doi: 10.1128/JVI.02485-08. Epub 2009 Mar 4.
Suppression of host gene expression by nsp1 proteins of group 2 bat coronaviruses
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- PMID:19264783
- PMCID: PMC2682096
- DOI: 10.1128/JVI.02485-08
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Suppression of host gene expression by nsp1 proteins of group 2 bat coronaviruses
Yukinobu Tohya et al. J Virol.2009 May.
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Abstract
nsp1 protein of severe acute respiratory syndrome coronavirus (SARS-CoV), a group 2b CoV, suppresses host gene expression by promoting host mRNA degradation and translation inhibition. The present study analyzed the activities of nsp1 proteins from the group 2 bat CoV strains Rm1, 133, and HKU9-1, belonging to groups 2b, 2c, and 2d, respectively. The host mRNA degradation and translational suppression activities of nsp1 of SARS-CoV and Rm1 nsp1 were similar and stronger than the activities of the nsp1 proteins of 133 and HKU9-1. Rm1 nsp1 expression in trans strongly inhibited the induction of type I interferon (IFN-I) and IFN-stimulated genes in cells infected with an IFN-inducing SARS-CoV mutant, while 133 and HKU9-1 nsp1 proteins had relatively moderate IFN-inhibitory activities. The results of our studies suggested a conserved function among nsp1 proteins of SARS-CoV and group 2 bat CoVs.
Figures
Unrooted phylogenetic tree of CoV nsp1 proteins and amino acid sequence variations among group 2b CoV nsp1 proteins. (A) The unrooted phylogenetic tree of CoV nsp1 proteins was constructed by the neighbor-joining method and the Treeview program and based on 100 bootstrapped data sets. The names of virus strains with nsp1 proteins that were analyzed in this study are underlined. The virus strains used in the phylogenetic analysis are as follows: Urbani, human isolate of SARS-CoV; Rm1 and 279, bat isolates fromR. macrotis (horseshoe bat); Rf1, bat isolate fromR. ferrumequinum; Rp3, bat isolate fromR. pearsoni; 133 and HKU4-1, bat isolates fromTylonycteris pachypus (lesser bamboo bat); HKU5-5, bat isolate fromPipistrellus abramus (Japanese pipistrelle); HKU9-1, HKU9-2, HKU9-3, and HKU9-4, bat isolates fromRousettus leschenaulti (Leschenault's rousette); MHV strain A59; and feline infectious peritonitis virus (FIPV) strain WSU-79/1146. The amino acid (aa) lengths of nsp1 proteins of group 2b, 2c, and 2d CoVs are indicated in parentheses. The scale bar indicates the estimated number of substitutions per 10 amino acids. (B) The full-length SARS-CoV Urbani nsp1 amino acid sequence is represented. Only the amino acid residues that differ between SARS-CoV nsp1 and other group 2b CoV nsp1 proteins are shown. In addition to the group 2b CoV amino acid sequences used for the phylogenetic tree, those of GZ02 (human isolate of SARS-CoV) and HKU3-1 (bat isolate fromR. sinicus) were compared. The total number of amino acid differences in each nsp1 protein compared with SARS-CoV nsp1 is indicated in parentheses. The number at the end of each line of the SARS-CoV Urbani nsp1 sequence indicates the amino acid position.
Effects of expression of bat CoV nsp1 on reporter gene expression. 293 cells were independently cotransfected with pRL-SV40 and one of the following plasmids: pCAGGS (empty vector [EV]), pCAGGS-Nsp1-WT (WT), pCAGGS-Nsp1-mt (mt), pCAGGS-Rm1 (Rm1), pCAGGS-133 (133), or pCAGGS-HKU9-1 (HKU9-1). At 20 h posttransfection, cell extracts were prepared. (A) Western blot analysis using anti-myc monoclonal antibody (Upstate) and anti-β-actin antibody (Santa Cruz Biotechnology) demonstrated the accumulation of expressed nsp1 proteins and β-actin, respectively. The data are representative of results from three independent experiments. (B) RLuc activities were measured and represented as the average of results from three independent experiments. Activities are expressed in relative light units. (C) The abundance of expressed RLuc RNA transcripts from pRL-SV40 was examined by Northern blot analysis using an RLuc probe. The data are representative of results from three independent experiments. The abundance of 28S and 18S rRNAs in each sample is also shown.
Effect of bat CoV nsp1 expression on host mRNA stability and protein synthesis. 293 cells were independently transfected with in vitro-synthesized RNA transcripts encoding CAT (CAT), SARS-CoV nsp1 (WT), SCoVnsp1-mt (mt), Rm1 nsp1 (Rm1), 133 nsp1 (133), or HKU9-1 nsp1 (HKU9-1) protein. One hour after RNA transfection, cells were incubated in the absence of ActD (ActD −) or the presence of ActD (ActD +). (A) At 8 h posttransfection, total proteins were extracted. Western blot analysis was performed using anti-myc or anti-β-actin antibodies. (B) Total RNAs were extracted at 8 h posttransfection and used for Northern blot analysis with riboprobes specific for GAPDH, β-actin, and cyclophilin. The abundance of 28S and 18S rRNAs is shown at the bottom. (C and D) Cells were radiolabeled with 20 μCi/ml of [35S]methionine from 6.5 to 7 h after the ActD addition. Equivalent amounts of intracellular proteins were analyzed by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis. (C) The gel was exposed to X-ray film. Phosphorimager analysis was performed to determine the level of host protein synthesis, and the numbers below the lanes represent the percentages of radioactivity relative to that for cells transfected with CAT RNA (% of CAT). The boxes indicate the regions of the gel used for phosphorimager analysis. Representative data from two independent experiments are shown. (D) The gel was stained with colloid Coomassie blue.
Effect of bat CoV nsp1 expression on IFN-I and ISG induction in SARS-CoV-mt-infected cells. 293/ACE2 cells were either mock infected or infected with SARS-CoV-mt at a multiplicity of infection of 3. One hour after infection, cells were independently transfected with RNA transcripts encoding CAT (CAT), SARS-CoV nsp1 (WT), SCoVnsp1-mt (mt), Rm1 nsp1 (Rm1), 133 nsp1 (133), or HKU9-1 nsp1 (HKU9-1) protein. At 18 h p.i., total intracellular proteins and RNAs were extracted. (A) Western blot analysis was performed using anti-myc (α-myc), anti-nsp1, and anti-β-actin antibodies to demonstrate the accumulations of nsp1 proteins expressed from the RNA transcripts in transfected cells, the virally encoded nsp1-mt protein, and the host β-actin protein, respectively. (B) The viral mRNAs were detected by Northern blot analysis using a probe that binds to the 3′ end of the SARS-CoV genome (nucleotides 29,084 to 29,608) (14). +, infection; −, mock infection. (C) The accumulation of endogenous IFN-β, ISG15, and ISG56 mRNAs was evaluated by Northern blot analysis using riboprobes specific for the IFN-β gene, ISG15, and ISG56, respectively. (D) Western blot analysis was performed using anti-ISG15 protein polyclonal antibody (Cell Signaling Technology) to detect ISG15 protein. (E) At 43 h p.i., culture supernatants were collected from mock-infected cells (mock) and the SARS-CoV-mt-infected cells that were transfected with the indicated RNA transcripts. After complete inactivation of viruses by60Co irradiation (2 × 106 rad) (8), the samples were subjected to acid treatment (14). After neutralization of the samples, the human IFN-I activity was measured by a standard plaque reduction assay using Sindbis virus on Vero cells (5). The units of IFN are expressed as the reciprocal of the dilution that inhibited the formation of 50% of the viral plaques. The data are the averages of results from two independent experiments. IU, international units.
References
- Atasoy, U., S. L. Curry, I. López de Silances, A.-B. Shyu, V. Casolaro, M. Gorospe, and C. Stellato. 2003. Regulation of eotaxin gene expression by TNF-α and IL-4 through mRNA stabilization: involvement of the RNA-binding protein HuR. J. Immunol. 1714369-4378. - PubMed
- Condeelis, J., and R. H. Singer. 2005. How and why does β-actin mRNA target? Biol. Cell 9797-110. - PubMed
- Devaraj, S. G., N. Wang, Z. Chen, Z. Chen, M. Tseng, N. Barretto, R. Lin, C. J. Peters, C.-T. K. Tseng, S. C. Bake, and K. Li. 2007. Regulation of IRF-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus. J. Biol. Chem. 28232208-32221. - PMC - PubMed
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