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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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In vitro translation of RV proteins from cohorts of cDNA-derived and DLP-derived ssRNAs.In vitro translation of RV DLP derived ssRNAs. 1 µg of capped DLP ssRNA was incubated in a RRL as described, electrophoresed on a 15% SDS-PAGE alongside PageRuler™ protein markers (in kDa) and exposed to X-ray film for 4 days. Lane 1: no ssRNA; lane 2: XEF ssRNA; lanes 3 & 4: S1–11 post-capped or co-capped ssRNA respectively; lane 5: RV ssRNAs from RV RF strain DLPs. The sizes of protein markers run alongside SDS-PAGE are indicated in kDa to the right hand side and were used to predict the location of RV proteins.
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pone-0074328-g005: In vitro translation of RV proteins from cohorts of cDNA-derived and DLP-derived ssRNAs.In vitro translation of RV DLP derived ssRNAs. 1 µg of capped DLP ssRNA was incubated in a RRL as described, electrophoresed on a 15% SDS-PAGE alongside PageRuler™ protein markers (in kDa) and exposed to X-ray film for 4 days. Lane 1: no ssRNA; lane 2: XEF ssRNA; lanes 3 & 4: S1–11 post-capped or co-capped ssRNA respectively; lane 5: RV ssRNAs from RV RF strain DLPs. The sizes of protein markers run alongside SDS-PAGE are indicated in kDa to the right hand side and were used to predict the location of RV proteins.

Mentions: We also tested the translation capacity of the DLP-derived ssRNAs of the strains we had used. We incubated DLP-derived transcripts and mixtures of all 11 segments from co-capped or post-capped ssRNAs of the RF strain, in RRL. Figure 5, lanes 3 and 4 demonstrate that cohorts of the post-capped ssRNAs are translated with greater efficiency than cohorts of co-capped RNAs. Comparison between lanes 3, 4 and 5 reveals highly similar protein translation profiles for the cDNA-derived and DLP-derived ssRNAs of the same strain (RF). While the presence of every RV protein would require complex Western blot analysis, the striking resemblance between the protein profiles of synthetically generated and DLP-derived ssRNA cohorts provides compelling evidence that the ssRNAs are functional templates for protein synthesis.


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)

In vitro translation of RV proteins from cohorts of cDNA-derived and DLP-derived ssRNAs.In vitro translation of RV DLP derived ssRNAs. 1 µg of capped DLP ssRNA was incubated in a RRL as described, electrophoresed on a 15% SDS-PAGE alongside PageRuler™ protein markers (in kDa) and exposed to X-ray film for 4 days. Lane 1: no ssRNA; lane 2: XEF ssRNA; lanes 3 & 4: S1–11 post-capped or co-capped ssRNA respectively; lane 5: RV ssRNAs from RV RF strain DLPs. The sizes of protein markers run alongside SDS-PAGE are indicated in kDa to the right hand side and were used to predict the location of RV proteins.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760874&req=5

pone-0074328-g005: In vitro translation of RV proteins from cohorts of cDNA-derived and DLP-derived ssRNAs.In vitro translation of RV DLP derived ssRNAs. 1 µg of capped DLP ssRNA was incubated in a RRL as described, electrophoresed on a 15% SDS-PAGE alongside PageRuler™ protein markers (in kDa) and exposed to X-ray film for 4 days. Lane 1: no ssRNA; lane 2: XEF ssRNA; lanes 3 & 4: S1–11 post-capped or co-capped ssRNA respectively; lane 5: RV ssRNAs from RV RF strain DLPs. The sizes of protein markers run alongside SDS-PAGE are indicated in kDa to the right hand side and were used to predict the location of RV proteins.
Mentions: We also tested the translation capacity of the DLP-derived ssRNAs of the strains we had used. We incubated DLP-derived transcripts and mixtures of all 11 segments from co-capped or post-capped ssRNAs of the RF strain, in RRL. Figure 5, lanes 3 and 4 demonstrate that cohorts of the post-capped ssRNAs are translated with greater efficiency than cohorts of co-capped RNAs. Comparison between lanes 3, 4 and 5 reveals highly similar protein translation profiles for the cDNA-derived and DLP-derived ssRNAs of the same strain (RF). While the presence of every RV protein would require complex Western blot analysis, the striking resemblance between the protein profiles of synthetically generated and DLP-derived ssRNA cohorts provides compelling evidence that the ssRNAs are functional templates for protein synthesis.

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