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Experimental pathways towards developing a rotavirus reverse genetics system: synthetic full length rotavirus ssRNAs are neither infectious nor translated in permissive cells.

Richards JE, Desselberger U, Lever AM - PLoS ONE (2013)

Bottom Line: Transfected RNAs were not infectious since a viral cytopathic effect was not observed after infection of MA104 cells with lysates from transfected cells.Co-expression of plasmids encoding NSP2 and NSP5 using a fowlpox T7 polymerase recombinant virus revealed viroplasm-like structure formation, but this did not enable the translation of transfected RV ssRNAs.Attempts to recover RV from ssRNAs transcribed intracellularly from transfected cDNAs were also unsuccessful and suggested that these RNAs were also not translated, in contrast to successful translation from a transfected cDNA encoding an eGFP mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom.

ABSTRACT
At present the ability to create rationally engineered mutant rotaviruses is limited because of the lack of a tractable helper virus-free reverse genetics system. Using the cell culture adapted bovine RV RF strain (G6P6 [1]), we have attempted to recover infectious RV by co-transfecting in vitro transcribed ssRNAs which are identical in sequence to the positive sense strand of each of the 11 dsRNA genomic segments of the RF strain. The RNAs were produced either from cDNAs cloned by a target sequence-independent procedure, or from purified double layered RV particles (DLPs). We have validated their translational function by in vitro synthesis of (35)S-labelled proteins in rabbit reticulocyte lysates; all 11 proteins encoded by the RV genome were expressed. Transfection experiments with DLP- or cDNA-derived ssRNAs suggested that the RNAs do not act independently as mRNAs for protein synthesis, once delivered into various mammalian cell lines, and exhibit cytotoxicity. Transfected RNAs were not infectious since a viral cytopathic effect was not observed after infection of MA104 cells with lysates from transfected cells. By contrast, an engineered mRNA encoding eGFP was expressed when transfected under identical conditions into the same cell lines. Co-expression of plasmids encoding NSP2 and NSP5 using a fowlpox T7 polymerase recombinant virus revealed viroplasm-like structure formation, but this did not enable the translation of transfected RV ssRNAs. Attempts to recover RV from ssRNAs transcribed intracellularly from transfected cDNAs were also unsuccessful and suggested that these RNAs were also not translated, in contrast to successful translation from a transfected cDNA encoding an eGFP mRNA.

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eGFP ssRNA species produced in vitro.ssRNAs were synthesised from PCR-derived amplicons with a T7 Pol promoter introduced at the 5′ end to facilitate in vitro transcription. PCR amplicons were digested with BsmBI to define the 3′ end of the transcription cassette. Templates, either 500 ng or 1 µg, were incubated with T7 Pol in the absence or presence of a cap analogue using MEGAscript® or Mmessage Mmachine®. Uncapped ssRNAs were purified and post-transcriptionally capped using ScriptCap™ m7G Capping System. ssRNA was polyadenylated using E. coli polyadenylation polymerase (ePAP) Ambion. Lane R: RiboRuler™ High Range; lanes 1–6: approximately 250 ng each of eGFP ssRNA; uncapped, post-capped, uncapped polyadenylated, post-capped polyadenylated, co-capped, co-capped polyadenylated, respectively. 1.5% TBE AGE 60 V for –90 min. The polyadenylated RNA bands are less sharp as the molecules differ in the numbers of A residues added at the 3′end.
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pone-0074328-g003: eGFP ssRNA species produced in vitro.ssRNAs were synthesised from PCR-derived amplicons with a T7 Pol promoter introduced at the 5′ end to facilitate in vitro transcription. PCR amplicons were digested with BsmBI to define the 3′ end of the transcription cassette. Templates, either 500 ng or 1 µg, were incubated with T7 Pol in the absence or presence of a cap analogue using MEGAscript® or Mmessage Mmachine®. Uncapped ssRNAs were purified and post-transcriptionally capped using ScriptCap™ m7G Capping System. ssRNA was polyadenylated using E. coli polyadenylation polymerase (ePAP) Ambion. Lane R: RiboRuler™ High Range; lanes 1–6: approximately 250 ng each of eGFP ssRNA; uncapped, post-capped, uncapped polyadenylated, post-capped polyadenylated, co-capped, co-capped polyadenylated, respectively. 1.5% TBE AGE 60 V for –90 min. The polyadenylated RNA bands are less sharp as the molecules differ in the numbers of A residues added at the 3′end.

Mentions: We used a commercial vector (pEGFP-N1; Clontech) to create an in vitro transcribed mRNA so that the autofluorescence of expressed enhanced GFP (eGFP) could be used as a marker for the efficiency of RNA transfection into mammalian cells. The requirement for a reporter ssRNA relying on conventional mammalian translation machinery rather than utilising RV UTRs was essential as it has been shown that a transfected luciferase reporter gene flanked with the UTR of gene 6 was only expressed efficiently in cells already infected with RV [37]. Furthermore a recent publication has demonstrated that ssRNA transfection of a chimeric mRNA also utilising RV segment 6 UTRs to control translation was less efficient than a globin control [38]. Using similar technology to that described above, the eGFP ORF was amplified using PCR with primers which introduced a T7 Pol promoter fused to the 5′ end of the transcriptional start point and a BsmBI site to define the 3′ end. BsmBI-digested T7 eGFP amplicons were used as a template for invitro transcription as described, and transcripts were additionally polyadenylated using E. coli poly (A) polymerase (Ambion) to resemble mammalian mRNAs. Samples of uncapped, post-transcriptionally capped (‘post-capped’) and co-transcriptionally capped (‘co-capped’) eGFP ssRNA were analysed by AGE (Figure 3, lanes 1, 2 and 5, respectively). Samples of uncapped, post-capped and co-capped polyadenylated eGFP ssRNAs were also analysed (Figure 3, lanes 3, 4 and 6, respectively). Figure 3 shows that uncapped, post-capped and co-capped ssRNAs all migrate at the same rate and that the three respective polyadenylated RNA species also co-migrated, although at an expected slower rate.


Experimental pathways towards developing a rotavirus reverse genetics system: synthetic full length rotavirus ssRNAs are neither infectious nor translated in permissive cells.

Richards JE, Desselberger U, Lever AM - PLoS ONE (2013)

eGFP ssRNA species produced in vitro.ssRNAs were synthesised from PCR-derived amplicons with a T7 Pol promoter introduced at the 5′ end to facilitate in vitro transcription. PCR amplicons were digested with BsmBI to define the 3′ end of the transcription cassette. Templates, either 500 ng or 1 µg, were incubated with T7 Pol in the absence or presence of a cap analogue using MEGAscript® or Mmessage Mmachine®. Uncapped ssRNAs were purified and post-transcriptionally capped using ScriptCap™ m7G Capping System. ssRNA was polyadenylated using E. coli polyadenylation polymerase (ePAP) Ambion. Lane R: RiboRuler™ High Range; lanes 1–6: approximately 250 ng each of eGFP ssRNA; uncapped, post-capped, uncapped polyadenylated, post-capped polyadenylated, co-capped, co-capped polyadenylated, respectively. 1.5% TBE AGE 60 V for –90 min. The polyadenylated RNA bands are less sharp as the molecules differ in the numbers of A residues added at the 3′end.
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Related In: Results  -  Collection

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pone-0074328-g003: eGFP ssRNA species produced in vitro.ssRNAs were synthesised from PCR-derived amplicons with a T7 Pol promoter introduced at the 5′ end to facilitate in vitro transcription. PCR amplicons were digested with BsmBI to define the 3′ end of the transcription cassette. Templates, either 500 ng or 1 µg, were incubated with T7 Pol in the absence or presence of a cap analogue using MEGAscript® or Mmessage Mmachine®. Uncapped ssRNAs were purified and post-transcriptionally capped using ScriptCap™ m7G Capping System. ssRNA was polyadenylated using E. coli polyadenylation polymerase (ePAP) Ambion. Lane R: RiboRuler™ High Range; lanes 1–6: approximately 250 ng each of eGFP ssRNA; uncapped, post-capped, uncapped polyadenylated, post-capped polyadenylated, co-capped, co-capped polyadenylated, respectively. 1.5% TBE AGE 60 V for –90 min. The polyadenylated RNA bands are less sharp as the molecules differ in the numbers of A residues added at the 3′end.
Mentions: We used a commercial vector (pEGFP-N1; Clontech) to create an in vitro transcribed mRNA so that the autofluorescence of expressed enhanced GFP (eGFP) could be used as a marker for the efficiency of RNA transfection into mammalian cells. The requirement for a reporter ssRNA relying on conventional mammalian translation machinery rather than utilising RV UTRs was essential as it has been shown that a transfected luciferase reporter gene flanked with the UTR of gene 6 was only expressed efficiently in cells already infected with RV [37]. Furthermore a recent publication has demonstrated that ssRNA transfection of a chimeric mRNA also utilising RV segment 6 UTRs to control translation was less efficient than a globin control [38]. Using similar technology to that described above, the eGFP ORF was amplified using PCR with primers which introduced a T7 Pol promoter fused to the 5′ end of the transcriptional start point and a BsmBI site to define the 3′ end. BsmBI-digested T7 eGFP amplicons were used as a template for invitro transcription as described, and transcripts were additionally polyadenylated using E. coli poly (A) polymerase (Ambion) to resemble mammalian mRNAs. Samples of uncapped, post-transcriptionally capped (‘post-capped’) and co-transcriptionally capped (‘co-capped’) eGFP ssRNA were analysed by AGE (Figure 3, lanes 1, 2 and 5, respectively). Samples of uncapped, post-capped and co-capped polyadenylated eGFP ssRNAs were also analysed (Figure 3, lanes 3, 4 and 6, respectively). Figure 3 shows that uncapped, post-capped and co-capped ssRNAs all migrate at the same rate and that the three respective polyadenylated RNA species also co-migrated, although at an expected slower rate.

Bottom Line: Transfected RNAs were not infectious since a viral cytopathic effect was not observed after infection of MA104 cells with lysates from transfected cells.Co-expression of plasmids encoding NSP2 and NSP5 using a fowlpox T7 polymerase recombinant virus revealed viroplasm-like structure formation, but this did not enable the translation of transfected RV ssRNAs.Attempts to recover RV from ssRNAs transcribed intracellularly from transfected cDNAs were also unsuccessful and suggested that these RNAs were also not translated, in contrast to successful translation from a transfected cDNA encoding an eGFP mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom.

ABSTRACT
At present the ability to create rationally engineered mutant rotaviruses is limited because of the lack of a tractable helper virus-free reverse genetics system. Using the cell culture adapted bovine RV RF strain (G6P6 [1]), we have attempted to recover infectious RV by co-transfecting in vitro transcribed ssRNAs which are identical in sequence to the positive sense strand of each of the 11 dsRNA genomic segments of the RF strain. The RNAs were produced either from cDNAs cloned by a target sequence-independent procedure, or from purified double layered RV particles (DLPs). We have validated their translational function by in vitro synthesis of (35)S-labelled proteins in rabbit reticulocyte lysates; all 11 proteins encoded by the RV genome were expressed. Transfection experiments with DLP- or cDNA-derived ssRNAs suggested that the RNAs do not act independently as mRNAs for protein synthesis, once delivered into various mammalian cell lines, and exhibit cytotoxicity. Transfected RNAs were not infectious since a viral cytopathic effect was not observed after infection of MA104 cells with lysates from transfected cells. By contrast, an engineered mRNA encoding eGFP was expressed when transfected under identical conditions into the same cell lines. Co-expression of plasmids encoding NSP2 and NSP5 using a fowlpox T7 polymerase recombinant virus revealed viroplasm-like structure formation, but this did not enable the translation of transfected RV ssRNAs. Attempts to recover RV from ssRNAs transcribed intracellularly from transfected cDNAs were also unsuccessful and suggested that these RNAs were also not translated, in contrast to successful translation from a transfected cDNA encoding an eGFP mRNA.

Show MeSH
Related in: MedlinePlus