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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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Transfection of COS-7 cells with in vitro transcribed ssRNAs encoding RV ssRNAs and DLP derived ssRNAs.COS-7 cells at 80% confluence were transfected with ssRNAs encoding RV proteins using Mirus transfection reagent. Cells were fixed at 24 hours post transfection and stained with NSP2 and NSP5-specific antibodies (Table S4). Images were analysed by confocal microscopy. Panel A: transfection with cohorts of in vitro transcribed ssRNAs, 1 µg of S1– S11, post-capped (PC) or co-capped (CC) respectively, stained for both NSP2 and NSP5. Panel B: 1 µg of DLP derived ssRNAs from RV strains RF, 125 and 128 respectively, stained for both NSP2 and NSP5. Panel C: individual or co-transfection of 500 ng of ssRNAs S8, S11 or both stained for NSP2, NSP5 or both, respectively. Panel D: immunofluorescence of control MA104 cells infected with RF RV, stained for NSP2 and NSP5, transfection control with 1 µg of eGFP ssRNAs (autofluorescence), mock transfection stained for NSP2 and NSP5. Cell nuclei were stained with Hoechst 33342 in all panels. Scale bars: 20 µm.
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pone-0074328-g007: Transfection of COS-7 cells with in vitro transcribed ssRNAs encoding RV ssRNAs and DLP derived ssRNAs.COS-7 cells at 80% confluence were transfected with ssRNAs encoding RV proteins using Mirus transfection reagent. Cells were fixed at 24 hours post transfection and stained with NSP2 and NSP5-specific antibodies (Table S4). Images were analysed by confocal microscopy. Panel A: transfection with cohorts of in vitro transcribed ssRNAs, 1 µg of S1– S11, post-capped (PC) or co-capped (CC) respectively, stained for both NSP2 and NSP5. Panel B: 1 µg of DLP derived ssRNAs from RV strains RF, 125 and 128 respectively, stained for both NSP2 and NSP5. Panel C: individual or co-transfection of 500 ng of ssRNAs S8, S11 or both stained for NSP2, NSP5 or both, respectively. Panel D: immunofluorescence of control MA104 cells infected with RF RV, stained for NSP2 and NSP5, transfection control with 1 µg of eGFP ssRNAs (autofluorescence), mock transfection stained for NSP2 and NSP5. Cell nuclei were stained with Hoechst 33342 in all panels. Scale bars: 20 µm.

Mentions: We transfected COS-7, MA104 and 293T cells with cohorts of the in vitro transcribed ssRNAs in Opti-MEM I, using the Mirus transfection reagent. Cells were transfected with cohorts of ssRNAs, ranging in mass from 10 ng –2 µg (each segment representing 1/11th of the total mass) or equimolar concentrations of each ssRNA. Protein expression or nascent viral formation was sought. All three cells lines showed significant cytopathic effects after 24 hours, progressing to almost total cell destruction (60 - 80%) at day 3 post transfection. Media or frozen and thawed lysates from transfected cells were inoculated onto fresh MA104 monolayers to test for infectious virus but none was detected at 7 days. It was recently shown that BTV rescue was enhanced by double transfection of ssRNAs [45]; we thus transfected two cohorts (S1–S11) of RV ssRNA segments 16 hours apart. We hypothesised that, S1, S2, S3, S6, S8 and S11, encoding VP1, VP2, VP3, VP6, NSP2 and NSP5, respectively, might form the minimum essential group of proteins for initial viroplasm and DLP formation. The second transfection step contained all 11 ssRNAs. (We also added the SA11 RNA 3 to the RNAs of the RF RV strain in some experiments). Viable viral progeny was not detected (in MA104 cells) from the cell extracts of these experiments. Transfections (single or double) of cells with DLP-derived ssRNAs of five RV strains (RF, SA11, OSU, 125 and 128) were similarly unsuccessful (unpublished data). To ascertain whether RV ssRNA transcripts had actually entered cells, the cohort of S1–S11 ssRNAs was mixed with eGFP ssRNA and transfected as an ensemble of 12 ssRNA transcripts. eGFP expression was observed in many cells, however the supernatant from this experiment yielded no progeny virus (unpublished data). We hypothesised that the full length RV ssRNAs were not autonomously infectious, but it was unclear whether this was due to a lack of protein translation, potent innate immune responses, a high degree of cytotoxicity exerted by the transfected RNAs or a combination of more than one of these factors. To investigate this we transfected cells and sought RV protein expression using specific antibodies. Figure 7 shows the results for individual and ensemble ssRNA transfection experiments in COS-7 cells. Cells were transfected with varying amounts of a variety of ssRNAs, either cDNA- or DLP-derived, and incubated for 24 hours prior to fixing and staining with RV specific antibodies (for details see legend of Figure 7). There was no detectable NSP2 or NSP5 expression in any of the transfection experiments (and consequently no VLS formation). Similar observations were made in MA104 and 293T cells, VP1 expression was similarly undetectable (unpublished data). We then hypothesised that expressing RV proteins in cells prior to RV RNA transfection might be necessary to permit RV protein synthesis from ssRNAs under these conditions. Co-expression in trans of NSP2 and NSP5 six hours before, or simultaneously with, RV ssRNA transfection failed to yield infectious progeny virus (unpublished data). An MA104 cell line constitutively expressing NSP5-eGFP [46], was transfected with S8 ssRNA encoding NSP2, but we saw no VLS formation by immunofluorescence (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)

Transfection of COS-7 cells with in vitro transcribed ssRNAs encoding RV ssRNAs and DLP derived ssRNAs.COS-7 cells at 80% confluence were transfected with ssRNAs encoding RV proteins using Mirus transfection reagent. Cells were fixed at 24 hours post transfection and stained with NSP2 and NSP5-specific antibodies (Table S4). Images were analysed by confocal microscopy. Panel A: transfection with cohorts of in vitro transcribed ssRNAs, 1 µg of S1– S11, post-capped (PC) or co-capped (CC) respectively, stained for both NSP2 and NSP5. Panel B: 1 µg of DLP derived ssRNAs from RV strains RF, 125 and 128 respectively, stained for both NSP2 and NSP5. Panel C: individual or co-transfection of 500 ng of ssRNAs S8, S11 or both stained for NSP2, NSP5 or both, respectively. Panel D: immunofluorescence of control MA104 cells infected with RF RV, stained for NSP2 and NSP5, transfection control with 1 µg of eGFP ssRNAs (autofluorescence), mock transfection stained for NSP2 and NSP5. Cell nuclei were stained with Hoechst 33342 in all panels. Scale bars: 20 µm.
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Related In: Results  -  Collection

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pone-0074328-g007: Transfection of COS-7 cells with in vitro transcribed ssRNAs encoding RV ssRNAs and DLP derived ssRNAs.COS-7 cells at 80% confluence were transfected with ssRNAs encoding RV proteins using Mirus transfection reagent. Cells were fixed at 24 hours post transfection and stained with NSP2 and NSP5-specific antibodies (Table S4). Images were analysed by confocal microscopy. Panel A: transfection with cohorts of in vitro transcribed ssRNAs, 1 µg of S1– S11, post-capped (PC) or co-capped (CC) respectively, stained for both NSP2 and NSP5. Panel B: 1 µg of DLP derived ssRNAs from RV strains RF, 125 and 128 respectively, stained for both NSP2 and NSP5. Panel C: individual or co-transfection of 500 ng of ssRNAs S8, S11 or both stained for NSP2, NSP5 or both, respectively. Panel D: immunofluorescence of control MA104 cells infected with RF RV, stained for NSP2 and NSP5, transfection control with 1 µg of eGFP ssRNAs (autofluorescence), mock transfection stained for NSP2 and NSP5. Cell nuclei were stained with Hoechst 33342 in all panels. Scale bars: 20 µm.
Mentions: We transfected COS-7, MA104 and 293T cells with cohorts of the in vitro transcribed ssRNAs in Opti-MEM I, using the Mirus transfection reagent. Cells were transfected with cohorts of ssRNAs, ranging in mass from 10 ng –2 µg (each segment representing 1/11th of the total mass) or equimolar concentrations of each ssRNA. Protein expression or nascent viral formation was sought. All three cells lines showed significant cytopathic effects after 24 hours, progressing to almost total cell destruction (60 - 80%) at day 3 post transfection. Media or frozen and thawed lysates from transfected cells were inoculated onto fresh MA104 monolayers to test for infectious virus but none was detected at 7 days. It was recently shown that BTV rescue was enhanced by double transfection of ssRNAs [45]; we thus transfected two cohorts (S1–S11) of RV ssRNA segments 16 hours apart. We hypothesised that, S1, S2, S3, S6, S8 and S11, encoding VP1, VP2, VP3, VP6, NSP2 and NSP5, respectively, might form the minimum essential group of proteins for initial viroplasm and DLP formation. The second transfection step contained all 11 ssRNAs. (We also added the SA11 RNA 3 to the RNAs of the RF RV strain in some experiments). Viable viral progeny was not detected (in MA104 cells) from the cell extracts of these experiments. Transfections (single or double) of cells with DLP-derived ssRNAs of five RV strains (RF, SA11, OSU, 125 and 128) were similarly unsuccessful (unpublished data). To ascertain whether RV ssRNA transcripts had actually entered cells, the cohort of S1–S11 ssRNAs was mixed with eGFP ssRNA and transfected as an ensemble of 12 ssRNA transcripts. eGFP expression was observed in many cells, however the supernatant from this experiment yielded no progeny virus (unpublished data). We hypothesised that the full length RV ssRNAs were not autonomously infectious, but it was unclear whether this was due to a lack of protein translation, potent innate immune responses, a high degree of cytotoxicity exerted by the transfected RNAs or a combination of more than one of these factors. To investigate this we transfected cells and sought RV protein expression using specific antibodies. Figure 7 shows the results for individual and ensemble ssRNA transfection experiments in COS-7 cells. Cells were transfected with varying amounts of a variety of ssRNAs, either cDNA- or DLP-derived, and incubated for 24 hours prior to fixing and staining with RV specific antibodies (for details see legend of Figure 7). There was no detectable NSP2 or NSP5 expression in any of the transfection experiments (and consequently no VLS formation). Similar observations were made in MA104 and 293T cells, VP1 expression was similarly undetectable (unpublished data). We then hypothesised that expressing RV proteins in cells prior to RV RNA transfection might be necessary to permit RV protein synthesis from ssRNAs under these conditions. Co-expression in trans of NSP2 and NSP5 six hours before, or simultaneously with, RV ssRNA transfection failed to yield infectious progeny virus (unpublished data). An MA104 cell line constitutively expressing NSP5-eGFP [46], was transfected with S8 ssRNA encoding NSP2, but we saw no VLS formation by immunofluorescence (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