Engineering the splice acceptor for improved gene expression and viral titer in an MLV-based retroviral vector

Jt, Lee; Yu, Shiying; Han, Eunyoung; Kim, S

doi:10.1038/sj.gt.3302138

Cited by 17 publications

(11 citation statements)

References 30 publications

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“…pEQ273 contains the full genomic sequence of IE1, including the HCMV major immediate-early promoter, while pEQ336 having only the promoter and lacking the coding sequence as described (Biegalke and Geballe, 1991). The retroviral vector, MT5-cIE1, was constructed by inserting the cDNA sequence of IE1 into retroviral vector MT5 (Lee et al, 2004). The reporter plasmid, pCN-chloramphenicol acetyl transferase (CAT), were constructed by inserting the CAT gene into expression vector pCN (Lee et al, 2000).…”

Section: Materials and Methods Cell Cultures And Plasmidsmentioning

confidence: 99%

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Downregulation of GFAP, TSP‐1, and p53 in human glioblastoma cell line, U373MG, by IE1 protein from human cytomegalovirus

Lee

Jeon

Kim

et al. 2005

Glia

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Human cytomegalovirus (HCMV) is a member of the b-herpesvirus family, which has tropism for glial cells. It was recently reported that HCMV might play important roles in the pathogenesis of malignant glioma. In this study, we investigated the effects of the HCMV IE1 protein on the gene expression profile in the human glioblastoma cell line, U373MG by employing cDNA microarray technology. Using DNA chips containing approximately 1,000 human cDNAs, RNA samples from U373MG cells stably expressing IE1 were compared with those from the control cells lacking IE1 cDNA. Fluorescence intensities of 13 genes were significantly decreased in IE1-expressing cells, while one gene was found to be upregulated. Among these 14 genes, we chose to work further on glial fibrillary acidic protein (GFAP), thrombospondin-1 (TSP-1), and p53, because of their previously known involvement in tumorigenesis. The mRNA levels of all these genes were found to be decreased in IE1-expressing glioblastoma cells by real-time quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) as well as Northern blot analysis. The decreased expression of these genes was also observed at protein levels as measured by immunocytochemistry or fluorescence-activated cell sorting (FACS) analysis. Our data strongly suggested that HCMV IE1 could modulate the expression of cellular genes that might play important roles in the pathogenesis of glial tumors. '

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Section: Materials and Methods Cell Cultures And Plasmidsmentioning

confidence: 99%

“…6A). The cDNA sequence of IE1 was inserted into retroviral vector MT5 (Lee et al, 2004). Cell-free retroviral vectors were prepared by the three-plasmid transfection method and used to transduce U373MG cells.…”

Section: Effects Of Transient Expression Of Ie1 Proteinmentioning

confidence: 99%

Downregulation of GFAP, TSP‐1, and p53 in human glioblastoma cell line, U373MG, by IE1 protein from human cytomegalovirus

Lee

Jeon

Kim

et al. 2005

Glia

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“…21 Briefly, an HIV-1-LTR-MazF-polyA cassette was introduced in the direction opposite of the MoMLV-LTR at the multiple cloning site of the retroviral vector plasmid pMT. 36 The Δ LNGFR gene 37 was introduced into the retrovirus vector as a surface marker. The Δ LNGFR gene is under the control of the human phosphoglycerate kinase (PGK) promoter.…”

Section: Methodsmentioning

confidence: 99%

CD4+ T Cells Modified by the Endoribonuclease MazF Are Safe and Can Persist in SHIV-infected Rhesus Macaques

Saito

Chono

Shibata

et al. 2014

Molecular Therapy - Nucleic Acids

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MazF, an endoribonuclease encoded by Escherichia coli, specifically cleaves the ACA (adenine–cytosine–adenine) sequence of single-stranded RNAs. Conditional expression of MazF under the control of the HIV-1 LTR promoter rendered CD4+ T cells resistant to HIV-1 replication without affecting cell growth. To investigate the safety, persistence and efficacy of MazF-modified CD4+ T cells in a nonhuman primate model in vivo, rhesus macaques were infected with a pathogenic simian/human immunodeficiency virus (SHIV) and transplanted with autologous MazF-modified CD4+ T cells. MazF-modified CD4+ T cells were clearly detected throughout the experimental period of more than 6 months. The CD4+ T cell count values increased in all four rhesus macaques. Moreover, the transplantation of the MazF-modified CD4+ T cells was not immunogenic, and did not elicit cellular or humoral immune responses. These data suggest that the autologous transplantation of MazF-modified CD4+ T cells in the presence of SHIV is effective, safe and not immunogenic, indicating that this is an attractive strategy for HIV-1 gene therapy.

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“…Such a relatively strong PPT can substitute for either a weak branch point or poor sequence conservation of the actual 3Јss (35). Moreover, introducing a very efficient cellular 3Јss into MLV vectors did not fully prevent the formation of genomic RNA (15). Finally, when constructing a new generation of retroviral self-inactivating vectors, we placed the internal promoter 18 bp downstream of the PBS and thus 24 bp upstream of the 5Јss.…”

Section: Discussionmentioning

confidence: 99%

“…The polypyrimidine tract (PPT), a second key feature of all 3Јss, is of suboptimal length (10 nucleotides long when compared with 13 residues in average) but not interrupted by weakening purines as most PPTs of HIV (14). Interestingly, a recent study showed that in the context of MLV-based retroviral vectors, the 3Јss can be replaced by very efficient counterparts derived from the human EF1␣ gene (15). Although the infectious titer was reduced by about 1 order of magnitude, unspliced genomic RNA was still formed, arguing for the existence of additional splice inhibitory sequences.…”

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confidence: 99%

Murine Leukemia Virus Regulates Alternative Splicing through Sequences Upstream of the 5· Splice Site

Kraunus

Zychlinski

Heise

et al. 2006

Journal of Biological Chemistry

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Alternative splicing of the primary transcript plays a key role in retroviral gene expression. In contrast to all known mechanisms that mediate alternative splicing in retroviruses, we found that in murine leukemia virus, distinct elements located upstream of the 5 splice site either inhibited or activated splicing of the genomic RNA. Detailed analysis of the first untranslated exon showed that the primer binding site (PBS) activates splicing, whereas flanking sequences either downstream or upstream of the PBS are inhibitory. This new function of the PBS was independent of its orientation and primer binding but associated with a particular destabilizing role in a proposed secondary structure. On the contrary, all sequences surrounding the PBS that are involved in stem formation of the first exon were found to suppress splicing. Targeted mutations that destabilized the central stem and compensatory mutations of the counter strand clearly validated the concept that murine leukemia virus attenuates its 5 splice site by forming an inhibitory stem-loop in its first exon. Importantly, this mode of splice regulation was conserved in a complete proviral clone. Some of the mutants that increase splicing revealed an opposite effect on translation, implying that the first exon also regulates this process. Together, these findings suggest that sequences upstream of the 5 splice site play an important role in splice regulation of simple retroviruses, directly or indirectly attenuating the efficiency of splicing.A characteristic feature of all retroviruses is the process of reverse transcription of the RNA genome into double-stranded DNA. Following integration into the host genome, the proviral DNA functions as one expression unit, which is transcribed by cellular RNA polymerase II, yielding a single polycistronic primary transcript that serves as genomic RNA for progeny virus. Productive infection and formation of new retroviral particles require the well balanced expression of all viral genes. This is accomplished by a combination of alternative splicing (intron retention) and regulated nuclear export of the primary transcript on the RNA processing level and proteolytic cleavage and translational read-through on the post-translational level (reviewed in Refs. 1-4).The genomic organization of all retroviruses is similar (Fig. 1A). The gag-pol open reading frame (ORF) 3 encoding the inner structural proteins (Gag), and the replication enzymes (Pol) is located in the 5Ј half of the transcript and expressed from the unspliced genomic RNA after nuclear export. The gag-pol ORF in all primary retroviral transcripts is defined as an intron through the presence of a preceding 5Ј splice site (ss) in the 5Ј-untranslated region and a functional 3Јss located toward the end of the polymerase ORF (Fig. 1A). To express the glycoproteins (Env), which are encoded in the 3Ј half of the genomic RNA, the gag-pol ORF is removed by a single splice event for subsequent export of the fully spliced RNA (1, 3, 4). This onesplice event strategy cre...

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Engineering the splice acceptor for improved gene expression and viral titer in an MLV-based retroviral vector

Cited by 17 publications

References 30 publications

Downregulation of GFAP, TSP‐1, and p53 in human glioblastoma cell line, U373MG, by IE1 protein from human cytomegalovirus

Downregulation of GFAP, TSP‐1, and p53 in human glioblastoma cell line, U373MG, by IE1 protein from human cytomegalovirus

CD4+ T Cells Modified by the Endoribonuclease MazF Are Safe and Can Persist in SHIV-infected Rhesus Macaques

Murine Leukemia Virus Regulates Alternative Splicing through Sequences Upstream of the 5· Splice Site

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