Towards the Development of a Minigenome Assay for Species A Rotaviruses

Abstract

RNA virus polymerases carry out multiple functions necessary for successful genome replication and transcription. A key tool for molecular studies of viral RNA-dependent RNA polymerases (RdRps) is a 'minigenome' or 'minireplicon' assay, in which viral RdRps are reconstituted in cells in the absence of full virus infection. Typically, plasmids expressing the viral polymerase protein(s) and other co-factors are co-transfected, along with a plasmid expressing an RNA encoding a fluorescent or luminescent reporter gene flanked by viral untranslated regions containing cis-acting elements required for viral RdRp recognition. This reconstitutes the viral transcription/replication machinery and allows the viral RdRp activity to be measured as a correlate of the reporter protein signal. Here, we report on the development of a 'first-generation' plasmid-based minigenome assay for species A rotavirus using a firefly luciferase reporter gene.

Keywords: RNA-dependent RNA polymerase; minigenome; reporter assay; rotavirus.

Figure 1

Minigenome reporter gene construct design. (A) Schematic of the reporter gene construct for the minigenome assay. Plasmids under a T7 promoter (T7P) encoding the Fluc gene in either the positive (5′-reporter) or negative sense (3′-reporter) flanked by the 5′- and 3′-UTRs. The plasmids included HDV ribozyme and T7 terminator sequences (T7T). Pink boxes, UTRs; yellow box, firefly luciferase ORF; blue box, HDV ribozyme sequence. (B) Dose-dependent titration of the 5′- and 3′-reporter plasmids. The pVR1255 plasmid expressing the Fluc gene was used as a positive control (denoted as ‘+ve’). The mock sample contained transfection reagent only. Data are the mean ± SEM from four independent experiments.

Figure 2

Reporter expression by rotavirus polymerase. (A) Schematic of the proposed minigenome assay. RV plasmids encoding each bovine RF stain gene were co-transfected with T7 reporter plasmids expressing the Fluc gene in either the positive (5′-reporter) or negative sense (3′-reporter). Luciferase activity was measured after 48 h post transfection. Dose-dependent titration of 11 RV plasmids with 200 ng of the 5′-reporter in (B) and with 200 ng of the 3′-reporter in (C), with the RLU values for the reporter-only signal (‘background’) subtracted. As in reverse genetics, the amount of plasmids expressing NSP2 and NSP5 genes was increased to scale.

Figure 3

Generation of inactive polymerase. (A) VP1 shown as a linear schematic and coloured according to the domain organisation, with amino acid numbers labelled above. Adapted from [27], with alignments for RVH-RVL updated/added based on their reference sequences (KT962027.1, NC_026825.2, NC_055268.1, OQ934016.1, OM101015.1). The N-terminal and C-terminal domains (deep blue) flank the core domain containing the fingers (pale blue), palm (mid blue) and thumb (orange). Sequence-based alignment of RdRps across RV species and the related reovirus showing the conserved catalytic ‘GDD’ site. Dots indicate amino acid conservation. Highlighted in yellow are the two conserved aspartic acid residues targeted for mutagenesis. (B) Mutagenesis strategy for evolutionarily conserved aspartic acid residues in the VP1 catalytic domain. (C) Viral titres of WT RF and of VP1 mutants. (D) Minigenome assay for VP1 mutants. In all cases, all 11 RG plasmids were transfected (with 3.125×X amounts of NSP2 and NSP5 plasmids) along with 200 ng 5′-reporter. pVR1255 plasmid expressing Fluc gene was used as a positive control (denoted as ‘+ve’). The RLU values for the reporter-only signal (‘background’) were subtracted. * p < 0.05; *** p < 0.001.

Figure 4

Measuring RdRp activity. Luciferase activity following co-transfection of RV plasmids with 200 ng of 5′-reporter. WT RF denotes co-transfection of all 11 RG plasmids (with 3.125× amounts of NSP2 and NSP5 plasmids) with the reporter. The pVR1255 plasmid expressing the Fluc gene was used as a positive control (denoted as ‘+ve’). The RLU values for the reporter-only signal (‘background’) were subtracted. (A) To test whether the number of RG plasmids expressed could be reduced, 4 plasmids corresponding to VP1, VP2, VP3 and VP6 were co-transfected with the 5′-reporter (‘4 plasmids + 5′ rep). To test whether the 4-plasmid system could be improved upon, the amount of the VP2 plasmid was increased 11-fold (‘VP1:VP2 ratio’). (B) To test the dependency of the system on VP1 and NSP3, polymerase assays were undertaken either by co-transfecting reporter with VP1 or NSP3 alone, or with ten segments minus either VP1 or NSP3. * p < 0.05; **** p < 0.0001; n.s, not significant.