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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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FPV-T7-driven intracellular transcription and translation from transfected linear DNA templates of NSP2 and NSP5.COS-7 cells infected with FPV-T7 Pol for 1 hour and transfected with 1 µg of linear PCR amplicon encoding either RV segments 8 or 11 or EGFP. pcDNA3-NSP2 and pT7V-NSP5 were used as protein controls. Cells were incubated for 24 hours, prior to fixing and staining for RV proteins as previously described. Cells transfected with EGFP were only stained with Hoechst 33342. Samples were imaged using a fluorescence microscope. All panels were exposed to UV light, panels A, B, C, and D were exposed to blue and panels E and F were exposed to green wavelengths of light. Panel A: mock transfection; panel B: transfection with T7EGFP; panel C: transfection with pcDNA3-NSP2; panel D: transfection with T7RFS8; panel E: transfection with pT7V-NSP5; panel F: transfection with T7RFS11. Cell nuclei were stained with Hoechst 33342. Scale bars: 100 µm.
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pone-0074328-g009: FPV-T7-driven intracellular transcription and translation from transfected linear DNA templates of NSP2 and NSP5.COS-7 cells infected with FPV-T7 Pol for 1 hour and transfected with 1 µg of linear PCR amplicon encoding either RV segments 8 or 11 or EGFP. pcDNA3-NSP2 and pT7V-NSP5 were used as protein controls. Cells were incubated for 24 hours, prior to fixing and staining for RV proteins as previously described. Cells transfected with EGFP were only stained with Hoechst 33342. Samples were imaged using a fluorescence microscope. All panels were exposed to UV light, panels A, B, C, and D were exposed to blue and panels E and F were exposed to green wavelengths of light. Panel A: mock transfection; panel B: transfection with T7EGFP; panel C: transfection with pcDNA3-NSP2; panel D: transfection with T7RFS8; panel E: transfection with pT7V-NSP5; panel F: transfection with T7RFS11. Cell nuclei were stained with Hoechst 33342. Scale bars: 100 µm.

Mentions: Once we had validated the experimental protocol with the T7-eGFP amplicon, we examined whether intracellular transcription of RV positive sense RNAs from transfected cDNA could enhance protein expression, or produce infectious RV particles. We transfected cDNA templates of segments 11 (T7RF11) and segment 8 (T7RFS8) into COS-7 cells. Figure 9 shows the absence of detectable protein expression when linear DNA templates encoding either NSP2 (panel D) or NSP5 (panel F) were transfected. Several cells (<20 in total) in panel D (transfected with S8 [NSP2] cDNA) showed green fluorescence. However, this was also seen in the FPV-T7-only infected control (Panel A). We attributed this effect to non-specific binding of NSP2 antibody to apoptotic cells. These artifacts were not observed with the NSP5 specific antibody (panel F). When the cohort of 11 RV cDNAs was transfected into FPV-T7 infected cells, no infectious RV progeny was rescued (unpublished data). It was reasonable to assume that the cDNA amplicons were transfected successfully into cells and probably acted as templates for intracellular transcription, as eGFP was expressed from a transfected linear amplicon however the RV ssRNA transcripts were apparently not competent for efficient translation.


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)

FPV-T7-driven intracellular transcription and translation from transfected linear DNA templates of NSP2 and NSP5.COS-7 cells infected with FPV-T7 Pol for 1 hour and transfected with 1 µg of linear PCR amplicon encoding either RV segments 8 or 11 or EGFP. pcDNA3-NSP2 and pT7V-NSP5 were used as protein controls. Cells were incubated for 24 hours, prior to fixing and staining for RV proteins as previously described. Cells transfected with EGFP were only stained with Hoechst 33342. Samples were imaged using a fluorescence microscope. All panels were exposed to UV light, panels A, B, C, and D were exposed to blue and panels E and F were exposed to green wavelengths of light. Panel A: mock transfection; panel B: transfection with T7EGFP; panel C: transfection with pcDNA3-NSP2; panel D: transfection with T7RFS8; panel E: transfection with pT7V-NSP5; panel F: transfection with T7RFS11. Cell nuclei were stained with Hoechst 33342. Scale bars: 100 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760874&req=5

pone-0074328-g009: FPV-T7-driven intracellular transcription and translation from transfected linear DNA templates of NSP2 and NSP5.COS-7 cells infected with FPV-T7 Pol for 1 hour and transfected with 1 µg of linear PCR amplicon encoding either RV segments 8 or 11 or EGFP. pcDNA3-NSP2 and pT7V-NSP5 were used as protein controls. Cells were incubated for 24 hours, prior to fixing and staining for RV proteins as previously described. Cells transfected with EGFP were only stained with Hoechst 33342. Samples were imaged using a fluorescence microscope. All panels were exposed to UV light, panels A, B, C, and D were exposed to blue and panels E and F were exposed to green wavelengths of light. Panel A: mock transfection; panel B: transfection with T7EGFP; panel C: transfection with pcDNA3-NSP2; panel D: transfection with T7RFS8; panel E: transfection with pT7V-NSP5; panel F: transfection with T7RFS11. Cell nuclei were stained with Hoechst 33342. Scale bars: 100 µm.
Mentions: Once we had validated the experimental protocol with the T7-eGFP amplicon, we examined whether intracellular transcription of RV positive sense RNAs from transfected cDNA could enhance protein expression, or produce infectious RV particles. We transfected cDNA templates of segments 11 (T7RF11) and segment 8 (T7RFS8) into COS-7 cells. Figure 9 shows the absence of detectable protein expression when linear DNA templates encoding either NSP2 (panel D) or NSP5 (panel F) were transfected. Several cells (<20 in total) in panel D (transfected with S8 [NSP2] cDNA) showed green fluorescence. However, this was also seen in the FPV-T7-only infected control (Panel A). We attributed this effect to non-specific binding of NSP2 antibody to apoptotic cells. These artifacts were not observed with the NSP5 specific antibody (panel F). When the cohort of 11 RV cDNAs was transfected into FPV-T7 infected cells, no infectious RV progeny was rescued (unpublished data). It was reasonable to assume that the cDNA amplicons were transfected successfully into cells and probably acted as templates for intracellular transcription, as eGFP was expressed from a transfected linear amplicon however the RV ssRNA transcripts were apparently not competent for efficient translation.

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