China
| | - Shuai Hou
- Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China
| | - Mo Chen
- Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China
| | - Haixin Lei
- Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China
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4
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Beemon KL. Retroviral RNA Processing. Viruses 2022; 14:v14051113. [PMID: 35632854 PMCID: PMC9143442 DOI: 10.3390/v14051113] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2022] [Revised: 05/20/2022] [Accepted: 05/20/2022] [Indexed: 01/27/2023] Open
Abstract
This review is an accompaniment to a Special Issue on “Retroviral RNA Processing”. It discusses post-transcriptional regulation of retroviruses, ranging from the ancient foamy viruses to more modern viruses, such as HIV-1, HTLV-1, Rous sarcoma virus, murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus. This review is not comprehensive. However, it tries to address some of the major questions in the field with examples of how different retroviruses express their genes. It is amazing that a single primary RNA transcript can have so many possible fates: genomic RNA, unspliced mRNA, and up to 50 different alternatively spliced mRNAs. This review will discuss the sorting of RNAs for packaging or translation, RNA nuclear export mechanisms, splicing, translation, RNA modifications, and avoidance of nonsense-mediated RNA decay.
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Affiliation(s)
- Karen L Beemon
- Biology Department, Johns Hopkins University, Baltimore, MD 21218, USA
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5
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Khan M, Hou S, Azam S, Lei H. Sequence-dependent recruitment of SRSF1 and SRSF7 to intronless lncRNA NKILA promotes nuclear export via the TREX/TAP pathway. Nucleic Acids Res 2021; 49:6420-6436. [PMID: 34096602 PMCID: PMC8216466 DOI: 10.1093/nar/gkab445] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2021] [Revised: 05/03/2021] [Accepted: 05/07/2021] [Indexed: 12/19/2022] Open
Abstract
The TREX-TAP pathway is vital for mRNA export. For spliced mRNA, the TREX complex is recruited during splicing; however, for intronless mRNA, recruitment is sequence dependent. However, the export of cytoplasmic long noncoding RNA (lncRNA) is poorly characterized. We report the identification of a cytoplasmic accumulation region (CAR-N) in the intronless lncRNA, NKILA. CAR-N removal led to strong nuclear retention of NKILA, and CAR-N insertion promoted the export of cDNA transcripts. In vitro RNP purification via CAR-N, mass spectrometry, and siRNA screening revealed that SRSF1 and SRSF7 were vital to NKILA export, and identified a cluster of SRSF1/7 binding sites within a 55 nucleotide sequence in CAR-N. Significant nuclear enrichment of NKILA was observed for NKILA lacking CAR-N or the cluster of binding sites in knock-in models. Depletion of TREX-TAP pathway components resulted in strong nuclear retention of NKILA. RNA and protein immunoprecipitation verified that SRSF1/7 were bound to NKILA and interacted with UAP56 and ALYREF. Moreover, NKILA lacking CAR-N was unable to inhibit breast cancer cell migration. We concluded that the binding of SRSF1/7 to clustered motifs in CAR-N facilitated TREX recruitment, promoting the export of NKILA, and confirmed the importance of NKILA localization to its function.
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Affiliation(s)
- Misbah Khan
- Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, 9 West Section, Lvshun South Rd, Dalian 116044, P.R. China
| | - Shuai Hou
- Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, 9 West Section, Lvshun South Rd, Dalian 116044, P.R. China.,School of Food Science and Technology, Dalian Polytechnic University, Dalian 1160343, P.R. China
| | - Sikandar Azam
- Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, 9 West Section, Lvshun South Rd, Dalian 116044, P.R. China.,Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan, USA
| | - Haixin Lei
- Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, 9 West Section, Lvshun South Rd, Dalian 116044, P.R. China
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6
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Guha S, Bhaumik SR. Viral regulation of mRNA export with potentials for targeted therapy. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2020; 1864:194655. [PMID: 33246183 DOI: 10.1016/j.bbagrm.2020.194655] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Received: 07/04/2020] [Revised: 10/15/2020] [Accepted: 11/05/2020] [Indexed: 12/12/2022]
Abstract
Eukaryotic gene expression begins with transcription in the nucleus to synthesize mRNA (messenger RNA), which is subsequently exported to the cytoplasm for translation to protein. Like transcription and translation, mRNA export is an important regulatory step of eukaryotic gene expression. Various factors are involved in regulating mRNA export, and thus gene expression. Intriguingly, some of these factors interact with viral proteins, and such interactions interfere with mRNA export of the host cell, favoring viral RNA export. Hence, viruses hijack host mRNA export machinery for export of their own RNAs from nucleus to cytoplasm for translation to proteins for viral life cycle, suppressing host mRNA export (and thus host gene expression and immune/antiviral response). Therefore, the molecules that can impair the interactions of these mRNA export factors with viral proteins could emerge as antiviral therapeutic agents to suppress viral RNA transport and enhance host mRNA export, thereby promoting host gene expression and immune response. Thus, there has been a number of studies to understand how virus hijacks mRNA export machinery in suppressing host gene expression and promoting its own RNA export to the cytoplasm for translation to proteins required for viral replication/assembly/life cycle towards developing targeted antiviral therapies, as concisely described here.
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Affiliation(s)
- Shalini Guha
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA
| | - Sukesh R Bhaumik
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA.
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7
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Gales JP, Kubina J, Geldreich A, Dimitrova M. Strength in Diversity: Nuclear Export of Viral RNAs. Viruses 2020; 12:E1014. [PMID: 32932882 PMCID: PMC7551171 DOI: 10.3390/v12091014] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2020] [Revised: 09/03/2020] [Accepted: 09/09/2020] [Indexed: 12/11/2022] Open
Abstract
The nuclear export of cellular mRNAs is a complex process that requires the orchestrated participation of many proteins that are recruited during the early steps of mRNA synthesis and processing. This strategy allows the cell to guarantee the conformity of the messengers accessing the cytoplasm and the translation machinery. Most transcripts are exported by the exportin dimer Nuclear RNA export factor 1 (NXF1)-NTF2-related export protein 1 (NXT1) and the transcription-export complex 1 (TREX1). Some mRNAs that do not possess all the common messenger characteristics use either variants of the NXF1-NXT1 pathway or CRM1, a different exportin. Viruses whose mRNAs are synthesized in the nucleus (retroviruses, the vast majority of DNA viruses, and influenza viruses) exploit both these cellular export pathways. Viral mRNAs hijack the cellular export machinery via complex secondary structures recognized by cellular export factors and/or viral adapter proteins. This way, the viral transcripts succeed in escaping the host surveillance system and are efficiently exported for translation, allowing the infectious cycle to proceed. This review gives an overview of the cellular mRNA nuclear export mechanisms and presents detailed insights into the most important strategies that viruses use to export the different forms of their RNAs from the nucleus to the cytoplasm.
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Affiliation(s)
- Jón Pol Gales
- Institut de Biologie Moléculaire des Plantes, The French National Center for Scientific Research (CNRS) UPR2357, Université de Strasbourg, F-67084 Strasbourg, France; (J.P.G.); (J.K.); (A.G.)
| | - Julie Kubina
- Institut de Biologie Moléculaire des Plantes, The French National Center for Scientific Research (CNRS) UPR2357, Université de Strasbourg, F-67084 Strasbourg, France; (J.P.G.); (J.K.); (A.G.)
- SVQV UMR-A 1131, INRAE, Université de Strasbourg, F-68000 Colmar, France
| | - Angèle Geldreich
- Institut de Biologie Moléculaire des Plantes, The French National Center for Scientific Research (CNRS) UPR2357, Université de Strasbourg, F-67084 Strasbourg, France; (J.P.G.); (J.K.); (A.G.)
| | - Maria Dimitrova
- Institut de Biologie Moléculaire des Plantes, The French National Center for Scientific Research (CNRS) UPR2357, Université de Strasbourg, F-67084 Strasbourg, France; (J.P.G.); (J.K.); (A.G.)
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8
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Rekosh D, Hammarskjold ML. Intron retention in viruses and cellular genes: Detention, border controls and passports. WILEY INTERDISCIPLINARY REVIEWS-RNA 2018; 9:e1470. [PMID: 29508942 DOI: 10.1002/wrna.1470] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/02/2017] [Revised: 01/04/2018] [Accepted: 01/24/2018] [Indexed: 02/06/2023]
Abstract
Intron retention (IR), where one or more introns remain in the RNA after splicing, was long thought to be rare in mammalian cells, albeit common in plants and some viruses. Largely due to the development of better methods for RNA analysis, it has now been recognized that IR is much more common than previously thought and that this mechanism is likely to play an important role in mammalian gene regulation. To date, most publications and reviews about IR have described the resulting mRNAs as "dead end" products, with no direct consequence for the proteome. However, there are also many reports of mRNAs with retained introns giving rise to alternative protein isoforms. Although this was originally revealed in viral systems, there are now numerous examples of bona fide cellular proteins that are translated from mRNAs with retained introns. These new isoforms have sometimes been shown to have important regulatory functions. In this review, we highlight recent developments in this area and the research on viruses that led the way to the realization of the many ways in which mRNAs with retained introns can be regulated. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing RNA Export and Localization > Nuclear Export/Import RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.
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Affiliation(s)
- David Rekosh
- The Myles H. Thaler Center for AIDS and Human Retrovirus Research and the Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia.,Department of Microbiology, University of Venda, Thohoyandou, South Africa
| | - Marie-Louise Hammarskjold
- The Myles H. Thaler Center for AIDS and Human Retrovirus Research and the Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia.,Department of Microbiology, University of Venda, Thohoyandou, South Africa
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9
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Pocock GM, Zimdars LL, Yuan M, Eliceiri KW, Ahlquist P, Sherer NM. Diverse activities of viral cis-acting RNA regulatory elements revealed using multicolor, long-term, single-cell imaging. Mol Biol Cell 2017; 28:476-487. [PMID: 27903772 PMCID: PMC5341730 DOI: 10.1091/mbc.e16-08-0612] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2016] [Revised: 11/22/2016] [Accepted: 11/23/2016] [Indexed: 02/06/2023] Open
Abstract
Cis-acting RNA structural elements govern crucial aspects of viral gene expression. How these structures and other posttranscriptional signals affect RNA trafficking and translation in the context of single cells is poorly understood. Herein we describe a multicolor, long-term (>24 h) imaging strategy for measuring integrated aspects of viral RNA regulatory control in individual cells. We apply this strategy to demonstrate differential mRNA trafficking behaviors governed by RNA elements derived from three retroviruses (HIV-1, murine leukemia virus, and Mason-Pfizer monkey virus), two hepadnaviruses (hepatitis B virus and woodchuck hepatitis virus), and an intron-retaining transcript encoded by the cellular NXF1 gene. Striking behaviors include "burst" RNA nuclear export dynamics regulated by HIV-1's Rev response element and the viral Rev protein; transient aggregations of RNAs into discrete foci at or near the nuclear membrane triggered by multiple elements; and a novel, pulsiform RNA export activity regulated by the hepadnaviral posttranscriptional regulatory element. We incorporate single-cell tracking and a data-mining algorithm into our approach to obtain RNA element-specific, high-resolution gene expression signatures. Together these imaging assays constitute a tractable, systems-based platform for studying otherwise difficult to access spatiotemporal features of viral and cellular gene regulation.
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MESH Headings
- Active Transport, Cell Nucleus/physiology
- Cell Nucleus/metabolism
- Gene Expression Regulation, Viral
- Gene Products, rev/metabolism
- Genes, env/physiology
- HIV-1
- Mason-Pfizer monkey virus
- Molecular Imaging/methods
- RNA Processing, Post-Transcriptional/physiology
- RNA, Messenger/metabolism
- RNA, Viral
- Regulatory Sequences, Nucleic Acid/genetics
- Regulatory Sequences, Nucleic Acid/physiology
- Regulatory Sequences, Ribonucleic Acid/genetics
- Regulatory Sequences, Ribonucleic Acid/physiology
- Single-Cell Analysis/methods
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Affiliation(s)
- Ginger M Pocock
- McArdle Laboratory for Cancer Research and Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI 53706
- Morgridge Institute for Research, University of Wisconsin-Madison, Madison, WI 53706
| | - Laraine L Zimdars
- McArdle Laboratory for Cancer Research and Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI 53706
| | - Ming Yuan
- Morgridge Institute for Research, University of Wisconsin-Madison, Madison, WI 53706
- Department of Statistics, University of Wisconsin-Madison, Madison, WI 53706
| | - Kevin W Eliceiri
- Morgridge Institute for Research, University of Wisconsin-Madison, Madison, WI 53706
- Laboratory for Optical and Computational Instrumentation and Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706
| | - Paul Ahlquist
- McArdle Laboratory for Cancer Research and Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI 53706
- Morgridge Institute for Research, University of Wisconsin-Madison, Madison, WI 53706
- Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, WI 53706
| | - Nathan M Sherer
- McArdle Laboratory for Cancer Research and Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI 53706
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10
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HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors. PLoS Pathog 2016; 12:e1005565. [PMID: 27070420 PMCID: PMC4829213 DOI: 10.1371/journal.ppat.1005565] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/25/2015] [Accepted: 03/21/2016] [Indexed: 12/15/2022] Open
Abstract
Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent "burst-like" transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm.
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11
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Ge Z, Quek BL, Beemon KL, Hogg JR. Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway. eLife 2016; 5. [PMID: 26744779 PMCID: PMC4764554 DOI: 10.7554/elife.11155] [Citation(s) in RCA: 118] [Impact Index Per Article: 13.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2015] [Accepted: 01/07/2016] [Indexed: 12/26/2022] Open
Abstract
The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD. DOI:http://dx.doi.org/10.7554/eLife.11155.001 Genes are used as templates to create molecules of messenger RNA (mRNA) that contain all the information needed to make a protein. This information begins with a 'start site' and ends with a 'stop site.' The regions of the mRNA outside of the start and stop sites are called untranslated regions. Not all mRNAs are correctly made, and cells combat this problem by detecting and destroying faulty mRNAs before they are translated into protein. One way cells do this is by recognizing and destroying mRNAs that include long untranslated regions, which can indicate that the mRNA might have a stop site too early in its sequence. A key problem with this mechanism, however, is that long untranslated regions also serve important roles in the cell: for example, by determining where and when mRNA molecules are read to make protein. How then do mRNAs with long but important untranslated regions escape detection and degradation? Ge et al. have now investigated this question using an approach that allows a 'handle' to be attached to particular RNA molecules. This allows the RNA and any proteins bound to it to be purified away from all other RNAs and proteins in the cell, and the proteins can then be identified by a technique called mass spectrometry. Ge at al. found that mRNAs can recruit a protein called PTBP1 to part of the RNA sequence near the stop site. This prevents an RNA decay protein recognizing and triggering the degradation of the mRNA, even if the mRNA has a long untranslated region. Thus, PTBP1 plays a crucial role in protecting human RNAs with long untranslated regions from destruction by the nonsense-mediated decay pathway. Some viral RNAs are also able to evade decay, and so Ge et al. hypothesize that the virus stole this method for maintaining its RNAs from host cells. A future goal is to understand whether this system works the same way in all cell types or protects different RNAs in different cells. DOI:http://dx.doi.org/10.7554/eLife.11155.002
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Affiliation(s)
- Zhiyun Ge
- Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, United States
| | - Bao Lin Quek
- Department of Biology, Johns Hopkins University, Baltimore, United States
| | - Karen L Beemon
- Department of Biology, Johns Hopkins University, Baltimore, United States
| | - J Robert Hogg
- Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, United States
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12
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Identification of the alternative splicing of the UL49 locus of human cytomegalovirus. BIOMED RESEARCH INTERNATIONAL 2015; 2015:280276. [PMID: 25866769 PMCID: PMC4383306 DOI: 10.1155/2015/280276] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/31/2014] [Accepted: 09/06/2014] [Indexed: 12/04/2022]
Abstract
The UL49 ORF of human cytomegalovirus (HCMV) is essential for viral replication; conserved among all herpes viruses; however, the function is unclear. Once the UL49 ORF was precisely deleted from the start to stop codon, the mutant did not yield infectious progeny. In this study, we find out many alternatively processed ESTs in UL49 locus in HCMV-infected cells, in which there are two novel transcription termination sites in UL49 locus. Most of these ESTs are rare transcripts that contain directed repeat sequences in the intron splicing regions. There is a typical GU-AG intron splicing site in UL49Y transcripts. The 1847 bp UL49Y cDNA spans an ORF from 335 to 1618 and encodes a putative protein of 427 amino acids with a predicted molecular mass of 47.1 kDa. All the new EST sequences and UL49Y cDNA sequence have been deposited in the GenBank database (GenBank Accession nos. GW314860-GW314900 and GU376796). This study provides us with very important clues for revealing the importance of the UL49 locus alternative splicing.
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13
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Bartels H, Luban J. Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag-encoded RNA. Retrovirology 2014; 11:73. [PMID: 25212909 PMCID: PMC4174252 DOI: 10.1186/s12977-014-0073-0] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2014] [Accepted: 08/11/2014] [Indexed: 11/21/2022] Open
Abstract
Background All retroviruses synthesize essential proteins via alternatively spliced mRNAs. Retrovirus genera, though, exploit different mechanisms to coordinate the synthesis of proteins from alternatively spliced mRNAs. The best studied of these retroviral, post-transcriptional effectors are the trans-acting Rev protein of lentiviruses and the cis-acting constitutive transport element (CTE) of the betaretrovirus Mason-Pfizer monkey virus (MPMV). How members of the gammaretrovirus genus translate protein from unspliced RNA has not been elucidated. Results The mechanism by which two gammaretroviruses, XMRV and MLV, synthesize the Gag polyprotein (Pr65Gag) from full-length, unspliced mRNA was investigated here. The yield of Pr65Gag from a gag–only expression plasmid was found to be at least 30-fold less than that from an otherwise isogenic gag-pol expression plasmid. A frameshift mutation disrupting the pol open reading frame within the gag-pol expression plasmid did not decrease Pr65Gag production and 398 silent nucleotide changes engineered into gag rendered Pr65Gag synthesis pol-independent. These results are consistent with pol-encoded RNA acting in cis to promote Pr65Gag translation. Two independently-acting pol fragments were identified by screening 17 pol deletion mutations. To determine the mechanism by which pol promoted Pr65Gag synthesis, gag RNA in total and cytoplasmic fractions was quantitated by northern blot and by RT-PCR. The pol sequences caused, maximally, three-fold increase in total or cytoplasmic gag mRNA. Instead, pol sequences increased gag mRNA association with polyribosomes ~100-fold, a magnitude sufficient to explain the increase in Pr65Gag translation efficiency. The MPMV CTE, an NXF1-binding element, substituted for pol in promoting Pr65Gag synthesis. A pol RNA stem-loop resembling the CTE promoted Pr65Gag synthesis. Over-expression of NXF1 and NXT, host factors that bind to the MPMV CTE, synergized with pol to promote gammaretroviral gag RNA loading onto polysomes and to increase Pr65Gag synthesis. Conversely, Gag polyprotein synthesis was decreased by NXF1 knockdown. Finally, overexpression of SRp20, a shuttling protein that binds to NXF1 and promotes NXF1 binding to RNA, also increased gag RNA loading onto polysomes and increased Pr65Gag synthesis. Conclusion These experiments demonstrate that gammaretroviral pol sequences act in cis to recruit NXF1 and SRp20 to promote polysome loading of gag RNA and, thereby license the synthesis of Pr65Gag from unspliced mRNA.
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Affiliation(s)
| | - Jeremy Luban
- Department of Microbiology and Molecular Medicine, University of Geneva, Geneva 1205, Switzerland.
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Pilkington GR, Purzycka KJ, Bear J, Le Grice SFJ, Felber BK. Gammaretrovirus mRNA expression is mediated by a novel, bipartite post-transcriptional regulatory element. Nucleic Acids Res 2014; 42:11092-106. [PMID: 25190459 PMCID: PMC4176177 DOI: 10.1093/nar/gku798] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
Post-transcriptional regulatory mechanisms of several complex and simple retroviruses and retroelements have been elucidated, with the exception of the gammaretrovirus family. We found that, similar to the other retroviruses, gag gene expression of MuLV and XMRV depends on post-transcriptional regulation mediated via an RNA sequence overlapping the pro-pol open reading frame, termed the Post-Transcriptional Element (PTE). PTE function can be replaced by heterologous RNA export elements, e.g. CTE of simian type D retroviruses. Alternatively, Gag particle production is achieved using an RNA/codon optimized gag gene. PTE function is transferable and can replace HIV Rev-RRE-regulated expression of HIV gag. Analysis of PTE by SHAPE revealed a highly structured RNA comprising seven stem-loop structures, with the 5′ and 3′ stem-loops forming an essential bipartite signal. MuLV and XMRV PTE share 98% identity and have highly similar RNA structures, with changes mostly located to single-stranded regions. PTE identification strongly suggests that all retroviruses and retroelements share common strategies of post-transcriptional gene regulation to produce Gag. Expression depends on complex RNA structures embedded within retroviral mRNA, in coding regions or the 3′ untranslated region. These specific structures serve as recognition signals for either cellular or viral proteins.
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Affiliation(s)
- Guy R Pilkington
- Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA
| | - Katarzyna J Purzycka
- RT Biochemistry Section, Drug Resistance Program, National Cancer Institute at Frederick, Frederick, MD 21702, USA Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, 61-704, Poland
| | - Jenifer Bear
- Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA
| | - Stuart F J Le Grice
- RT Biochemistry Section, Drug Resistance Program, National Cancer Institute at Frederick, Frederick, MD 21702, USA
| | - Barbara K Felber
- Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA
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Murine leukemia virus uses NXF1 for nuclear export of spliced and unspliced viral transcripts. J Virol 2014; 88:4069-82. [PMID: 24478440 DOI: 10.1128/jvi.03584-13] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
UNLABELLED Intron-containing mRNAs are subject to restricted nuclear export in higher eukaryotes. Retroviral replication requires the nucleocytoplasmic transport of both spliced and unspliced RNA transcripts, and RNA export mechanisms of gammaretroviruses are poorly characterized. Here, we report the involvement of the nuclear export receptor NXF1/TAP in the nuclear export of gammaretroviral RNA transcripts. We identified a conserved cis-acting element in the pol gene of gammaretroviruses, including murine leukemia virus (MLV) and xenotropic murine leukemia virus (XMRV), named the CAE (cytoplasmic accumulation element). The CAE enhanced the cytoplasmic accumulation of viral RNA transcripts and the expression of viral proteins without significantly affecting the stability, splicing, or translation efficiency of the transcripts. Insertion of the CAE sequence also facilitated Rev-independent HIV Gag expression. We found that the CAE sequence interacted with NXF1, whereas disruption of NXF1 ablated CAE function. Thus, the CAE sequence mediates the cytoplasmic accumulation of gammaretroviral transcripts in an NXF1-dependent manner. Disruption of NXF1 expression impaired cytoplasmic accumulations of both spliced and unspliced RNA transcripts of XMRV and MLV, resulting in their nuclear retention or degradation. Thus, our results demonstrate that gammaretroviruses use NXF1 for the cytoplasmic accumulation of both spliced and nonspliced viral RNA transcripts. IMPORTANCE Murine leukemia virus (MLV) has been studied as one of the classic models of retrovirology. Although unspliced host messenger RNAs are rarely exported from the nucleus, MLV actively exports unspliced viral RNAs to the cytoplasm. Despite extensive studies, how MLV achieves this difficult task has remained a mystery. Here, we have studied the RNA export mechanism of MLV and found that (i) the genome contains a sequence which supports the efficient nuclear export of viral RNAs, (ii) the cellular factor NXF1 is involved in the nuclear export of both spliced and unspliced viral RNAs, and, finally, (iii) depletion of NXF1 results in nuclear retention or degradation of viral RNAs. Our study provides a novel insight into MLV nuclear export.
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Molecular events accompanying rous sarcoma virus rescue from rodent cells and the role of viral gene complementation. J Virol 2014; 88:3505-15. [PMID: 24403579 DOI: 10.1128/jvi.02761-13] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
UNLABELLED Transformation of rodent cells with avian Rous sarcoma virus (RSV) opened new ways to studying virus integration and expression in nonpermissive cells. We were interested in (i) the molecular changes accompanying fusion of RSV-transformed mammalian cells with avian cells leading to virus rescue and (ii) enhancement of this process by retroviral gene products. The RSV-transformed hamster RSCh cell line was characterized as producing only a marginal amount of env mRNA, no envelope glycoprotein, and a small amount of unprocessed Gag protein. Egress of viral unspliced genomic RNA from the nucleus was hampered, and its stability decreased. Cell fusion of the chicken DF-1 cell line with RSCh cells led to production of env mRNA, envelope glycoprotein, and processed Gag and virus-like particle formation. Proteosynthesis inhibition in DF-1 cells suppressed steps leading to virus rescue. Furthermore, new aberrantly spliced env mRNA species were found in the RSCh cells. Finally, we demonstrated that virus rescue efficiency can be significantly increased by complementation with the env gene and the highly expressed gag gene and can be increased the most by a helper virus infection. In summary, Env and Gag synthesis is increased after RSV-transformed hamster cell fusion with chicken fibroblasts, and both proteins provided in trans enhance RSV rescue. We conclude that the chicken fibroblast yields some factor(s) needed for RSV replication, particularly Env and Gag synthesis, in nonpermissive rodent cells. IMPORTANCE One of the important issues in retrovirus heterotransmission is related to cellular factors that prevent virus replication. Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, is able to infect and transform mammalian cells; however, such transformed cells do not produce infectious virus particles. Using the well-defined model of RSV-transformed rodent cells, we established that the lack of virus replication is due to the absence of chicken factor(s), which can be supplemented by cell fusion. Cell fusion with permissive chicken cells led to an increase in RNA splicing and nuclear export of specific viral mRNAs, as well as synthesis of respective viral proteins and production of virus-like particles. RSV rescue by cell fusion can be potentiated by in trans expression of viral genes in chicken cells. We conclude that rodent cells lack some chicken factor(s) required for proper viral RNA processing and viral protein synthesis.
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17
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Nuclear trafficking of retroviral RNAs and Gag proteins during late steps of replication. Viruses 2013; 5:2767-95. [PMID: 24253283 PMCID: PMC3856414 DOI: 10.3390/v5112767] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2013] [Revised: 10/31/2013] [Accepted: 11/12/2013] [Indexed: 11/16/2022] Open
Abstract
Retroviruses exploit nuclear trafficking machinery at several distinct stages in their replication cycles. In this review, we will focus primarily on nucleocytoplasmic trafficking events that occur after the completion of reverse transcription and proviral integration. First, we will discuss nuclear export of unspliced viral RNA transcripts, which serves two essential roles: as the mRNA template for the translation of viral structural proteins and as the genome for encapsidation into virions. These full-length viral RNAs must overcome the cell's quality control measures to leave the nucleus by co-opting host factors or encoding viral proteins to mediate nuclear export of unspliced viral RNAs. Next, we will summarize the most recent findings on the mechanisms of Gag nuclear trafficking and discuss potential roles for nuclear localization of Gag proteins in retrovirus replication.
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18
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Avian retroviral replication. Curr Opin Virol 2013; 3:664-9. [PMID: 24011707 DOI: 10.1016/j.coviro.2013.08.008] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2013] [Revised: 08/14/2013] [Accepted: 08/15/2013] [Indexed: 12/17/2022]
Abstract
Avian retroviruses were originally identified as cancer-inducting filterable agents in chicken neoplasms at the beginning of the 20th century. Since their discovery, the study of these simple retroviruses has contributed greatly to our understanding of viral replication and cancer. Avian retroviruses continue to evolve and have great economic importance in the poultry industry worldwide. The aim of this review is to provide a broad overview of the genome, pathology, and replication of avian retroviruses. Notable gaps in our current knowledge are highlighted, and areas where avian retroviruses differ from other retroviruses are emphasized.
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Freitas N, Cunha C. Searching for nuclear export elements in hepatitis D virus RNA. World J Virol 2013; 2:123-135. [PMID: 24255883 PMCID: PMC3832856 DOI: 10.5501/wjv.v2.i3.123] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/06/2013] [Revised: 07/26/2013] [Accepted: 08/09/2013] [Indexed: 02/05/2023] Open
Abstract
AIM: To search for the presence of cis elements in hepatitis D virus (HDV) genomic and antigenomic RNA capable of promoting nuclear export.
METHODS: We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid pDM138. Twenty cDNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pDM138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by real-time polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export.
RESULTS: Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter mRNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway.
CONCLUSION: A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.
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Leblanc J, Weil J, Beemon K. Posttranscriptional regulation of retroviral gene expression: primary RNA transcripts play three roles as pre-mRNA, mRNA, and genomic RNA. WILEY INTERDISCIPLINARY REVIEWS-RNA 2013; 4:567-80. [PMID: 23754689 DOI: 10.1002/wrna.1179] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/28/2013] [Revised: 05/09/2013] [Accepted: 05/11/2013] [Indexed: 12/11/2022]
Abstract
After reverse transcription of the retroviral RNA genome and integration of the DNA provirus into the host genome, host machinery is used for viral gene expression along with viral proteins and RNA regulatory elements. Here, we discuss co-transcriptional and posttranscriptional regulation of retroviral gene expression, comparing simple and complex retroviruses. Cellular RNA polymerase II synthesizes full-length viral primary RNA transcripts that are capped and polyadenylated. All retroviruses generate a singly spliced env mRNA from this primary transcript, which encodes the viral glycoproteins. In addition, complex viral RNAs are alternatively spliced to generate accessory proteins, such as Rev, which is involved in posttranscriptional regulation of HIV-1 RNA. Importantly, the splicing of all retroviruses is incomplete; they must maintain and export a fraction of their primary RNA transcripts. This unspliced RNA functions both as the major mRNA for Gag and Pol proteins and as the packaged genomic RNA. Different retroviruses export their unspliced viral RNA from the nucleus to the cytoplasm by either Tap-dependent or Rev/CRM1-dependent routes. Translation of the unspliced mRNA involves frame-shifting or termination codon suppression so that the Gag proteins, which make up the capsid, are expressed more abundantly than the Pol proteins, which are the viral enzymes. After the viral polyproteins assemble into viral particles and bud from the cell membrane, a viral encoded protease cleaves them. Some retroviruses have evolved mechanisms to protect their unspliced RNA from decay by nonsense-mediated RNA decay and to prevent genome editing by the cellular APOBEC deaminases.
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Affiliation(s)
- Jason Leblanc
- Biology Department, Johns Hopkins University, Baltimore, MD, USA
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21
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Checkley MA, Mitchell JA, Eizenstat LD, Lockett SJ, Garfinkel DJ. Ty1 gag enhances the stability and nuclear export of Ty1 mRNA. Traffic 2013; 14:57-69. [PMID: 22998189 PMCID: PMC3548082 DOI: 10.1111/tra.12013] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2012] [Revised: 09/19/2012] [Accepted: 09/21/2012] [Indexed: 11/28/2022]
Abstract
Retrotransposon and retroviral RNA delivery to particle assembly sites is essential for their replication. mRNA and Gag from the Ty1 retrotransposon colocalize in cytoplasmic foci, which are required for transposition and may be the sites for virus-like particle (VLP) assembly. To determine which Ty1 components are required to form mRNA/Gag foci, localization studies were performed in a Ty1-less strain expressing galactose-inducible Ty1 plasmids (pGTy1) containing mutations in GAG or POL. Ty1 mRNA/Gag foci remained unaltered in mutants defective in Ty1 protease (PR) or deleted for POL. However, Ty1 mRNA containing a frameshift mutation (Ty1fs) that prevents the synthesis of all proteins accumulated in the nucleus. Ty1fs RNA showed a decrease in stability that was mediated by the cytoplasmic exosome, nonsense-mediated decay (NMD) and the processing body. Localization of Ty1fs RNA remained unchanged in an nmd2Δ mutant. When Gag and Ty1fs mRNA were expressed independently, Gag provided in trans increased Ty1fs RNA level and restored localization of Ty1fs RNA in cytoplasmic foci. Endogenously expressed Gag also localized to the nuclear periphery independent of RNA export. These results suggest that Gag is required for Ty1 mRNA stability, efficient nuclear export and localization into cytoplasmic foci.
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Affiliation(s)
- Mary Ann Checkley
- Gene Regulation and Chromosome Biology Laboratory, Frederick National Laboratory for Cancer Research, Frederick, MD 21702
| | - Jessica A. Mitchell
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602
| | - Linda D. Eizenstat
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602
| | | | - David J. Garfinkel
- Gene Regulation and Chromosome Biology Laboratory, Frederick National Laboratory for Cancer Research, Frederick, MD 21702
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602
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A 205-nucleotide deletion in the 3' untranslated region of avian leukosis virus subgroup J, currently emergent in China, contributes to its pathogenicity. J Virol 2012; 86:12849-60. [PMID: 22993155 DOI: 10.1128/jvi.01113-12] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
In the past 5 years, an atypical clinical outbreak of avian leukosis virus subgroup J (ALV-J), which contains a unique 205-nucleotide deletion in its 3' untranslated region (3'UTR), has become epidemic in chickens in China. To determine the role of the 205-nucleotide deletion in the pathogenicity of ALV-J, a pair of viruses were constructed and rescued. The first virus was an ALV-J Chinese isolate (designated HLJ09SH01) containing the 205-nucleotide deletion in its 3'UTR. The second virus was a chimeric clone in which the 3'UTR contains a 205-nucleotide sequence corresponding to a region of the ALV-J prototype virus. The replication and pathogenicity of the rescued viruses (rHLJ09SH01 and rHLJ09SH01A205) were investigated. Compared to rHLJ09SH01A205, rHLJ09SH01 showed a moderate growth advantage in vitro and in vivo, in addition to exhibiting a higher oncogenicity rate and lethality rate in layers and broilers. Increased vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth receptor subtype 2 (VEGFR-2) expression was induced by rHLJ09SH01 more so than by rHLJ09SH01A205 during early embryonic vascular development, but this increased expression disappeared when the expression levels were normalized to the viral levels. This finding suggests that the expression of VEGF-A and VEGFR-2 is associated with viral replication and may also represent a novel molecular mechanism underlying the oncogenic potential of ALV-J. Overall, our findings not only indicate that the unique 205-nucleotide deletion in the ALV-J genome occurred naturally in China and contributes to increased pathogenicity but also point to the possible mechanism of ALV-J-induced oncogenicity.
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Human intronless genes: Functional groups, associated diseases, evolution, and mRNA processing in absence of splicing. Biochem Biophys Res Commun 2012; 424:1-6. [DOI: 10.1016/j.bbrc.2012.06.092] [Citation(s) in RCA: 66] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2012] [Accepted: 06/18/2012] [Indexed: 11/18/2022]
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Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag. J Virol 2012; 86:2990-3002. [PMID: 22258250 DOI: 10.1128/jvi.06874-11] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.
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Hohenadl C, Gunzburg WH, Salmons B, Indik S. The 5' leader sequence of mouse mammary tumor virus enhances expression of the envelope and reporter genes. J Gen Virol 2011; 93:308-318. [PMID: 22113011 DOI: 10.1099/vir.0.035196-0] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Mouse mammary tumor virus (MMTV) is a complex betaretrovirus, which utilizes a Rev-like auxiliary protein Rem to export the unspliced viral RNA from the nucleus. MMTV env mRNA appears to be exported via a distinct, Rem-independent, mechanism. Here, we analysed the effect of an extensively folded region coinciding with the 5' leader sequence on env gene expression. We found that the presence of the 5' leader stimulates expression of the envelope protein. Enhanced Env production was accompanied by increased cytoplasmic levels of env mRNA. The 5' leader promotes nucleocytoplasmic translocation and increases stability of env mRNA. The region responsible for this effect was mapped to the distal part of the 5' leader. Furthermore, the 5' leader inserted in the sense orientation into a heterologous luciferase expression construct increased luciferase activity.
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Affiliation(s)
- Christine Hohenadl
- Institute of Virology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria
| | - Walter H Gunzburg
- Christian Doppler Laboratory for Gene Therapeutic Vector Development, Vienna, Austria.,Institute of Virology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria
| | | | - Stanislav Indik
- Institute of Virology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria
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Jaagsiekte sheep retrovirus biology and oncogenesis. Viruses 2010; 2:2618-48. [PMID: 21994634 PMCID: PMC3185594 DOI: 10.3390/v2122618] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2010] [Revised: 11/22/2010] [Accepted: 11/23/2010] [Indexed: 11/19/2022] Open
Abstract
Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a lung cancer in sheep known as ovine pulmonary adenocarcinoma (OPA). The disease has been identified around the world in several breeds of sheep and goats, and JSRV infection typically has a serious impact on affected flocks. In addition, studies on OPA are an excellent model for human lung carcinogenesis. A unique feature of JSRV is that its envelope (Env) protein functions as an oncogene. The JSRV Env-induced transformation or oncogenesis has been studied in a variety of cell systems and in animal models. Moreover, JSRV studies have provided insights into retroviral genomic RNA export/expression mechanisms. JSRV encodes a trans-acting factor (Rej) within the env gene necessary for the synthesis of Gag protein from unspliced viral RNA. This review summarizes research pertaining to JSRV-induced pathogenesis, Env transformation, and other aspects of JSRV biology.
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Withers JB, Beemon KL. Structural features in the Rous sarcoma virus RNA stability element are necessary for sensing the correct termination codon. Retrovirology 2010; 7:65. [PMID: 20687936 PMCID: PMC2925335 DOI: 10.1186/1742-4690-7-65] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2010] [Accepted: 08/05/2010] [Indexed: 02/05/2023] Open
Abstract
Background Nonsense-mediated mRNA decay (NMD) is an mRNA quality control mechanism that selectively recognizes and targets for degradation mRNAs containing premature termination codons. Retroviral full-length RNA is presented to the host translation machinery with characteristics rarely observed among host cell mRNAs: a long 3' UTR, retained introns, and multiple open reading frames. As a result, the viral RNA is predicted to be recognized by the host NMD machinery and degraded. In the case of the Rous sarcoma virus (RSV), we identified a stability element (RSE), which resides immediately downstream of the gag termination codon and facilitates NMD evasion. Results We defined key RNA features of the RSE through directed mutagenesis of the virus. These data suggest that the minimal RSE is 155 nucleotides (nts) and functions independently of the nucleotide sequence of the stop codon or the first nucleotide following the stop codon. Further data suggested that the 3'UTRs of the RSV pol and src may also function as stability elements. Conclusions We propose that these stability elements in RSV may be acting as NMD insulators to mask the preceding stop codon from the NMD machinery.
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Affiliation(s)
- Johanna B Withers
- Department of Biology, Johns Hopkins University, 3400 N, Charles St., Baltimore, MD 21218, USA
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28
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Analysis of bovine foamy virus btas mRNA transcripts during persistent infection. Virus Genes 2009; 40:84-93. [PMID: 19911263 DOI: 10.1007/s11262-009-0422-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2009] [Accepted: 11/01/2009] [Indexed: 12/11/2022]
Abstract
Foamy virus (FV) is an unconventional retrovirus that possesses a complex genome and a special mechanism for gene expression regulation. The genome encodes transcriptional protein Tas which is found to regulate both the internal promoter (IP) and the long terminal repeat promoter (LTR). However, the detailed mechanism of Tas-mediated gene expression remains unknown. In this study, we provided the first evidence for the temporal production and utilization of four different bovine foamy virus (BFV) btas mRNAs during persistent infection. These four forms of btas mRNA transcripts initiated either at BFV LTR or IP and spliced or unspliced have a differential ability to activate BFV promoters. Furthermore, by developing an MS2 translational operator/coat protein combined system to track mRNA exportation from the nucleus and distribution throughout the cytoplasm, we observed that the IP spliced transcript could be exported into the cytoplasm more efficiently than unspliced transcripts. These findings provide evidence for the hypothesis that the functional interplay of both promoters contributes to the temporal pattern of BFV transcription and suggest that a post-transcriptional regulation exist in BFV replication.
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Probing the HIV-1 genomic RNA trafficking pathway and dimerization by genetic recombination and single virion analyses. PLoS Pathog 2009; 5:e1000627. [PMID: 19834549 PMCID: PMC2757677 DOI: 10.1371/journal.ppat.1000627] [Citation(s) in RCA: 85] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2009] [Accepted: 09/21/2009] [Indexed: 11/19/2022] Open
Abstract
Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, we sought to delineate the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, we demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, we show that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag in trans, we have demonstrated that Gag is able to package RNA from either export pathway, irrespective of the transport pathway used by the gag mRNA. These findings provide unique insights into the process of RNA export in general, and more specifically, of HIV-1 genomic RNA trafficking. High genetic diversity of HIV-1 presents a difficult barrier for drug treatment and vaccine development. HIV-1 efficiently reassorts mutations in its genome by packaging two copies of RNA into one virion and using portions of each RNA as the template for DNA synthesis. Thus, a key to HIV-1's genetic diversity is how it packages two RNAs into one virion. We have previously shown that HIV-1 packages RNA as a dimer, rather than two monomers. In this report, we determined that HIV-1 RNA finds its copackaged dimer partner mostly in the cytoplasm, not the nucleus, of the infected cells. We also found that the host cell machinery used to transport the HIV-1 RNA out of the nucleus affects the ability of two RNAs to copackage into one virion: HIV-1 RNAs transported using the CRM-1 pathway does not copackage efficiently with RNA transported by the NXF1 pathway. This is also the first demonstration that RNAs transported by the two major cellular pathways, CRM-1 and NXF1, are partially segregated at some point in the cytoplasm. Therefore, this work provides novel insights to both HIV replication mechanisms and cellular RNA transport pathways.
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Jaagsiekte sheep retrovirus encodes a regulatory factor, Rej, required for synthesis of Gag protein. J Virol 2009; 83:12483-98. [PMID: 19776124 DOI: 10.1128/jvi.01747-08] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Retroviruses express Gag and Pol proteins by translation of unspliced genome-length viral RNA. For some retroviruses, transport of unspliced viral RNA to the cytoplasm is mediated by small regulatory proteins such as human immunodeficiency virus Rev, while other retroviruses contain constitutive transport elements in their RNAs that allow transport without splicing. In this study, we found that the betaretrovirus Jaagsiekte sheep retrovirus (JSRV) encodes within the env gene a trans-acting factor (Rej) necessary for the synthesis of Gag protein from unspliced viral RNA. Deletion of env sequences from a JSRV proviral expression plasmid (pTN3) abolished its ability to produce Gag polyprotein in transfected 293T cells, and Gag synthesis could be restored by cotransfection of an env expression plasmid (DeltaGP). Deletion analysis localized the complementing activity (Rej) to the putative Env signal peptide, and a signal peptide expression construct showed Rej activity. Two other betaretroviruses, mouse mammary tumor virus (MMTV) and human endogenous retrovirus type K, encode analogous factors (Rem and Rec, respectively) that are encoded from doubly spliced env mRNAs. Reverse transcriptase-PCR cloning and sequencing identified alternate internal splicing events in the 5' end of JSRV env that could signify analogous doubly spliced Rej mRNAs, and cDNA clones expressing two of them also showed Rej activity. The predicted Rej proteins contain motifs similar to those found in MMTV Rem and other analogous retroviral regulatory proteins. Interestingly, in most cell lines, JSRV expression plasmids with Rej deleted showed normal transport of unspliced JSRV RNA to the cytoplasm; however, in 293T cells Rej modestly enhanced export of unspliced viral RNA (2.8-fold). Metabolic labeling experiments with [(35)S]methionine indicated that JSRV Rej is required for the synthesis of viral Gag polyprotein. Thus, in most cell lines, the predominant function of Rej is to facilitate translation of unspliced viral mRNA.
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31
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Mertz JA, Chadee AB, Byun H, Russell R, Dudley JP. Mapping of the functional boundaries and secondary structure of the mouse mammary tumor virus Rem-responsive element. J Biol Chem 2009; 284:25642-52. [PMID: 19632991 DOI: 10.1074/jbc.m109.012476] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Mouse mammary tumor virus (MMTV) is a complex retrovirus that encodes at least three regulatory and accessory proteins, including Rem. Rem is required for nuclear export of unspliced viral RNA and efficient expression of viral proteins. Our previous data indicated that sequences at the envelope-3' long terminal repeat junction are required for proper export of viral RNA. To further map the Rem-responsive element (RmRE), reporter vectors containing various portions of the viral envelope gene and the 3' long terminal repeat were tested in the presence and absence of Rem in transient transfection assays. A 476-bp fragment that spans the envelope-long terminal repeat junction had activity equivalent to the entire 3'-end of the mouse mammary tumor virus genome, but further deletions at the 5'- or 3'-ends reduced Rem responsiveness. RNase structure mapping of the full-length RmRE and a 3'-truncation suggested multiple domains with local base pairing and intervening single-stranded segments. A secondary structure model constrained by these data is reminiscent of the RNA response elements of other complex retroviruses, with numerous local stem-loops and long-range base pairs near the 5'- and 3'-boundaries, and differs substantially from an earlier model generated without experimental constraints. Covariation analysis provides limited support for basic features of our model. Reporter assays in human and mouse cell lines revealed similar boundaries, suggesting that the RmRE does not require cell type-specific proteins to form a functional structure.
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Affiliation(s)
- Jennifer A Mertz
- Section of Molecular Genetics and Microbiology, The University of Texas, Austin, Texas 78712-0162, USA
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32
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Genetic evidence for a connection between Rous sarcoma virus gag nuclear trafficking and genomic RNA packaging. J Virol 2009; 83:6790-7. [PMID: 19369339 DOI: 10.1128/jvi.00101-09] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
The packaging of retroviral genomic RNA (gRNA) requires cis-acting elements within the RNA and trans-acting elements within the Gag polyprotein. The packaging signal psi, at the 5' end of the viral gRNA, binds to Gag through interactions with basic residues and Cys-His box RNA-binding motifs in the nucleocapsid. Although specific interactions between Gag and gRNA have been demonstrated previously, where and when they occur is not well understood. We discovered that the Rous sarcoma virus (RSV) Gag protein transiently localizes to the nucleus, although the roles of Gag nuclear trafficking in virus replication have not been fully elucidated. A mutant of RSV (Myr1E) with enhanced plasma membrane targeting of Gag fails to undergo nuclear trafficking and also incorporates reduced levels of gRNA into virus particles compared to those in wild-type particles. Based on these results, we hypothesized that Gag nuclear entry might facilitate gRNA packaging. To test this idea by using a gain-of-function genetic approach, a bipartite nuclear localization signal (NLS) derived from the nucleoplasmin protein was inserted into the Myr1E Gag sequence (generating mutant Myr1E.NLS) in an attempt to restore nuclear trafficking. Here, we report that the inserted NLS enhanced the nuclear localization of Myr1E.NLS Gag compared to that of Myr1E Gag. Also, the NLS sequence restored gRNA packaging to nearly wild-type levels in viruses containing Myr1E.NLS Gag, providing genetic evidence linking nuclear trafficking of the retroviral Gag protein with gRNA incorporation.
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33
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A premature termination codon mutation at the C terminus of foamy virus Gag downregulates the levels of spliced pol mRNA. J Virol 2007; 82:1656-64. [PMID: 18057244 DOI: 10.1128/jvi.00990-07] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Foamy viruses (FV) comprise a subfamily of retroviruses. Orthoretroviruses, such as human immunodeficiency virus type 1, synthesize Gag and Pol from unspliced genomic RNA. However, FV Pol is expressed from a spliced mRNA independently of Gag. FV pol splicing uses a 3' splice site located at the 3' end of gag, resulting in a shared exon between gag and pol. Previously, our laboratory showed that C-terminal Gag premature termination codon (PTC) mutations in the 3' shared exon led to greatly decreased levels of Pol protein (C. R. Stenbak and M. L. Linial, J. Virol. 78:9423-9430, 2004). To further characterize these mutants, we quantitated the levels of unspliced gag and spliced pol mRNAs using a real-time PCR assay. In some of the PTC mutants, the levels of spliced pol mRNA were reduced as much as 30-fold, whereas levels of unspliced gag RNA were not affected. Substitutions of a missense codon in place of a PTC restored normal levels of spliced pol mRNA. Disrupting Upf proteins involved in nonsense-mediated mRNA decay (NMD) did not affect Pol protein expression. Introduction of an exonic splicing enhancer downstream of the PTC mutation restored pol splicing to the wild-type level. Taken together, our results show that the PTC mutation itself is responsible for decreased levels of pol mRNA but that mechanisms other than NMD might be involved in downregulating Pol expression. The results also suggest that normal pol splicing utilizes a suboptimal splice site seen for other spliced mRNAs in most retroviruses, in that introduced exonic enhancer elements can increase splicing efficiency.
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34
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LeBlanc JJ, Uddowla S, Abraham B, Clatterbuck S, Beemon KL. Tap and Dbp5, but not Gag, are involved in DR-mediated nuclear export of unspliced Rous sarcoma virus RNA. Virology 2007; 363:376-86. [PMID: 17328934 PMCID: PMC2564995 DOI: 10.1016/j.virol.2007.01.026] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2006] [Revised: 12/18/2006] [Accepted: 01/18/2007] [Indexed: 10/23/2022]
Abstract
All retroviruses must circumvent cellular restrictions on the export of unspliced RNAs from the nucleus. While the unspliced RNA export pathways for HIV and Mason-Pfizer monkey virus are well characterized, that of Rous sarcoma virus (RSV) is not. We have previously reported that the RSV direct repeat (DR) elements are involved in the cytoplasmic accumulation of unspliced viral RNA. Here, using fluorescent in situ hybridization (FISH), we demonstrate that unspliced viral RNAs bearing a single point mutation (G8863C) in the DR exhibit a restricted cellular localization in and around the nucleus. In contrast, wild type unspliced viral RNA had a diffuse localization throughout the nucleus and cytoplasm. Since the RSV Gag protein has a transient localization in the nucleus, we examined the effect of Gag over-expression on a DR-mediated reporter construct. While Gag did not enhance DR-mediated nuclear export, the dominant-negative expression of two cellular export factors, Tap and Dbp5, inhibited expression of the same reporter construct. Furthermore, FISH studies using the dominant-negative Dbp5 demonstrated that unspliced wild type RSV RNA was retained within the nucleus. Taken together, these results further implicate the DR in nuclear RNA export through interactions with Tap and Dbp5.
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Affiliation(s)
- Jason J LeBlanc
- Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA
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35
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Felber BK, Zolotukhin AS, Pavlakis GN. Posttranscriptional Control of HIV‐1 and Other Retroviruses and Its Practical Applications. ADVANCES IN PHARMACOLOGY 2007; 55:161-97. [PMID: 17586315 DOI: 10.1016/s1054-3589(07)55005-2] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Affiliation(s)
- Barbara K Felber
- Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702, USA
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36
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Cochrane AW, McNally MT, Mouland AJ. The retrovirus RNA trafficking granule: from birth to maturity. Retrovirology 2006; 3:18. [PMID: 16545126 PMCID: PMC1475878 DOI: 10.1186/1742-4690-3-18] [Citation(s) in RCA: 100] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2005] [Accepted: 03/17/2006] [Indexed: 11/10/2022] Open
Abstract
Post-transcriptional events in the life of an RNA including RNA processing, transport, translation and metabolism are characterized by the regulated assembly of multiple ribonucleoprotein (RNP) complexes. At each of these steps, there is the engagement and disengagement of RNA-binding proteins until the RNA reaches its final destination. For retroviral genomic RNA, the final destination is the capsid. Numerous studies have provided crucial information about these processes and serve as the basis for studies on the intracellular fate of retroviral RNA. Retroviral RNAs are like cellular mRNAs but their processing is more tightly regulated by multiple cis-acting sequences and the activities of many trans-acting proteins. This review describes the viral and cellular partners that retroviral RNA encounters during its maturation that begins in the nucleus, focusing on important events including splicing, 3' end-processing, RNA trafficking from the nucleus to the cytoplasm and finally, mechanisms that lead to its compartmentalization into progeny virions.
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Affiliation(s)
- Alan W Cochrane
- Department of Medical Genetics and Microbiology, University of Toronto, 1 King's College Circle, Toronto, Ontario, M5S 1A8, Canada
| | - Mark T McNally
- Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI, 53226, USA
| | - Andrew J Mouland
- HIV-1 RNA Trafficking Laboratory, Lady Davis Institute for Medical Research-Sir Mortimer B. Davis Jewish General Hospital and McGill University, 3755 Côte-Ste-Catherine Road, H3T 1E2, Canada
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37
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Kumar S, Saradhi M, Chaturvedi NK, Tyagi RK. Intracellular localization and nucleocytoplasmic trafficking of steroid receptors: an overview. Mol Cell Endocrinol 2006; 246:147-56. [PMID: 16388893 DOI: 10.1016/j.mce.2005.11.028] [Citation(s) in RCA: 72] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Subcellular compartmentalization and dynamic movements of steroid receptors are major steps in executing their transcription regulatory function. Though significant progress has been made in understanding the mechanisms underlying nuclear import of NLS-bearing proteins, our general and mechanistic understanding about the nuclear export processes has begun to emerge only recently. The discovery of most commonly utilized CRM1/exportin1 dependent nuclear export pathway is attributed to a potent nuclear export inhibitor leptomycin B that helped dissecting this and other nuclear export pathways. Simultaneously, utilization of green fluorescent protein (GFP)-tagged intracellular steroid receptors has contributed to not only resolving controversial issue of subcellular localization of unliganded hormone receptors but also provided further insight into finer details of receptor dynamics in living cells. With judicious use of leptomycin B and expression of GFP-tagged receptors in living cells, existence of exportin1/CRM1 independent pathway(s), nuclear export signals and receptors for bi-directional translocation that are unique to steroid receptor trafficking have been specified. Currently, we appear to be arriving at a consensus that steroid/nuclear receptors follow dynamic nucleocytoplasmic processes that deviate from the ones commonly utilized by majority of other proteins.
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Affiliation(s)
- Sanjay Kumar
- Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067, India
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38
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Smagulova F, Maurel S, Morichaud Z, Devaux C, Mougel M, Houzet L. The highly structured encapsidation signal of MuLV RNA is involved in the nuclear export of its unspliced RNA. J Mol Biol 2005; 354:1118-28. [PMID: 16289115 DOI: 10.1016/j.jmb.2005.10.021] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2005] [Revised: 10/03/2005] [Accepted: 10/05/2005] [Indexed: 11/22/2022]
Abstract
The encapsidation signal (Psi) of retroviruses is located in the 5' UTR of the viral genomic unspliced transcript and is highly structured. In the Psi of murine leukaemia virus (MuLV), four stem-loops, called A, B, C and D, promote dimerization and encapsidation of the MuLV unspliced RNA into virions. Through analysis of Psi-deleted transcripts, we found that the AB and CD motifs independently enhanced the cytoplasmic accumulation of RNAs. Furthermore, we showed that over-expression of the Psi sequence in the infected cells led to a competition with the nuclear export of unspliced MuLV transcripts, revealing a new function for these stem-loops in the transport of viral intron-containing RNAs from the nucleus to the cytoplasm.
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MESH Headings
- 5' Untranslated Regions
- Active Transport, Cell Nucleus
- Animals
- Capsid/metabolism
- Cell Fractionation
- Cell Nucleus/metabolism
- Cytoplasm/metabolism
- Dimerization
- Fluorescent Dyes
- In Situ Hybridization, Fluorescence
- Indoles
- Introns
- Mice
- Microscopy, Fluorescence
- Moloney murine leukemia virus/chemistry
- Moloney murine leukemia virus/genetics
- Mutation
- NIH 3T3 Cells
- Nucleic Acid Conformation
- RNA Splicing
- RNA, Ribosomal/analysis
- RNA, Ribosomal/chemistry
- RNA, Small Nucleolar/analysis
- RNA, Small Nucleolar/metabolism
- RNA, Transfer/analysis
- RNA, Transfer/chemistry
- RNA, Viral/analysis
- RNA, Viral/chemistry
- RNA, Viral/isolation & purification
- RNA, Viral/metabolism
- Transfection
- Virion/chemistry
- Virion/genetics
- Virus Replication/genetics
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Affiliation(s)
- Fatima Smagulova
- CNRS UMR5121-UM1, IFR122, Institut de Biologie, 34960 Montpellier, France
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39
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Hlavaty J, Schittmayer M, Stracke A, Jandl G, Knapp E, Felber BK, Salmons B, Günzburg WH, Renner M. Effect of posttranscriptional regulatory elements on transgene expression and virus production in the context of retrovirus vectors. Virology 2005; 341:1-11. [PMID: 16054668 DOI: 10.1016/j.virol.2005.06.037] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2005] [Revised: 05/27/2005] [Accepted: 06/22/2005] [Indexed: 11/20/2022]
Abstract
Ineffective transgene expression in a sufficient amount of target cells is still a limitation in retroviral vector mediated gene therapy. Thus, we systematically evaluated four genetic modulators, (i) the woodchuck posttranscriptional regulatory element (WPRE), (ii) the mouse RNA transport element (RTE), (iii) the constitutive transport element (CTE) of the simian retrovirus type 1 (SRV-1), and (iv) the 5' untranslated region of the human heat shock protein 70 (Hsp70 5'UTR), all of them involved in the posttranscriptional control of mRNA nucleo/cytoplasmatic transport, RNA stability, and translation efficiency, in an MLV-based retrovirus vector context. Insertion of the WPRE into the retrovirus vector resulted in enhancement of transgene expression (EGFP) both in transfected virus producing cells as well as in infected recipient cells irrespective of the location in the vector. The best effect was observed with two copies of the WPRE, 3' of the transgene and in the 3' untranslated region of the vector backbone. However, oligomerization of this element does not further increase transgene expression. Presence of the WPRE resulted also in an increase in virus production. Introduction of the CTE and/or RTE in the retroviral vector did not alter transgene expression and infectious particle production. Positive effects were observed only in vectors harboring the CTE and/or RTE in combination with the WPRE. The activity of the Hsp70 5'UTR as a translational enhancer was found to be negligible in the context of the retroviral vector. However, interference of the Hsp70 5'UTR strong secondary structure with the packaging sequence of the viral RNA was experimentally excluded as being the cause of this. These data suggest that only the WPRE is a suitable element for the improvement of transgene expression and oncoretroviral vector production.
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Affiliation(s)
- Juraj Hlavaty
- Research Institute of Virology and Biomedicine, University of Veterinary Medicine, A-1210 Vienna, Austria
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Smulevitch S, Michalowski D, Zolotukhin AS, Schneider R, Bear J, Roth P, Pavlakis GN, Felber BK. Structural and functional analysis of the RNA transport element, a member of an extensive family present in the mouse genome. J Virol 2005; 79:2356-65. [PMID: 15681436 PMCID: PMC546579 DOI: 10.1128/jvi.79.4.2356-2365.2005] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We previously identified an RNA transport element (RTE), present in a subclass of rodent intracisternal A particle retroelements (F. Nappi, R. Schneider, A. Zolotukhin, S. Smulevitch, D. Michalowski, J. Bear, B. Felber, and G. Pavlakis, J. Virol. 75:4558-4569, 2001), that is able to replace Rev-responsive element regulation in human immunodeficiency virus type 1. RTE-directed mRNA export is mediated by a still-unknown cellular factor(s), is independent of the CRM1 nuclear export receptor, and is conserved among vertebrates. Here we show that this RTE folds into an extended RNA secondary structure and thus does not resemble any known RTEs. Computer searches revealed the presence of 105 identical elements and more than 3,000 related elements which share at least 70% sequence identity with the RTE and which are found on all mouse chromosomes. These related elements are predicted to fold into RTE-like structures. Comparison of the sequences and structures revealed that the RTE and related elements can be divided into four groups. Mutagenesis of the RTE revealed that the minimal element contains four internal stem-loops, which are indispensable for function in mammalian cells. In contrast, only part of the element is essential to mediate RNA transport in microinjected Xenopus laevis oocyte nuclei. Importantly, the minimal RTE able to promote RNA transport has key structural features which are preserved in all the RTE-related elements, further supporting their functional importance. Therefore, RTE function depends on a complex secondary structure that is important for the interaction with the cellular export factor(s).
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Affiliation(s)
- Sergey Smulevitch
- Human Retrovirus Pathogenesis Section, NCI--Frederick, Frederick, MD 21702, USA
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Abstract
Herpesvirus saimiri (Saimiriine herpesvirus-2), a gamma2-herpesvirus (rhadinovirus) of non-human primates, causes T-lymphoproliferative diseases in susceptible organisms and transforms human and non-human T lymphocytes to continuous growth in vitro in the absence of stimulation. T cells transformed by H. saimiri retain many characteristics of intact T lymphocytes, such as the sensitivity to interleukin-2 and the ability to recognize the corresponding antigens. As a result, H. saimiri is widely used in immunobiology for immortalization of various difficult-to-obtain and/or -to-maintain T cells in order to obtain useful experimental models. In particular, H. saimiri-transformed human T cells are highly susceptible to infection with HIV-1 and -2. This makes them a convenient tool for propagation of poorly replicating strains of HIV, including primary clinical isolates. Therefore, the mechanisms mediating transformation of T cells by H. saimiri are of considerable interest. A single transformation-associated protein, StpA or StpB, mediates cell transformation by H. saimiri strains of group A or B, respectively. Strains of group C, which exhibit the highest oncogenic potential, have two proteins involved in transformation-StpC and Tip. Both proteins have been shown to dramatically affect signal transduction pathways leading to the activation of crucial transcription factors. This review is focused on the biological effects and molecular mechanisms of action of proteins involved in H. saimiri-dependent transformation.
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MESH Headings
- Animals
- Cell Transformation, Neoplastic/genetics
- Cell Transformation, Neoplastic/metabolism
- Gene Expression Regulation, Viral/genetics
- HIV-1/genetics
- HIV-1/metabolism
- Herpesviridae Infections/genetics
- Herpesviridae Infections/metabolism
- Herpesvirus 2, Saimiriine/genetics
- Herpesvirus 2, Saimiriine/metabolism
- Humans
- Lymphoma, T-Cell/genetics
- Lymphoma, T-Cell/metabolism
- Models, Biological
- Oncogene Proteins, Viral/biosynthesis
- Oncogene Proteins, Viral/genetics
- Tumor Virus Infections/genetics
- Tumor Virus Infections/metabolism
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Affiliation(s)
- Alexander Y Tsygankov
- Department of Microbiology and Immunology, Temple University School of Medicine, 3400 N. Broad Street, Philadelphia, PA 19104, USA.
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42
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Guang S, Mertz JE. Pre-mRNA processing enhancer (PPE) elements from intronless genes play additional roles in mRNA biogenesis than do ones from intron-containing genes. Nucleic Acids Res 2005; 33:2215-26. [PMID: 15843684 PMCID: PMC1083424 DOI: 10.1093/nar/gki506] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Most mRNA-encoding genes require introns for efficient expression in high eukaryotes. However, mRNAs can efficiently accumulate in the cytoplasm without intron excision if they contain cis-acting elements such as the post-transcriptional regulatory element (PRE) of hepatitis B virus (HBV), the constitutive transport element (CTE) of Mason–Pfizer monkey virus (MPMV), or the pre-mRNA processing enhancer (PPE) of herpes simplex virus' thymidine kinase (HSV-TK) gene. We compared the activities of these viral elements, the Rev-responsive element (RRE) of the human immunodeficiency virus (HIV), and the human c-Jun gene's enhancer (CJE), an element newly identified here, to enable expression of an intronless variant of the human β-globin gene. The PRE, PPE and CJE from naturally intronless genes, but not the CTE or RRE from intron-containing genes, significantly enhanced stability, 3′ end processing and cytoplasmic accumulation. When the transcripts included the β-globin gene's first intron, the PRE, PPE and CJE still enhanced mRNA biogenesis, in some cases without intron excision. Thus, elements enabling stability, 3′ end formation and nucleocytoplasmic export, not the presence of introns or their excision per se, are necessary for mRNA biogenesis. While the CTE and RRE primarily enhance nucleocytoplasmic export, PPE-like elements from naturally intronless genes facilitate polyadenylation as well.
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Affiliation(s)
| | - Janet E. Mertz
- To whom correspondence should be addressed. Tel: +1 608 262 2383; Fax: +1 608 262 2824;
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43
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O'Rourke JP, Olsen JC, Bunnell BA. Optimization of equine infectious anemia derived vectors for hematopoietic cell lineage gene transfer. Gene Ther 2005; 12:22-9. [PMID: 15550928 DOI: 10.1038/sj.gt.3302350] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Gene transfer into hematopoietic cells may allow correction of a variety of hematopoietic and metabolic disorders. Optimized HIV-1 based lentiviral vectors have been developed for improved gene transfer and transgene expression into hematopoietic cells. However, the use of HIV-1 based vectors for human gene therapy may be limited due to ethical and biosafety issues. We report that vectors based on the non-primate equine infectious anemia virus (EIAV) transduce a variety of human hematopoietic cell lines and primary blood cells. To investigate optimization of gene expression in hematopoietic cells, we compared a variety of post-transcriptional elements and promoters in the context of EIAV vectors. We observed cell specific increase in the number of transgene expressing cells with the different post-transcriptional elements, whereas the use of elongation factor alpha 1 (EFalpha1) promoter resulted in significant increases in both the number of transgene expressing cells and the level of transgene protein in all cell types tested. We then demonstrate increased transduction of hematopoietic cells using a second-generation EIAV vector containing a self-inactivating EIAV LTR and the EIAV central polypurine tract (cppt). These data suggest that optimized EIAV vectors may be a suitable alternative to HIV-1 vectors for use in hematopoietic gene therapy.
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Affiliation(s)
- J P O'Rourke
- Department of Molecular Medicine, Children's Research Institute, Columbus, OH, USA
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44
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LeBlanc JJ, Beemon KL. Unspliced Rous sarcoma virus genomic RNAs are translated and subjected to nonsense-mediated mRNA decay before packaging. J Virol 2004; 78:5139-46. [PMID: 15113896 PMCID: PMC400353 DOI: 10.1128/jvi.78.10.5139-5146.2004] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Retroviruses package full-length, unspliced RNAs into progeny virions as dimerized RNA genomes. They also use unspliced RNAs as mRNAs to produce the gag and pol gene products. We asked whether a single Rous sarcoma virus (RSV) RNA can be translated and subsequently packaged or whether genomic packaging requires a nontranslated population of RNAs. We addressed this issue by utilizing the translation-dependent nonsense-mediated mRNA decay (NMD) pathway. NMD is the selective destruction of mRNAs bearing premature termination codons (PTCs). The pathway has been shown to be associated with splicing in higher eukaryotes. Here, we demonstrate that both translation and the cellular factor Upf1 are required for the decay of unspliced, PTC-bearing RSV RNA by the NMD pathway. To address the relationship between RNA translation and packaging, we examined virus produced in cells cotransfected with PTC-bearing retroviral clones and wild-type viral clones. We observed that PTC-bearing transcripts are packaged into viral particles at levels three- to fivefold less than those of control RNAs. Since PTC-mediated degradation requires translation, we conclude that RSV can package progeny virion particles using previously translated RNAs.
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Affiliation(s)
- Jason J LeBlanc
- Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA
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Roberts TM, Boris-Lawrie K. Primary sequence and secondary structure motifs in spleen necrosis virus RU5 confer translational utilization of unspliced human immunodeficiency virus type 1 reporter RNA. J Virol 2003; 77:11973-84. [PMID: 14581534 PMCID: PMC254288 DOI: 10.1128/jvi.77.22.11973-11984.2003] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The 5' long terminal repeat (LTR) of spleen necrosis virus (SNV) contains a unique posttranscriptional control element that facilitates Rev/Rev-responsive element-independent expression of unspliced human immunodeficiency virus type 1 (HIV-1) gag reporter RNA. HIV-1 Gag expression is eliminated when SNV LTR is repositioned to the 3' untranslated region or when the RU5 region is positioned in the antisense orientation. RU5 corresponds to the 5' RNA terminus, and results presented here indicate that Gag production is sustained upon introduction of transcribed spacers that reposition SNV RU5 35 to 200 nucleotides downstream. Concordant results of deletion and point mutagenesis identified two functionally redundant and synergistic motifs (designated A and C) that are necessary and sufficient for SNV RU5 activity. Enzymatic analysis of SNV RU5 RNA structure determined that A and C correspond to stem-loop structures. Quantitative RNA and protein analysis of A and C mutants revealed that the structural integrity of A and C is necessary for protein production, and loss of function correlates with little change in steady-state level, splicing efficiency, or cytoplasmic accumulation of HIV-1 gag reporter RNA. Instead, the structural mutations eliminate cytoplasmic utilization as an mRNA template for Gag protein production. Point mutations of unpaired loop-and-bulge nucleotides that maintain the structure of A eliminate activity. The results show that the unpaired UUGU loop and U-rich bulges function together and are candidate SNV RU5 binding sites for the host cell protein(s) that directs cytoplasmic utilization of unspliced HIV-1 reporter RNA.
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Affiliation(s)
- Tiffiney M Roberts
- Center for Retrovirus Research, The Ohio State University, Columbus, Ohio 43210-1093, USA
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Muthumani K, Zhang D, Dayes NS, Hwang DS, Calarota SA, Choo AY, Boyer JD, Weiner DB. Novel engineered HIV-1 East African Clade-A gp160 plasmid construct induces strong humoral and cell-mediated immune responses in vivo. Virology 2003; 314:134-46. [PMID: 14517067 DOI: 10.1016/s0042-6822(03)00459-8] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
HIV-1 sequences are highly diverse due to the inaccuracy of the viral reverse transcriptase. This diversity has been studied and used to categorize HIV isolates into subtypes or clades, which are geographically distinct. To develop effective vaccines against HIV-1, immunogens representing different subtypes may be important for induction of cross-protective immunity, but little data exist describing and comparing the immunogenicity induced by different subtype-based vaccines. This issue is further complicated by poor expression of HIV structural antigens due to rev dependence. One costly approach is to codon optimize each subtype construct to be examined. Interestingly, cis-acting transcriptional elements (CTE) can also by pass rev restriction by a rev independent export pathway. We reasoned that rev+CTE constructs might have advantages for such expression studies. A subtype A envelope sequence from a viral isolate from east Africa was cloned into a eukaryotic expression vector under the control of the CMV-IE promoter. The utility of inclusion of the Mason-Pfizer monkey virus (MPV)-CTE with/without rev for driving envelope expression and immunogenicity was examined. Expression of envelope (gp120) was confirmed by immunoblot analysis and by pseudotype virus infectivity assays. The presence of rev and the CTE together increased envelope expression and viral infection. Furthermore the CTE+rev construct was significantly more immunogenic then CTE alone vector. Isotype analysis and cytokine profiles showed strong Th1 response in plasmid-immunized mice, which also demonstrated the superior nature of the rev+CTE construct. These responses were of similar or greater magnitude to a codon-optimized construct. The resulting cellular immune responses were highly cross-reactive with a HIV-1 envelope subtype B antigen. This study suggests a simple strategy for improving the expression and immunogenicity of HIV subtype-specific envelope antigens as plasmid or vector-borne immunogens.
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Affiliation(s)
- Karuppiah Muthumani
- Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
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Abstract
To maximize the production of progeny virions, several viruses have evolved mechanisms that promote the selective nuclear export of viral mRNA transcripts while, in some cases, inhibiting the export of cellular mRNAs. To achieve this goal, viruses have evolved regulatory proteins and cis-acting RNA elements that selectively interact with key cellular nuclear export factors. Efforts to identify the cellular targets of these viral proteins and RNA elements have led to the identification of Crm1 and Tap as essential human nuclear RNA-export factors and continue to provide insights into how mRNAs are selected for export
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Affiliation(s)
- Bryan R Cullen
- Howard Hughes Medical Institute and Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
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Suh D, Seguin B, Atkinson S, Ozdamar B, Staffa A, Emili A, Mouland A, Cochrane A. Mapping of determinants required for the function of the HIV-1 env nuclear retention sequence. Virology 2003; 310:85-99. [PMID: 12788633 DOI: 10.1016/s0042-6822(03)00073-4] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Control of HIV-1 RNA processing and transport are critical to the successful replication of the virus. In previous work, we identified a region within the HIV-1 env that is involved in mediating nuclear retention of unspliced viral RNA. To define this sequence further and identify elements required for function, deletion mutagenesis was carried out. Progressive 5' and 3' deletions map the nuclear retention sequence (NRS) within the intron between nts 8281 and 8381. While deletion of sequences comprising the 3'ss had no effect, removal of the 5'ss resulted in cytoplasmic accumulation of unspliced RNA. Sequence analysis determined that the region corresponding to the NRS is highly conserved among HIV-1 strains. To evaluate whether this NRS interacts with cellular factors, RNA electrophoretic mobility shift assays (REMSA) were performed. We show that the NRS specifically interacts with cellular factors present in HeLa nuclear extracts, and, by UV crosslinking, correlates with the binding of a 49-kDa protein. Immunoprecipitation of the UV crosslinked products determined that this 49-kDa protein corresponds to hnRNP C.
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Affiliation(s)
- Daniel Suh
- Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S-1A8, Canada
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O'Rourke JP, Hiraragi H, Urban K, Patel M, Olsen JC, Bunnell BA. Analysis of gene transfer and expression in skeletal muscle using enhanced EIAV lentivirus vectors. Mol Ther 2003; 7:632-9. [PMID: 12718906 DOI: 10.1016/s1525-0016(03)00074-1] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Skeletal muscle is an attractive target tissue for gene therapy involving both muscle and nonmuscle disorders. HIV-1-based vectors transduce mature skeletal muscle; however, the use of these vectors for human gene therapy may be limited by biosafety concerns. In this study, we investigated gene transfer using lentivirus vectors based on the equine infectious anemia virus (EIAV) in skeletal muscle in vitro and in vivo. EIAV vectors transduce proliferating and differentiating C2C12 mouse muscle cells; furthermore, the addition of the woodchuck hepatitis posttranscriptional element to EIAV vectors markedly increases gene expression in these cells. A single injection of EIAV vectors into skeletal muscle of adult mice led to detectable gene marking and gene expression for the duration of the 3-month study. Use of a second-generation EIAV self-inactivating vector (E-SIN) increased transduction in muscle cells in vitro, and injection of E-SIN vectors into skeletal muscle resulted in increased gene marking and gene expression compared to first-generation EIAV vectors.
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Affiliation(s)
- J P O'Rourke
- Department of Molecular Medicine, Children's Research Institute, Columbus, Ohio 43205, USA
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Abstract
SINEs and LINEs are short and long interspersed retrotransposable elements, respectively, that invade new genomic sites using RNA intermediates. SINEs and LINEs are found in almost all eukaryotes (although not in Saccharomyces cerevisiae) and together account for at least 34% of the human genome. The noncoding SINEs depend on reverse transcriptase and endonuclease functions encoded by partner LINEs. With the completion of many genome sequences, including our own, the database of SINEs and LINEs has taken a great leap forward. The new data pose new questions that can only be answered by detailed studies of the mechanism of retroposition. Current work ranges from the biochemistry of reverse transcription and integration invitro, target site selection in vivo, nucleocytoplasmic transport of the RNA and ribonucleoprotein intermediates, and mechanisms of genomic turnover. Two particularly exciting new ideas are that SINEs may help cells survive physiological stress, and that the evolution of SINEs and LINEs has been shaped by the forces of RNA interference. Taken together, these studies promise to explain the birth and death of SINEs and LINEs, and the contribution of these repetitive sequence families to the evolution of genomes.
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Affiliation(s)
- Alan M Weiner
- Department of Biochemistry, HSB J417, University of Washington, Box 357350, Seattle, WA 98195-7350, USA.
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