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1、Formation of stable homodimer via the C-terminal -helical domain of coronavirus nonstructural protein 9 is critical for its function in viral replication Bo Chen a, Shouguo Fangb, James P. Tama, Ding Xiang Liua,b, a School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, S
2、ingapore 637551, Singapore b Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore a b s t r a c ta r t i c l ei n f o Article history: Received 14 August 2008 Returned to author for revision 5 September 2008 Accepted 23 October 2008 Available online 20 Nov
3、ember 2008 Keywords: Coronavirus Non-structural protein 9 Dimerization RNA-binding Viral replication Coronaviruses devote more than three quarters of their coding capacity to encode two large polyproteins (1a and1abpolyproteins),whichareproteolyticallyprocessedinto1516mature,nonstructuralreplicasepr
4、oteins (nsp1to16).ThesecleavageproductsarebelievedtoplayessentialrolesinreplicationofthegiantRNAgenome of 30 kb and transcription of a nested set of 5 to 9 subgenomic RNA species by a unique discontinuous transcription mechanism. In this report, one of these replicase proteins, nsp9 of the coronavir
5、us infectious bronchitis virus (IBV) is systematically studied using both biochemical and reverse genetic approaches. The results showed that substitution mutation of a conserved Gly (G98) residue in the C-terminal -helix domain with an Asp greatly destabilized the IBV nsp9 homodimer and abolished i
6、ts RNA-binding activity. Introduction of the same mutation into an infectious IBV clone system showed that the mutation totally abolishes the transcription of subgenomic RNA and no infectious virus could be recovered. Mutation of a semi-conserved Ile (I95) residue in the same region showed moderatel
7、y destabilizing effect on the IBV nsp9 homodimer but minimal effect on its RNA-binding activity. Introduction of the mutation into the IBV infectious clone system showed recovery of a mutant virus with severe growth defects, supporting that dimerization is critical for the function of this replicase
8、 protein. Meanwhile, mutations of some positively charged residues in the -barrel regions of the IBV nsp9 protein signifi cantly reduced its RNA-binding activity, but with no obvious effect on dimerization of the protein. Introduction of these mutations into the viral genome showed only mild to mode
9、rate effects on the growth and infectivity of the rescued mutant viruses. 2008 Elsevier Inc. All rights reserved. Introduction Coronavirus is a family of RNA viruses with the largest RNA genome known so far. In cells infected with coronavirus, a nested set of genomic and subgenomic RNA species is pr
10、oduced and used for translation of viral structural and nonstructural proteins. Among these proteins, the 1a and 1ab polyproteins are post-translationally processed to smaller, mature proteins (Ziebuhr et al., 2000; Ziebuhr, 2005).It is generallybelieved thatreplicationof thelargeRNA genome and tran
11、scription of a nested set of 5 to 8 subgenomic RNA species are carried out by these replicase proteins. However, the exact functions of individual replicase proteins are largely unknown. In recent years, determination of the crystal structures of some of these proteins, including the proteinase doma
12、in of nsp3, nsp5, nsp7 and 8, nsp9, nsp10 andnsp15, led tothe revelationof manyimportantfunctionsfor these proteins (Saikatendu et al., 2005; Ratia et al., 2006; Peti et al., 2005; Zhai et al., 2005; Anand et al., 2002, 2003; Yang et al., 2003; Egloff et al., 2004; Sutton et al., 2004; Su et al., 20
13、06; Joseph et al., 2006; Ricagno et al., 2006), but most of these functions are not rigorously tested in a biologically relevant system. In this study, residues critical for dimerization and the RNA-binding activity of nsp9 protein from coronavirus infectious bronchitis virus (IBV) were systematical
14、ly studied by mutagenesis, based on the structural studies of SARS-CoV nsp9 protein. IBV is a group 3 coronavirus. Its genome contains a 27.6 kb single- stranded, positive-sense RNA. In the virus-infected cells, six mRNA species, including the genome-length mRNA 1 and fi ve subgenomic mRNAs (mRNA 26
15、), are produced, and each mRNA species possesses a 64 nucleotides leader sequence derived from the 5-end of the genome (Boursnell et al., 1987). Subgenomic mRNAs 2, 3, 4, and 6 encode the four structural proteins, i.e., spike glycoprotein (S), envelope protein (E), membrane protein (M) and nucleocap
16、sid protein (N). The 5-two-third region of mRNA 1 encodes the 1a and 1ab polyproteins, which are proteolytically cleaved by two virus-encoded proteinases, the papain-like and 3C-like proteinases, into at least 15 nonstructural proteins (nsp2nsp16) (Lim and Liu, 1998a, 1998b; Lim et al., 2000; Liu et al.,1995, 1997,1998; Ng and Liu,1998, 2000, 2002; Xu et al., 2001). A cluster of small proteins, i.e., nsp7, nsp8, nsp9 and nsp10, is located at the C-terminal region of the la polyprotein. The func
