was supported, in part, by funding from the American Asthma Foundation. == Footnotes == Published ahead of print on 3 November 2010. == REFERENCES ==. of 30 kb, are classified into the groups alpha, beta, and gamma. In infected cells, CoV gene expression is initiated by the translation of two large polyproteins encoded by gene 1 from the incoming viral genomic RNA. These polyproteins are processed into 16 mature proteins (nsp1 to nsp16) by two or three viral proteinases, in the case of alphacoronavirus and betacoronavirus (16,18). Most of the gene 1 proteins play critical roles in viral RNA synthesis (3-5,7-9,14,17,18,20), while some have other biological functions (1,6,11,12,19). The nsp1 protein of betacoronaviruses inhibits host gene expression. SCoV nsp1 uses a two-pronged strategy to inhibit host translation/gene expression by first binding to the 40S ribosomal subunit and then inactivating the translation activity of these 40S subunits (10). The nsp1-40S ribosome complex then induces GTS-21 (DMBX-A) the modification of the 5 regions of capped mRNA templates and renders these template RNAs translationally incompetent. Importantly, SCoV nsp1 suppresses host innate immune functions by inhibiting type I interferon (IFN) expression in infected cells (15) and host antiviral signaling pathways (23), suggesting its important role in SCoV virulence. Nsp1 proteins of bat CoVs also suppress host translation, while some exert host translational suppression without degrading host mRNAs (22). Mouse hepatitis virus (MHV) nsp1 protein also suppresses host gene expression. A recombinant MHV lacking the nsp1 gene is severely attenuated in infected mice, yet mutant virus replication and spread are restored to wild-type (wt) virus levels in type I IFN receptor-deficient mice (26), indicating that MHV nsp1 interferes efficiently with the type I IFN system. Alphacoronaviruses encode nsp1 proteins of 9 kDa, which are substantially smaller than the 20-kDa nsp1 proteins of betacoronavirus. The nsp1 proteins of alphacoronavirus and betacoronavirus have no amino acid sequence similarities to each other or with known host proteins. Nonetheless, the expression of nsp1 of human CoV 229E, an alphacoronavirus, suppresses the expression of reporter genes under the control of beta IFN (IFN-), GTS-21 (DMBX-A) IFN-stimulated response element, and simian virus 40 (SV40) promoters (26). Within the alphacoronaviruses, the amino acid sequence of the human CoV 229E nsp1 protein is moderately similar to those of nsp1 proteins of human CoV NL63 (60%) and porcine epidemic diarrhea virus (52%), but it is different from that of transmissible gastroenteritis virus (TGEV) (32%). In contrast, the TGEV nsp1 protein has 97% and 93% amino acid sequence identities with the nsp1 proteins of porcine respiratory OGN coronavirus and feline infectious peritonitis virus, respectively. The present study used TGEV nsp1 as a model system to explore how the nsp1 protein of alphacoronavirus suppresses host gene expression. To determine if TGEV nsp1 suppresses host gene expression, we cotransfected human embryonic kidney 293 cells or swine testis (ST) cells, the latter of which supporting TGEV replication, with the plasmid pRL-SV40 expressing the SV40 promoter-drivenRenillaluciferase (rLuc) gene, together with one of the following plasmids: pCAGGS carrying the gene for chloramphenicol acetyltransferase (CAT), the nsp1 protein of TGEV (Purdue strain), the SCoV nsp1 protein, or the SCoV nsp1 mutant (SCoV nsp1-mt) protein; the SCoV nsp1-mt does not suppress host gene expression (10,15). All proteins, GTS-21 (DMBX-A) except CAT, contained a GTS-21 (DMBX-A) C-terminal myc epitope tag. Both TGEV nsp1 and SCoV nsp1 efficiently suppressed the reporter gene expression in both cell lines (Fig.1A). The low-level accumulation of the nsp1 proteins of TGEV and SCoV suggested that these nsp1 proteins inhibited their own expression. Furthermore, transfection of RNA expressing the C-terminal myc epitope-tagged TGEV nsp1 or SCoV nsp1, but not CAT or SCoV nsp1-mt, in 293 cells resulted in potent suppression of host protein synthesis in the presence or absence of actinomycin D (Act D), an inhibitor of host transcription (Fig.1B). Replication of TGEV in ST cells caused cytopathic effects in 30% of cells and floating of 10% cells at 14 h postinfection. Following metabolic radiolabeling of infected cells and mock-infected cells with [35S]methionine.
