JOURNAL OF VIROLOGY, May 2009, p. 5282–5288 Vol. 83, No. 10 0022-538X/09/$08.00�0 doi:10.1128/JVI.02485-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved. Suppression of Host Gene Expression by nsp1 Proteins of Group 2 Bat Coronaviruses� Yukinobu Tohya,1,2 Krishna Narayanan,1 Wataru Kamitani,1 Cheng Huang,1 Kumari Lokugamage,1 and Shinji Makino1* Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019,1 and Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan2 Received 3 December 2008/Accepted 19 February 2009 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. Severe acute respiratory syndrome coronavirus (SARS- CoV) is the etiological agent of a newly emerged disease, SARS, which originated in southern China in 2002 and spread to various areas of the world in a 2003 epidemic (reviewed in reference 2). Bats are the natural reservoir of a variety of group 1 and 2 CoVs, including viruses closely related to SARS- CoV, SARS-like CoVs (SLCoVs) (11, 13, 21, 23). Considering the potential of bat CoVs as emerging pathogens for humans and animals, it is of the utmost importance to characterize these viruses. SARS-CoV nsp1 is a 180-amino-acid protein that is trans- lated from the most 5� coding region of the SARS-CoV ge- nome (19). SARS-CoV nsp1 protein induces host mRNA deg- radation and translational suppression both in nsp1-expressing cells and in SARS-CoV-infected cells (9, 14). Studies of the nsp1 proteins of mouse hepatitis virus (MHV) and SARS- CoV, which belong to CoV groups 2a and 2b, respectively, suggest that nsp1 plays important roles in the suppression of host innate immune functions and contributes to viral patho- genesis (9, 14, 22, 26). These observations and the fact that SARS-CoV nsp1 exhibits sequence similarity to the nsp1 pro- teins of other group 2 CoVs but not to those of group 1 CoVs (18) led us to hypothesize that nsp1 proteins of the newly identified group 2 bat CoVs have similar functions to suppress host gene expression and block host antiviral immune re- sponses. The present study explored the activities of nsp1 proteins of group 2 bat CoVs for the suppression of host gene expression. At the initiation of our studies, group 2 bat CoVs had been further divided tentatively into three subgroups, including 2b and the putative subgroups 2c and 2d (24). We have chosen to characterize the nsp1 proteins of three group 2 bat CoV strains, Rm1 (15), 133 (20), and HKU9-1 (24), belonging to the subgroups 2b, 2c, and 2d, respectively. The levels of amino acid sequence identity of Rm1, 133, and HKU9-1 nsp1 proteins to SARS-CoV nsp1 protein are 92.2, 19.7, and 30.9%, respec- tively. The low levels of amino acid sequence homology be- tween nsp1 proteins of group 2c and 2d CoVs and SARS-CoV nsp1 are comparable to the levels of amino acid sequence homology between nsp1 proteins of the group 2a CoVs, in- cluding MHV and bovine CoV, and SARS-CoV nsp1; the degrees of amino acid sequence identity of MHV nsp1 and bovine CoV nsp1 to SARS-CoV nsp1 are 20.6 and 17.3%, respectively (18). Figure 1A shows the phylogenetic relation- ships of the group 2 CoV nsp1 proteins, including the group 2 bat CoV nsp1 proteins analyzed in this study. To examine the effects of group 2 bat CoV nsp1 proteins on reporter gene expression, cDNAs were synthesized by Bio Ba- sic Inc. according to the nsp1 amino acid sequences of Rm1 (180 amino acids) (15), 133 (195 amino acids) (20), and HKU9-1 (175 amino acids) (24), with the appropriate codon modifications for optimized translations in human cells. The plasmids pCAGGS-Rm1, pCAGGS-133, and pCAGGS-HKU9-1 were constructed by inserting the Rm1 nsp1 open reading frame (ORF), the 133 nsp1 ORF, and the HKU9-1 nsp1 ORF, respectively, into pCAGGS-MCS, each with a sequence encod- ing a C-terminal myc tag. As controls, the parental plasmid pCAGGS; pCAGGS-Nsp1-WT, encoding the SARS-CoV nsp1 protein (9); and pCAGGS-Nsp1-mt, encoding a mutant form of SARS-CoV nsp1 (SCoVnsp1-mt) containing alanines in place of the positively charged amino acids K164 and H165 in nsp1 of SARS-CoV (14), were used; expressed SARS-CoV nsp1 protein, but not SCoVnsp1-mt protein, suppresses host gene expression (14). 293 cells grown in 24-well plates were cotransfected in trip- licate with 0.1 �g of pRL-SV40, in which the Renilla luciferase * Corresponding author. Mailing address: Department of Microbi- ology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, TX 77555-1019. Phone: (409) 772-2323. Fax: (409) 772-5065. E-mail: