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. 2013 Jan;87(2):1098–1104. doi: 10.1128/JVI.02627-12

Fig 4.

Fig 4

MVC NP1 mutants have a significant effect on RNA processing independent of genome replication. (A) RNA expression determined by an RNase protection assay (RPA). WRD cells were transfected with the wild-type infectious clone pIMVC (lane 1) or three distinct NP1 termination mutants, NP1-2735TAG, NP1-2740TAA, or NP1-2839TAA (lanes 3, 4, and 5). At 2 days posttransfection, total RNAs were isolated and probed for MVC-specific mRNAs using the (pA)p probe that is depicted in Fig. 3A. The sizes of protected bands are shown on the left, and the designated (pA)d or (pA)d RNA species are shown on the right. The RNA probe is shown in lane 1. (B) RNase protection assays using the (pA)p probe showing RNA expression of WRD cells transfected with WT pIMVC (lane 2) or the replication-deficient mutants RC WT, NP1-2735TAG, pIMVC-NS1−, and pIMVC-FD, as described in the text (lanes 3, 4, 5, and 6, respectively). The (pA)p probe is shown in lane 1. RNA species are designated (pA)d or (pA)p. (C) RNA expression assayed by RPAs using the (pA)p probe of WRD cells transfected with WT pIMVC (lane 2) or the NP1-lacking mutants ATGm (lane 4), 5XPro (lane 5), or NP1-2735TAG (lane 6) as described in the text. The (pA)p probe is shown in lane 1. RNA species are designated (pA)d or (pA)p. (D) RPA utilizing the cap gene probe as depicted in Fig. 3A of either virus-infected WRD cells (VI; lane 3) or WRD cells transfected with WT pIMVC (lane 4) or the third intron splice mutant 3Am (lane 5), as described in the text. The WT cap gene probe (lane 1) and the homologous 3A cap gene probe (lane 2) are shown. Individual RNA species and their relative sizes are designated on the right and left of the image, respectively. (E) Quantification of RPAs showing the average polyadenylation ratios [(pA)p/(pA)d] of RNA transcripts from WT pIMVC, 3Am, NP1-5XPro, or NP1-2735TAG in either a replicating infectious clone (TR+) or replicating-deficient (TR−) background. Error bars indicate the standard deviation from 3 independent experiments. (F) RPA utilizing the cap gene probe (lane 1) showing RNA expression and distinguishing individual transcripts from viral infection (lane 2), transfection of WT pIMVC (lane 3), or transfection of the NP1 mutants 2735TAG, 2740TAA, and 2839TAA (lanes 4, 5, and 6, respectively). The individual RNA species R1 to R6 are labeled on the right, and their respective sizes are shown on the left. (G) Expression of NP1, NS1, and capsid proteins. Immunoblots of 293T cell extracts either mock transfected or transfected with WT pIMVC, the NP1 termination mutant NP1-2735TAG, or the NP1 proline mutant NP1-5XPro, probed with anti-NS1, anti-NP1, or anti-capsid antibodies.