Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs
Figure 5
Minus-strand RNA accumulation is also modulated by mutations in nsp1.
(A–D) Analysis and quantification of EAV minus-strand accumulation by a two-cycle RNase protection assay. (A) Schematic representation of the nested set of viral minus-strand RNA [(−)RNA] species produced in EAV-infected cells. The anti-leader sequence is depicted in light green. The in vitro-transcribed plus-strand probes used for detection of (−)RNA1 (pRNA1), (−)RNA6 (pRNA6) and (−) RNA7 (pRNA7) are shown. pRNA6 and pRNA7 target the leader-body junction sequences of (−)RNA6 and (−)RNA7, respectively. Note that hybridization with pRNA1 results in the protection of a single fragment, while the probes for (−)RNAs 6 and 7 each protect three fragments – one derived from the full-length sg minus strand, and two fragments derived in part from partial hybridization of these probes to larger viral (−)RNAs in which the target sequences are noncontiguous (exemplified for pRNA6). For simplicity, non-EAV sequences present near the termini of the three probes were omitted from the scheme. (B) Viral (−)RNA accumulation was analyzed at 11 h post-transfection for the ZCH, A1 and A4 mutants, and a wt control. Protected fragments were resolved on denaturing 5% polyacrylamide/8M urea gels and visualized by phosphorimaging. The constructs analyzed are labeled above the lanes (M, mock-transfected cells; (−), no-RNase control that shows a band corresponding to 0.2 fmol of the full-length probe). Sizes (nt) of RNA markers have been indicated on the left. The single 327-nt protected fragment resulting from hybridization with the positive-sense probe for RNA1(−) is indicated. The probes for subgenome-length minus strands protected fragments derived from the full-length (−)RNA6 and (−)RNA7 (327 nt and 319 nt, respectively; denoted with LB), as well as from the (−)RNA6 and (−)RNA7 body sequences (188 nt and 180 nt, respectively; denoted with B) and the anti-leader sequence (139 nt; denoted with L). The presence of two bands in the size range of the anti-leader fragment has been described previously [55]. (C) The relative levels of minus-strand accumulation were quantified by phosphorimaging. For (−)RNAs 6 and 7, only the bands resulting from protection of full-length sg minus strands (denoted with LB in panel [B]) were quantified. The values correspond to the means from three independent transfections that were normalized to the level of accumulation of each minus-strand RNA in the wt control, which was set at 1. Intracellular RNA from the same transfection samples for which plus-strand accumulation was quantified (Fig. 4B) was used. Genomic minus-strand RNA levels are represented as dark blue bars. Error bars denote standard deviation. (D) The ratio of plus-strand to minus-strand accumulation for RNAs 1, 6 and 7 was calculated using the mean relative values obtained in Fig. 4B and Fig. 5C.