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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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ssRNA derived from several strains of RV DLPs.ssRNA were synthesized in vitro from purified RV DLPs. DLPs were incubated at 37°C for 2 hours with the necessary components for positive sense ssRNA synthesis. 1.5% TBE AGE 45 V for 120 min. Lane R: RiboRuler™ High Range (in bases); 1.5 µg ssRNA derived from DLPs of RV strains SA11, OSU, RF, 125 and 128, respectively. DLP-derived ssRNAs 2 and 3 comigrate in all samples., ssRNAS 7–9 comigrate in SA11, OSU and RF, but only ssRNAS 7 and 9 comigrate in 125 and 128. Arrows indicate the altered migration of ssRNAs synthesised from the templates of rearranged genomic segments (RS). RS8, RS11 rearranged segment 8 or 11, respectively, of RV strains 125 and 128.
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pone-0074328-g002: ssRNA derived from several strains of RV DLPs.ssRNA were synthesized in vitro from purified RV DLPs. DLPs were incubated at 37°C for 2 hours with the necessary components for positive sense ssRNA synthesis. 1.5% TBE AGE 45 V for 120 min. Lane R: RiboRuler™ High Range (in bases); 1.5 µg ssRNA derived from DLPs of RV strains SA11, OSU, RF, 125 and 128, respectively. DLP-derived ssRNAs 2 and 3 comigrate in all samples., ssRNAS 7–9 comigrate in SA11, OSU and RF, but only ssRNAS 7 and 9 comigrate in 125 and 128. Arrows indicate the altered migration of ssRNAs synthesised from the templates of rearranged genomic segments (RS). RS8, RS11 rearranged segment 8 or 11, respectively, of RV strains 125 and 128.

Mentions: Purified RV DLPs can transcribe positive ssRNA in vitro from the dsRNA genomic segments packaged in the particles [34], [35], [36]. RNAs extruding from DLPs are positive sense and should possess authentic in vivo structures to facilitate experiments aimed at recovering infectious RV. DLPs of five different RV strains were used; the yields of ssRNA were between 3 and 10 µg of purified ssRNA per µg of purified DLPs. Figure 2 shows the successful synthesis of ssRNAs from the DLPs of all strains used alongside a ssRNA marker. The AGE migration patterns in Figure 2, lanes 2–6 reveal the genome structure of each strain. Thus, it was possible to identify ssRNAs derived from DLPs containing rearranged genomes (lanes 4 and 5).


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)

ssRNA derived from several strains of RV DLPs.ssRNA were synthesized in vitro from purified RV DLPs. DLPs were incubated at 37°C for 2 hours with the necessary components for positive sense ssRNA synthesis. 1.5% TBE AGE 45 V for 120 min. Lane R: RiboRuler™ High Range (in bases); 1.5 µg ssRNA derived from DLPs of RV strains SA11, OSU, RF, 125 and 128, respectively. DLP-derived ssRNAs 2 and 3 comigrate in all samples., ssRNAS 7–9 comigrate in SA11, OSU and RF, but only ssRNAS 7 and 9 comigrate in 125 and 128. Arrows indicate the altered migration of ssRNAs synthesised from the templates of rearranged genomic segments (RS). RS8, RS11 rearranged segment 8 or 11, respectively, of RV strains 125 and 128.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760874&req=5

pone-0074328-g002: ssRNA derived from several strains of RV DLPs.ssRNA were synthesized in vitro from purified RV DLPs. DLPs were incubated at 37°C for 2 hours with the necessary components for positive sense ssRNA synthesis. 1.5% TBE AGE 45 V for 120 min. Lane R: RiboRuler™ High Range (in bases); 1.5 µg ssRNA derived from DLPs of RV strains SA11, OSU, RF, 125 and 128, respectively. DLP-derived ssRNAs 2 and 3 comigrate in all samples., ssRNAS 7–9 comigrate in SA11, OSU and RF, but only ssRNAS 7 and 9 comigrate in 125 and 128. Arrows indicate the altered migration of ssRNAs synthesised from the templates of rearranged genomic segments (RS). RS8, RS11 rearranged segment 8 or 11, respectively, of RV strains 125 and 128.
Mentions: Purified RV DLPs can transcribe positive ssRNA in vitro from the dsRNA genomic segments packaged in the particles [34], [35], [36]. RNAs extruding from DLPs are positive sense and should possess authentic in vivo structures to facilitate experiments aimed at recovering infectious RV. DLPs of five different RV strains were used; the yields of ssRNA were between 3 and 10 µg of purified ssRNA per µg of purified DLPs. Figure 2 shows the successful synthesis of ssRNAs from the DLPs of all strains used alongside a ssRNA marker. The AGE migration patterns in Figure 2, lanes 2–6 reveal the genome structure of each strain. Thus, it was possible to identify ssRNAs derived from DLPs containing rearranged genomes (lanes 4 and 5).

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