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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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Agarose gel electrophoresis of the in vitro transcribed ssRNAs of the 11 RF RV segments.In vitro transcribed viral positive sense RNA, transcribed in the presence of a cap analogue from linear templates with a RE digested 3′ end. 200 ng of each ssRNA transcript was loaded onto the gel; 1.5% TBE AGE 80 V for 45 min. Lane R: RiboRuler™ High Range RNA markers (in bases); lanes 1–11: positive sense co-capped RV RF ssRNAs corresponding to segments 1 to 11, respectively.
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pone-0074328-g001: Agarose gel electrophoresis of the in vitro transcribed ssRNAs of the 11 RF RV segments.In vitro transcribed viral positive sense RNA, transcribed in the presence of a cap analogue from linear templates with a RE digested 3′ end. 200 ng of each ssRNA transcript was loaded onto the gel; 1.5% TBE AGE 80 V for 45 min. Lane R: RiboRuler™ High Range RNA markers (in bases); lanes 1–11: positive sense co-capped RV RF ssRNAs corresponding to segments 1 to 11, respectively.

Mentions: Transcription templates were digested with the appropriate restriction enzyme (RE) to define the 3′ end of the RV segment and then purified prior to in vitro transcription with T7 Pol. The resulting ssRNA transcripts were analysed by TBE-AGE as shown in Figure 1. Nine of 11 templates produced a unique ssRNA band of the appropriate size. Transcription products of segment 3 and 6 cDNA templates each migrated as double bands, one of the expected full length ssRNA and a second smaller transcript. The same dual product was seen for both segments when PCR amplicons were used as transcription templates (unpublished data), strongly suggesting that the shorter ssRNA had resulted from premature termination of transcription [33]. To ensure the bands were not gel-related artifacts, samples were separated by denaturing urea PAGE, and the same profile was seen (unpublished data). BLAST analyses did not detect intragenic sequences similar to the T7 Pol promoter or terminator sequences of segment 3 and segment 6 (unpublished data).


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)

Agarose gel electrophoresis of the in vitro transcribed ssRNAs of the 11 RF RV segments.In vitro transcribed viral positive sense RNA, transcribed in the presence of a cap analogue from linear templates with a RE digested 3′ end. 200 ng of each ssRNA transcript was loaded onto the gel; 1.5% TBE AGE 80 V for 45 min. Lane R: RiboRuler™ High Range RNA markers (in bases); lanes 1–11: positive sense co-capped RV RF ssRNAs corresponding to segments 1 to 11, respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760874&req=5

pone-0074328-g001: Agarose gel electrophoresis of the in vitro transcribed ssRNAs of the 11 RF RV segments.In vitro transcribed viral positive sense RNA, transcribed in the presence of a cap analogue from linear templates with a RE digested 3′ end. 200 ng of each ssRNA transcript was loaded onto the gel; 1.5% TBE AGE 80 V for 45 min. Lane R: RiboRuler™ High Range RNA markers (in bases); lanes 1–11: positive sense co-capped RV RF ssRNAs corresponding to segments 1 to 11, respectively.
Mentions: Transcription templates were digested with the appropriate restriction enzyme (RE) to define the 3′ end of the RV segment and then purified prior to in vitro transcription with T7 Pol. The resulting ssRNA transcripts were analysed by TBE-AGE as shown in Figure 1. Nine of 11 templates produced a unique ssRNA band of the appropriate size. Transcription products of segment 3 and 6 cDNA templates each migrated as double bands, one of the expected full length ssRNA and a second smaller transcript. The same dual product was seen for both segments when PCR amplicons were used as transcription templates (unpublished data), strongly suggesting that the shorter ssRNA had resulted from premature termination of transcription [33]. To ensure the bands were not gel-related artifacts, samples were separated by denaturing urea PAGE, and the same profile was seen (unpublished data). BLAST analyses did not detect intragenic sequences similar to the T7 Pol promoter or terminator sequences of segment 3 and segment 6 (unpublished data).

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