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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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Viroplasm-like structure (VLS) formation.MA104 cells were infected with FPV-T7 for one hour before co-transfection with pT7V-NSP2 and pT7V-NSP5 plasmids using Lipofectamine 2000. Cells were fixed at 24 hours post transfection and stained for NSP2 and NSP5. Cell nuclei were stained with Hoechst 33342. VLS and mock panels are merged images of UV and the other two composite excitations for NSP2 or NSP5. Images were visualised by confocal microscopy. Arrows indicate VLS and location of enlarged inset images. Scale bars: 20 µm.
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pone-0074328-g010: Viroplasm-like structure (VLS) formation.MA104 cells were infected with FPV-T7 for one hour before co-transfection with pT7V-NSP2 and pT7V-NSP5 plasmids using Lipofectamine 2000. Cells were fixed at 24 hours post transfection and stained for NSP2 and NSP5. Cell nuclei were stained with Hoechst 33342. VLS and mock panels are merged images of UV and the other two composite excitations for NSP2 or NSP5. Images were visualised by confocal microscopy. Arrows indicate VLS and location of enlarged inset images. Scale bars: 20 µm.

Mentions: We reasoned that the creation of pre-existing VLS from co-expression of plasmids encoding NSP2 and NSP5, under the control of T7 Pol promoter, might create a cellular environment more favourable to RV protein translation from ssRNAs. We tested this hypothesis in MA104 cells where VLS were formed (Figure 10). Subsequent transfection with the cohort of ssRNAs or just segment 1 alone did not yield protein expression or rescue infectious virus. VLS were also formed in COS-7 and Caco-2 cells but upon RV ssRNA transfection we did not observe corresponding RV protein expression or rescue of infectious virus (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)

Viroplasm-like structure (VLS) formation.MA104 cells were infected with FPV-T7 for one hour before co-transfection with pT7V-NSP2 and pT7V-NSP5 plasmids using Lipofectamine 2000. Cells were fixed at 24 hours post transfection and stained for NSP2 and NSP5. Cell nuclei were stained with Hoechst 33342. VLS and mock panels are merged images of UV and the other two composite excitations for NSP2 or NSP5. Images were visualised by confocal microscopy. Arrows indicate VLS and location of enlarged inset images. Scale bars: 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0074328-g010: Viroplasm-like structure (VLS) formation.MA104 cells were infected with FPV-T7 for one hour before co-transfection with pT7V-NSP2 and pT7V-NSP5 plasmids using Lipofectamine 2000. Cells were fixed at 24 hours post transfection and stained for NSP2 and NSP5. Cell nuclei were stained with Hoechst 33342. VLS and mock panels are merged images of UV and the other two composite excitations for NSP2 or NSP5. Images were visualised by confocal microscopy. Arrows indicate VLS and location of enlarged inset images. Scale bars: 20 µm.
Mentions: We reasoned that the creation of pre-existing VLS from co-expression of plasmids encoding NSP2 and NSP5, under the control of T7 Pol promoter, might create a cellular environment more favourable to RV protein translation from ssRNAs. We tested this hypothesis in MA104 cells where VLS were formed (Figure 10). Subsequent transfection with the cohort of ssRNAs or just segment 1 alone did not yield protein expression or rescue infectious virus. VLS were also formed in COS-7 and Caco-2 cells but upon RV ssRNA transfection we did not observe corresponding RV protein expression or rescue of infectious virus (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