Interplay between coronavirus, a cytoplasmic RNA virus, and nonsense-mediated mRNA decay pathway

Figures

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  Fig. 1.. Enhancement of MHV replication from transfected genomic RNA in NMD-deficient cells. MHV genomic RNA (0.5 μg), extracted from purified MHV particles, was transfected into 17Cl-1 cells that had been treated with either control siRNA (siCtrl) or siRNAs for UPF1 (siUpf1), UPF2 (siUpf2), SMG5 (siSmg5), or SMG6 (siSmg6). (A) At 24 h posttransfection of the genomic RNA, the cells were stained by anti-M protein antibody (green) and DAPI (blue) (Left). (Scale bar: 100 μm.) The fold increase in M protein-positive cells was calculated (Right). (B) MHV titers in the culture fluid at 24 h postgenomic RNA transfection were determined by plaque assay. The data represent the mean with SEM of at least three independent experiments. Statistical analysis was done by ANOVA. *P < 0.05, **P < 0.01.

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  Fig. 2.. Effect of depletion of UPF1 and UPF2 on the stabilities of transfected MHV genomic RNA and subgenomiclike mRNA (rLucRNA3). (A, Left) Two micrograms per well of MHV genomic RNA was transfected into UPF1-depleted cells (siUpf1), UPF2-depleted cells (siUpf2), or cells treated with control siRNAs (siCtrl) in a 12-well plate. The levels of genomic RNA at 1, 3, 5, and 7 h posttransfection were determined by qRT-PCR and normalized to the 18S rRNA levels. (A, Right) The 17Cl-1 cells were transfected with MHV genomic RNA. After 1 h of incubation and washing to remove free genomic RNAs, total intracellular RNAs were extracted from one plate (“1 h”). The remaining plates were cultured in the presence of CHX, wortmannin (Wort), or DMSO, and the levels of genomic RNAs at 3 h posttransfection were determined by qRT-PCR and normalized to 18S rRNA levels. (B) Schematic diagram of subgenomic mRNA 3-like RNA (rLucRNA3) is shown on Top. Experiments were performed as described in A, except that 0.5 μg of capped and polyadenylated in vitro-synthesized rLucRNA3 was used in place of the MHV genomic RNA. The data represent the mean with SEM of at least three independent experiments. Statistical analysis…

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  Fig. 3.. Cytoplasmically synthesized rLucRNA3, but not GLA mRNA, is susceptible to NMD. (A) UPF1-depleted 17Cl-1 cells (siUpf1), UPF2-depleted 17Cl-1 cells (siUpf2), or 17Cl-1 cells treated with control siRNAs (siCtrl) were cotransfected with plasmids expressing T7 polymerase, rLucRNA3, vaccinia virus capping enzyme D1R, and vaccinia virus capping enzyme D12L. At 20 h after plasmid transfection, cells were treated with actD, and levels of expressed rLucRNA3 at 0, 2, and 4 h after actD treatment were determined by qRT-PCR and normalized to 18S rRNA levels. The data represent the mean with SEM of three independent experiments. Statistical analysis was done by ANOVA. **P < 0.01. (B) Experiments were performed as described in A, except that plasmid encoding GLA RNA was used in place of that encoding rLucRNA3. (C) The 17Cl-1 cells were cotransfected as described in A. At 1 h after actD addition, DMSO, CHX, or wortmannin (Wort) was added to the cells, and levels of rLucRNA3 at 1 and 3 h after actD addition were determined by qRT-PCR and normalized to 18S rRNA levels. The data represent the mean with SEM of at least three independent experiments. Statistical analysis was done by ANOVA. *P < 0.05.

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  Fig. 4.. MHV replication induces NMD inhibition. (A) NS39 reporter cells (stably expressing NS39 reporter transcripts) and WT reporter cells (stably expressing WT reporter transcripts) were mock-infected (Mock) or infected with MHV at a multiplicity of infection (MOI) of 3. At indicated times p.i., levels of WT transcripts in WT reporter cells and NS39 reporter transcripts in NS39 reporter cells were determined by qRT-PCR and normalized to 18S rRNA levels. NS39 transcript levels are shown relative to WT (set arbitrarily at 100). (B) The 17Cl-1 cells were mock-infected (Mock) or infected with MHV at an MOI of 3. Total RNAs were extracted at 7 h p.i. and subjected to qRT-PCR analysis to measure the levels of alternatively spliced rpL3 mRNA relative to that of canonically spliced rpL3 mRNA. The relative ratio of the former to the latter is shown in y axis. The data represent the mean with SEM of at least three independent experiments. Statistical analysis was done by ANOVA. **P < 0.01. (C) The 17Cl-1 cells were mock-infected (M) or infected with MHV (I) at an MOI of 3. At different times p.i., cells were incubated with medium containing [35S]methionine/cysteine for 30 min. Cell extracts were…

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  Fig. 5.. MHV N protein inhibits the NMD pathway. (A, Left) The 17Cl-1 cells were cotransfected with NS39 reporter plasmid or WT reporter plasmid along with the pCMV-fLuc plasmid expressing fLuc transcripts and plasmids encoding either CAT, M protein, N protein, E protein, or S protein. At 40 h posttransfection, the levels of WT reporter transcripts and NS39 reporter transcripts were determined by qRT-PCR and normalized to the levels of fLuc transcripts. NS39 transcript levels are shown relative to WT (set arbitrarily at 100). (A, Right) Cells were transfected as described above. Cell extracts were prepared at 40 h posttransfection and subjected to Western blot analysis to detect CAT, MHV structural proteins, and β-actin. β-Actin served as the loading control. WT, cells transfected with WT reporter plasmid; NS, cells transfected with NS39 reporter plasmid. (B) Cells were cotransfected as described in A, except that one extra sample included a plasmid expressing TGEV nsp1. Levels of fLuc transcripts (relative to 18S rRNA levels) at 40 h posttransfection (Top); fLuc reporter activities at 40 h posttransfection (Bottom). (C) Stable cell lines expressing either WT reporter transcripts or NS39…

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  Fig. 6.. MHV N protein expression inhibits the degradation of transfected MHV genomic and subgenomiclike RNAs. In a 12-well plate, 17Cl-1 cells were transfected with a plasmid encoding MHV N, CATmyc, or TGEV nsp1myc. At 24 h posttransfection, cells were transfected with 2 μg of MHV genomic RNA (A) or 0.5 μg of rLucRNA3 (B). At 1, 3, 5, and 7 h after RNA transfection, levels of MHV genomic RNA and rLucRNA3 were determined by qRT-PCR and normalized to 18S rRNA. The data represent the mean with SEM of at least three independent experiments. Statistical analysis was done by ANOVA. *P < 0.05, **P < 0.01. The half-life (t1/2) of each RNA was calculated from the slope of the trendlines.

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  Fig. 7.. NMD inhibition promotes the accumulation of MHV mRNAs early in infection, facilitating virus replication. (A) The 17Cl-1 cells were mock-infected (Mock) or infected with MHV at a multiplicity of infection (MOI) of 0.1. Accumulation of N protein, at the indicated times p.i., was examined by Western blot analysis. β-Actin served as the loading control. (B) UPF1-depleted 17Cl-1 cells (siUpf1), UPF2-depleted 17Cl-1 cells (siUpf2), or control siRNA-treated 17Cl-1 cells (siCtrl) were infected with MHV at an MOI of 0.1. The levels of genomic RNA/mRNA 1 (relative to 18S rRNA) at 1, 2, 3, and 5 h p.i. were determined by qRT-PCR. (C) Cells were infected with MHV as described in B. The levels of mRNA 3 (Left) and mRNA 7 (Right) (relative to 18S rRNA) at 5 h p.i. were determined by qRT-PCR. Because the levels of these mRNAs at 1, 2, or 3 h p.i. were below the limit of detection, we could not accurately determine their relative levels at these early time points. (D) siRNA-treated cells were infected with MHV as described in B. Virus titers in culture supernatant at 2, 4, 8, 12, and 16 h p.i. are shown. (E) The 17Cl-1 cells were infected with MHV at an MOI of 0.1. Cells were treated with 300…