Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mRNA degradation -- Kamitani et al. 103 (34): 12885 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Kamitani et al. 10.1073/pnas.0603144103.

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Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10
Supporting Figure 11

Supporting Figure 7 Fig. 7.
Northern blot analysis of SeV N mRNA. 293 cells were independently transfected with pCAGGS (EV), pCAGGS-nsp1 (nsp1), or pCAGGS-NSs (NSs). At 24 h posttransfection, cells were infected with 100 HA units/ml of SeV (+) or mock infected (-). At 16 h, intracellular RNAs were prepared. SeV N mRNA (SeV N) was detected by Northern blot analysis using a digoxigenin-labeled 1.2 kb-long RNA probe that hybridizes with the SeV N mRNA. The same RNA samples were separated on an agarose gel, and the 28S and 18S rRNAs were stained with ethidium bromide.

Supporting Figure 8 Fig. 8.
Effect of nsp1 expression on reporter gene expression from transfected plasmids. 293 (A and E), Hec1B (B and F), Vero (C and G), and Vero E6 (D and H) cells were cotransfected with a mixture of pCMV-b reporter plasmid and pCGAAS (EV), that of pCMV-b reporter plasmid and pCAGGS-nps1 (nsp1), that of pCMV-b reporter plasmid and pCAGGS-NSs (NSs), and that of pCMV-b reporter plasmid and pCAGGS-3a (3a) (A–D) or a mixture of pRL-SV40 reporter plasmid and each of pCGAAS (EV), pCAGGS-nps1 (nsp1), pCAGGS-NSs (NSs), or pCAGGS-3a (3a) (E–H) as indicated in each panel. At 36 h after transfection, cells were lysed in 150 ml of Renilla Luciferase assay lysis buffer (Promega, Madison, WI) for Renilla Luc activities or reporter lysis buffer for b-gal activities. Aliquots of lysate were used to measure Renilla Luc or b-gal activities.

Supporting Figure 9 Fig. 9.
Effect of nsp1 protein expression on reporter gene mRNA accumulation in Vero E6 cells. Vero E6 cells were independently cotransfected with pCMV-b and pCAGGS-nsp1 (A; nsp1) or pRL-SV40 and pCAGGS-nsp1 (B; nsp1). As controls, pCAGGS (EV), pCAGGS-NSs (NSs), or pCAGGS-3a (3a) were used in place of pCAGGS-nsp1. At 48 h posttransfection, total RNAs were extracted, and Northern blot analysis was performed using riboprobes specific for b-gal or Luc. The same RNA samples were separated on agarose electrophoretic gels, and the 28S and 18S rRNAs were stained with ethidium bromide.

Supporting Figure 10 Fig. 10.
Effect of nsp1 expression on stabilities of reporter gene RNA and host endogenous mRNAs. 293 cells were transfected with pRL-SV40. At 16 h posttransfection, cells were independently transfected with in vitro-synthesized CAT RNA transcripts (CAT), nsp1 RNA transcripts (nsp1), or NSs RNA transcripts (NSs). One hour after RNA transfection, cells were incubated with actD (ActD+) or absence of actD (ActD-). Total RNAs were extracted at 0 h (0 h) or 8 h (8 h) after actD addition. Abundance of expressed Luc RNA and endogenous GAPDH and b-actin mRNAs were examined by using Northern blot analysis.

Supporting Figure 11 Fig. 11.
Effect of actD on SCoV replication. 293/ACE2 cells that had been transfected with pCMV-b were mock infected (Mock) or infected with SCoV (SCoV) at a multiplicity of infection (moi) of 3, as described in the text. After virus adsorption, cells were incubated in the presence of actD (ActD+) or in the absence of actD (ActD-). Intracellular RNAs or cell extracts were prepared at 14 h after actD addition. (A) Digoxigenin-labeled DNA probe that hybridizes with the 3'-end 0.5-kb region of the SCoV genome was used to detect all SCoV mRNAs in the Northern blot analysis. Numbers 1 to 9 represent SCoV mRNAs 1 to 9. (B) Western blot analysis was performed to detect nsp1 protein and actin by using anti-nsp1 peptide antibody and anti-actin antibody, respectively.