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Simian virus 40 strains with novel properties generated by replacing the viral enhancer with synthetic oligonucleotides.

ABSTRACT: Typical enhancers of viral or cellular genes are approximately 100 to 400 bp long and contain several transcription factor binding sites. Previously, we have shown that simian virus 40 (SV40) genomic DNA that lacks its own enhancer can be used as an "enhancer trap" since it reacquires infectivity upon incorporation of heterologous enhancers. Here, we show that SV40 infectivity can be restored with synthetic enhancers that are assembled by the host cell. We found that several oligonucleotides, cotransfected with enhancerless SV40 DNA into host cells, were incorporated into the viral genome via cellular DNA end joining. The oligonucleotides tested included metal response elements (MREs), the binding sites for the transcription factor MTF-1, which induces gene activity in response to heavy metals. These recombinant SV40 strains showed preferential growth on cells overloaded with zinc or cadmium. We also cotransfected enhancerless SV40 DNA with oligonucleotides corresponding to enhancer motifs of human and mouse cytomegalovirus (HCMV and MCMV, respectively). In contrast to SV40 wild type, the viruses with cytomegalovirus-derived patchwork enhancers strongly expressed T-antigen in human HEK293 cells, accompanied by viral DNA replication. Occasionally, we also observed the assembly of functional viral genomes by incorporation of fragments of bovine DNA, an ingredient of the fetal calf serum in the medium. These fragments contained, among other sites, binding sites for AP-1 and CREB transcription factors. Taken together, our studies show that viruses with novel properties can be generated by intracellular incorporation of synthetic enhancer DNA motifs.

SUBMITTER: Gunther V

PROVIDER: S-EPMC3302306 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Simian virus 40 strains with novel properties generated by replacing the viral enhancer with synthetic oligonucleotides.

Günther Viola V Strassen Till T Lindert Uschi U Dagani Patrizia P Waldvogel Dominique D Georgiev Oleg O Schaffner Walter W Bethge Tobias T

Journal of virology 20120111 6

Typical enhancers of viral or cellular genes are approximately 100 to 400 bp long and contain several transcription factor binding sites. Previously, we have shown that simian virus 40 (SV40) genomic DNA that lacks its own enhancer can be used as an "enhancer trap" since it reacquires infectivity upon incorporation of heterologous enhancers. Here, we show that SV40 infectivity can be restored with synthetic enhancers that are assembled by the host cell. We found that several oligonucleotides, co ...[more]

PMID: 22238322

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Project description:The transcription activity of the tumor-specific promoter may be increased using specific DNA sequences such as simian virus 40 (SV40). Human heparanase (HPSE) gene promoter is also considered a tumor-specific promoter. However, whether or not the SV40 enhancer affects the tumor specificity of HPSE remains to be determined. The SV40 enhancer sequence, 237 bp in length, was amplified and correctly inserted into the assigned multiple clone sites (MCS) of the eukaryotic expression vector pEGFP-Hp, which was constructed in advance. The recombinant plasmid pEGFP-Hp-SV40e was consistent with the anticipated Genbank data and transfected into human umbilical vein endothelial cell (ECV) and tumor cell lines, including hepatoma carcinoma (HepG2), laryngeal carcinoma (Hep2) and chronic myelogenous leukemia cell lines (K562) using lipofectamine, respectively. The expression of the reporter gene, green fluorescent protein (GFP), was detected using fluorescence microscopy and flow cytometry. The length of the amplified SV40 enchancer was 237 bp and the sequence was in accordance with the GenBank data. The recombinant plasmid pEGFP-Hp-SV40 was consistent with the anticipated results. Fluorimetric analysis showed that the fluorescence of pEGFP-Hp-SV40e in ECV cells was as dim as pEGFP-Hp, and obviously weaker than pEGFPN1. In tumor cells including HepG2, Hep2 and K562 cells, the fluorescence of pEGFP-Hp-SV40e was similar to that of pEGFP-N1, which was clearly brighter than pEGFP-Hp. The average transfecion rates in the 4 types of cells were 4.1, 17.2, 8.8 and 6.4% in the pEGFP-Hp; 18.3, 29.3, 17.0 and 13.0% in the pEGFP-Nl and 4.3, 28.8, 16.4 and 11.7% in the pEGFP-Hp-SV40e groups, respectively. The ratio of pEGFP-Hp-SV40e to pEGFP-Hp in all cells was 1.05, 1.67, 1.86 and 1.83, respectively. In conclusion, the inserted SV40 enhancer sequence is able to improve the transcriptional activity of the human HPSE gene promoter, but does not affect its tumor specificity.

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Simian virus 40 strains with novel properties generated by replacing the viral enhancer with synthetic oligonucleotides.

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Simian virus 40 strains with novel properties generated by replacing the viral enhancer with synthetic oligonucleotides.

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