RU5 of Mason-Pfizer Monkey Virus 5′ Long Terminal Repeat Enhances Cytoplasmic Expression of Human Immunodeficiency Virus Type 1 gag-pol and Nonviral Reporter RNA

ABSTRACT Retroviruses utilize an unspliced version of their primary transcription product as an RNA template for synthesis of viral Gag and Pol structural and enzymatic proteins. Cytoplasmic expression of the gag-pol RNA is achieved despite the lack of intron removal and the presence of a long and highly structured 5′ untranslated region that inhibits efficient ribosome scanning. In this study, we have identified for the first time that the 5′ long terminal repeat (LTR) of Mason-Pfizer monkey virus (MPMV) facilitates Rev/Rev-responsive element-independent expression of HIV-1 gag-pol reporter RNA. The MPMV RU5 region of the LTR is necessary and directs functional interaction with cellular posttranscriptional modulators present in human 293 and monkey COS cells but not in quail QT-6 cells and does not require any viral protein. Deletion of MPMV RU5 decreases the abundance of spliced mRNA but has little effect on cytoplasmic accumulation of unspliced gag-pol RNA despite complete elimination of detectable Gag protein production. MPMV RU5 also exerts a positive effect on the cytoplasmic expression of intronless luc RNA, and ribosomal profile analysis demonstrates that MPMV RU5 directs subcellular localization of the luc transcript to polyribosomes. Our findings have a number of similarities with those of reports on 5′ terminal posttranscriptional control elements in spleen necrosis virus and human foamy virus RNA and support the model that divergent retroviruses share 5′ terminal RNA elements that interact with host proteins to program retroviral RNA for productive cytoplasmic expression.

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Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential

Journal of Virology ◽

10.1128/jvi.01639-17 ◽

2017 ◽

Vol 92 (1) ◽

Cited By ~ 6

Author(s):

Michael Aaron Goodman ◽

Paritha Arumugam ◽

Devin Marie Pillis ◽

Anastacia Loberg ◽

Mohammed Nasimuzzaman ◽

...

Keyword(s):

Gene Therapy ◽

Long Terminal Repeat ◽

Transgene Expression ◽

Viral Vectors ◽

Foamy Virus ◽

Terminal Repeat ◽

Genotoxic Potential ◽

Binding Factor ◽

Activation Assay ◽

Lentivirus Vectors

ABSTRACTStrong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in “enhancerless” self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into theLMO2gene and then measuringLMO2expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay,LMO2expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required.IMPORTANCEUnderstanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy.

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The R Region Found in the Human Foamy Virus Long Terminal Repeat Is Critical for both Gag and Pol Protein Expression

Journal of Virology ◽

10.1128/jvi.75.15.6817-6824.2001 ◽

2001 ◽

Vol 75 (15) ◽

pp. 6817-6824 ◽

Cited By ~ 24

Author(s):

Rebecca A. Russell ◽

Yan Zeng ◽

Otto Erlwein ◽

Bryan R. Cullen ◽

Myra O. McClure

Keyword(s):

Protein Expression ◽

Long Terminal Repeat ◽

Splice Site ◽

Foamy Virus ◽

Noncoding Region ◽

Terminal Repeat ◽

Mrna Levels ◽

Human Foamy Virus ◽

Noncoding Sequence ◽

R Region

ABSTRACT It has been suggested that sequences located within the 5′ noncoding region of human foamy virus (HFV) are critical for expression of the viral Gag and Pol structural proteins. Here, we identify a discrete ∼151-nucleotide sequence, located within the R region of the HFV long terminal repeat, that activates HFV Gag and Pol expression when present in the 5′ noncoding region but that is inactive when inverted or when placed in the 3′ noncoding region. Sequences that are critical for the expression of both Gag and Pol include not only the 5′ splice site positioned at +51 in the R region, which is used to generate the spliced pol mRNA, but also intronic R sequences located well 3′ to this splice site. Analysis of total cellular gag andpol mRNA expression demonstrates that deletion of the R region has little effect on gag mRNA levels but that R deletions that would be predicted to leave thepol 5′ splice site intact nevertheless inhibit the production of the spliced pol mRNA. Gag expression can be largely rescued by the introduction of an intron into the 5′ noncoding sequence in place of the R region but not by an intron or any one of several distinct retroviral nuclear RNA export sequences inserted into the mRNA 3′ noncoding sequence. Neither the R element nor the introduced 5′ intron markedly affects the cytoplasmic level of HFV gag mRNA. The poor translational utilization of these cytoplasmic mRNAs when the R region is not present incis also extended to a cat indicator gene linked to an internal ribosome entry site introduced into the 3′ noncoding region. Together these data imply that the HFV R region acts in the nucleus to modify the cytoplasmic fate of target HFV mRNA. The close similarity between the role of the HFV R region revealed in this study and previous data (M. Butsch, S. Hull, Y. Wang, T. M. Roberts, and K. Boris-Lawrie, J. Virol. 73:4847–4855, 1999) demonstrating a critical role for the R region in activating gene expression in the unrelated retrovirus spleen necrosis virus suggests that several distinct retrovirus families may utilize a common yet novel mechanism for the posttranscriptional activation of viral structural protein expression.

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Increase in the basal transcriptional activity of the human foamy virus internal promoter by the homologous long terminal repeat promoter in cis

Nucleic Acids Research ◽

10.1093/nar/21.18.4226 ◽

1993 ◽

Vol 21 (18) ◽

pp. 4226-4230 ◽

Cited By ~ 19

Author(s):

Martin Loöchelt ◽

Mordechai Aboud ◽

Rolf M. Fluügel

Keyword(s):

Long Terminal Repeat ◽

Transcriptional Activity ◽

Foamy Virus ◽

Terminal Repeat ◽

Human Foamy Virus ◽

Long Terminal Repeat Promoter ◽

Internal Promoter ◽

In Cis ◽

Basal Transcriptional Activity

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BEL-1 Transactivator Responsive Sequences in the Long Terminal Repeat of Human Foamy Virus

Virology ◽

10.1006/viro.1993.1474 ◽

1993 ◽

Vol 196 (1) ◽

pp. 256-268 ◽

Cited By ~ 27

Author(s):

Otto Erlwein ◽

Axel Rethwilm

Keyword(s):

Long Terminal Repeat ◽

Foamy Virus ◽

Terminal Repeat ◽

Human Foamy Virus

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The 5′ Untranslated Region and Gag product of Idefix, a Long Terminal Repeat-Retrotransposon from Drosophila melanogaster, Act Together To Initiate a Switch between Translated and Untranslated States of the Genomic mRNA

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.8246-8254.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 8246-8254 ◽

Cited By ~ 14

Author(s):

Carine Meignin ◽

Jean-Luc Bailly ◽

Frédérick Arnaud ◽

Bernard Dastugue ◽

Chantal Vaury

Keyword(s):

Drosophila Melanogaster ◽

Long Terminal Repeat ◽

Untranslated Region ◽

Terminal Repeat ◽

Entry Site ◽

Independent Manner ◽

Gag Protein ◽

Genomic Arrangement

ABSTRACT Idefix is a long terminal repeat (LTR)-retrotransposon present in Drosophila melanogaster which shares similarities with vertebrates retroviruses both in its genomic arrangement and in the mechanism of transposition. Like in retroviruses, its two LTRs flank a long 5′ untranslated region (5′UTR) and three open reading frames referred to as the gag, pol, and env genes. Here we report that its 5′UTR, located upstream of the gag gene, can fold into highly structured domains that are known to be incompatible with efficient translation by ribosome scanning. Using dicistronic plasmids analyzed by both (i) in vitro transcription and translation in rabbit reticulocyte or wheat germ lysates and (ii) in vivo expression in transgenic flies, we show that the 5′UTR of Idefix exhibits an internal ribosome entry site (IRES) activity that is able to promote translation of a downstream cistron in a cap-independent manner. The functional state of this novel IRES depends on eukaryotic factors that are independent of their host origin. However, in vivo, its function can be down-regulated by trans-acting factors specific to tissues or developmental stages of its host. We identify one of these trans-acting factors as the Gag protein encoded by Idefix itself. Our data support a model in which nascent Gag is able to block translation initiated from the viral mRNA and thus its own translation. These data highlight the fact that LTR-retrotransposons may autoregulate their replication cycle through their Gag production.

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A Nucleocapsid Functionality Contained within the Amino Terminus of the Ty1 Protease That Is Distinct and Separable from Proteolytic Activity

Journal of Virology ◽

10.1128/jvi.76.1.346-354.2002 ◽

2002 ◽

Vol 76 (1) ◽

pp. 346-354 ◽

Cited By ~ 12

Author(s):

Joseph F. Lawler ◽

Gennady V. Merkulov ◽

Jef D. Boeke

Keyword(s):

Life Cycle ◽

Long Terminal Repeat ◽

Proteolytic Activity ◽

Reverse Transcription ◽

Protein S ◽

Terminal Repeat ◽

Amino Terminus ◽

Carboxy Terminus ◽

Gag Protein ◽

Virus Like Particles

ABSTRACT Ty1 is the most successful of the five endogenous yeast retrotransposons. The life cycle of Ty1 dictates that a number of nucleocapsid (NC)-facilitated events occur although the protein(s) responsible for these events has not been identified. The positioning of the NC peptide is conserved at the carboxy terminus of the Gag protein among most long terminal repeat (LTR)-containing retroelements. An analogous region of Ty1 that simultaneously encodes part of Gag, protease (PR), and the C-terminal p4 peptide was mutagenized. Some of these mutations result in smaller-than-normal virus-like particles (VLPs). The mutants were also found to impair an NC-like functionality contained within the amino terminus of the protease that is distinct and separable from its proteolytic activity. Remarkably, these mutants have distinct defects in reverse transcription.

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A 36-kilodalton cellular transcription factor mediates an indirect interaction of human T-cell leukemia/lymphoma virus type I TAX1 with a responsive element in the viral long terminal repeat

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.4192-4201.1990 ◽

1990 ◽

Vol 10 (8) ◽

pp. 4192-4201

Author(s):

S J Marriott ◽

P F Lindholm ◽

K M Brown ◽

S D Gitlin ◽

J F Duvall ◽

...

Keyword(s):

T Cell ◽

Long Terminal Repeat ◽

Virus Type ◽

Terminal Repeat ◽

Type I ◽

Indirect Interaction ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Human T Cell ◽

Responsive Element

The human T-cell leukemia/lymphoma virus type I (HTLV-I) trans activator, TAX1, interacts indirectly with a TAX1-responsive element, TRE-2, located at positions -117 to -163 in the viral long terminal repeat. This report describes the characterization of a 36-kilodalton (kDa) protein identified in HeLa nuclear extract which mediates the interaction of TAX1 with TRE-2. Purification of the protein was achieved by zinc chelate chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The renatured 36-kDa protein bound specifically to a TRE-2 oligonucleotide but not to nonfunctional base substitution mutant probes in a gel retardation assay. Renatured proteins of differing molecular weights were unable to form this complex. In addition, the 36-kDa protein specifically activated transcription from the HTLV-I promoter in vitro. Purified TAX1 protein formed a complex with the TRE-2 oligonucleotide in the presence of the 36-kDa protein, suggesting that indirect interaction of TAX1 with the viral long terminal repeat may be one of the mechanisms by which HTLV-I transcription is regulated.

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A Long Terminal Repeat-Containing Retrotransposon of Schizosaccharomyces pombe Expresses a Gag-Like Protein That Assembles into Virus-Like Particles Which Mediate Reverse Transcription

Journal of Virology ◽

10.1128/jvi.77.9.5451-5463.2003 ◽

2003 ◽

Vol 77 (9) ◽

pp. 5451-5463 ◽

Cited By ~ 19

Author(s):

Laure Teysset ◽

Van-Dinh Dang ◽

Min Kyung Kim ◽

Henry L. Levin

Keyword(s):

Electron Microscopy ◽

Long Terminal Repeat ◽

Schizosaccharomyces Pombe ◽

Reverse Transcription ◽

Terminal Repeat ◽

Terminal Deletion ◽

Rna Packaging ◽

Gag Protein ◽

Virus Like Particles ◽

Gag Proteins

ABSTRACT The Tf1 element of Schizosaccharomyces pombe is a long terminal repeat-containing retrotransposon that encodes functional protease, reverse transcriptase, and integrase proteins. Although these proteins are known to be necessary for protein processing, reverse transcription, and integration, respectively, the function of the protein thought to be Gag has not been determined. We present here the first electron microscopy of Tf1 particles. We tested whether the putative Gag of Tf1 was required for particle formation, packaging of RNA, and reverse transcription. We generated deletions of 10 amino acids in each of the four hydrophilic domains of the protein and found that all four mutations reduced transposition activity. The N-terminal deletion removed a nuclear localization signal and inhibited nuclear import of the transposon. The two mutations in the center of Gag destabilized the protein and resulted in no virus-like particles. The C-terminal deletion caused a defect in RNA packaging and, as a result, low levels of cDNA. The electron microscopy of cells expressing a truncated Tf1 showed that Gag alone was sufficient for the formation of virus-like particles. Taken together, these results indicate that Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses.

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