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

2001 ◽  
Vol 75 (15) ◽  
pp. 6817-6824 ◽  
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.

scholarly journals Long Terminal Repeat U3 Length Polymorphism of Human Foamy Virus

Virology ◽  
1997 ◽  
Vol 230 (2) ◽  
pp. 167-178 ◽  
Author(s):  
Martina Schmidt ◽  
Ottmar Herchenröder ◽  
Jonathan Heeney ◽  
Axel Rethwilm
Keyword(s):  
Long Terminal Repeat ◽  
Length Polymorphism ◽  
Foamy Virus ◽  
Terminal Repeat ◽  
Human Foamy Virus

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

2017 ◽  
Vol 92 (1) ◽  
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.

scholarly journals 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

2002 ◽  
Vol 76 (20) ◽  
pp. 10211-10218 ◽  
Author(s):  
Stacey Hull ◽  
Kathleen Boris-Lawrie
Keyword(s):  
Long Terminal Repeat ◽  
Profile Analysis ◽  
Foamy Virus ◽  
Terminal Repeat ◽  
Posttranscriptional Control ◽  
Gag Protein ◽  
Cytoplasmic Expression ◽  
Virus Rna ◽  
Intron Removal ◽  
Responsive Element

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.

scholarly journals Latency of Viral Expression In Vivo Is Not Related to CpG Methylation in the U3 Region and Part of the R Region of the Long Terminal Repeat of Bovine Leukemia Virus

2003 ◽  
Vol 77 (7) ◽  
pp. 4423-4430 ◽  
Author(s):  
Shigeru Tajima ◽  
Masako Tsukamoto ◽  
Yoko Aida
Keyword(s):  
Long Terminal Repeat ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Cpg Methylation ◽  
Terminal Repeat ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Methylation Of Dna ◽  
R Region

ABSTRACT Bovine leukemia virus (BLV) is silent in most cells detectable in vivo, and the repression of its expression allows BLV to evade the host's immune response. In this study, we examined whether CpG methylation of DNA might be involved in the regulation of the expression of BLV in vivo. To investigate the effects of CpG methylation on the activity of the long terminal repeat (LTR) of BLV, we measured the transactivation activity of this region after treatment with the CpG methyltransferase SssI by using a luciferase reporter system. The activity of methylated LTR was significantly lower than that of nonmethylated LTR. Therefore, we examined the extent of CpG methylation of the U3 region and part of the R region of the LTR in BLV-infected cattle and in experimentally BLV-infected sheep at various clinical stages by the bisulfite genomic sequencing method. We detected no or minimal CpG methylation at all stages examined in cattle and sheep, and our results indicate that CpG methylation probably does not participate in the silencing of BLV in vivo.

scholarly journals Upstream Stimulatory Factors Binding to an E Box Motif in the R Region of the Bovine Leukemia Virus Long Terminal Repeat Stimulates Viral Gene Expression

2001 ◽  
Vol 277 (11) ◽  
pp. 8775-8789 ◽  
Author(s):  
Claire Calomme ◽  
Thi Liên-Anh Nguyên ◽  
Yvan de Launoit ◽  
Véronique Kiermer ◽  
Louis Droogmans ◽  
...  
Keyword(s):  
Gene Expression ◽  
Long Terminal Repeat ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Viral Gene Expression ◽  
Terminal Repeat ◽  
Viral Gene ◽  
E Box ◽  
R Region
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