title: Functional and genetic studies of the substrate specificity of coronavirus infectious bronchitis virus 3C-like proteinase.
abstract: Coronavirus (CoV) 3C-like proteinase (3CLpro), located in nonstructural protein 5 (nsp5), processes the replicase polyproteins 1a and 1ab (pp1a and pp1ab) at 11 specific sites to produce 12 mature nonstructural proteins (nsp5 to nsp16). Structural and biochemical studies suggest that a conserved Gln residue at the P1 position is absolutely required for efficient cleavage. Here, we investigate the effects of amino acid substitution at the P1 position of 3CLpro cleavage sites of infectious bronchitis virus (IBV) on the cleavage efficiency and viral replication by in vitro cleavage assays and reverse genetic approaches. Our results demonstrated that a P1-Asn substitution at the nsp4-5/Q2779, nsp5-6/Q3086, nsp7-8/Q3462, nsp8-9/Q3672, and nsp9-10/Q3783 sites, a P1-Glu substitution at the nsp8-9/Q3672 site, and a P1-His substitution at the nsp15-16/Q6327 site were tolerated and allowed recovery of infectious mutant viruses, albeit with variable degrees of growth defects. In contrast, a P1-Asn substitution at the nsp6-7/Q3379, nsp12-13/Q4868, nsp13-14/Q5468, and nsp14-15/Q5989 sites, as well as a P1-Pro substitution at the nsp15-16/Q6327 site, abolished 3CLpro-mediated cleavage at the corresponding position and blocked the recovery of infectious viruses. Analysis of the effects of these lethal mutations on RNA synthesis suggested that processing intermediates, such as the nsp6-7, nsp12-13, nsp13-14, nsp14-15, and nsp15-16 precursors, may function in negative-stranded genomic RNA replication, whereas mature proteins may be required for subgenomic RNA (sgRNA) transcription. More interestingly, a mutant 3CLpro with either a P166S or P166L mutation was selected when an IBV infectious cDNA clone carrying the Q6327N mutation at the nsp15-16 site was introduced into cells. Either of the two mutations was proved to enhance significantly the 3CLpro-mediated cleavage efficiency at the nsp15-16 site with a P1-Asn substitution and compensate for the detrimental effects on recovery of infectious virus.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
authors.forename: Shouguo
authors.initials: S
authors.lastname: Fang
authors.forename: Hongyuan
authors.initials: H
authors.lastname: Shen
authors.forename: Jibin
authors.initials: J
authors.lastname: Wang
authors.forename: Felicia P L
authors.initials: FP
authors.lastname: Tay
authors.forename: Ding Xiang
authors.initials: DX
authors.lastname: Liu
mesh_terms: D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D001351:Infectious bronchitis virus / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases
references.citation: J Biol Chem. 2004 Jan 16;279(3):1637-42
references.pmid: 14561748
references.citation: Structure. 2004 Feb;12(2):341-53
references.pmid: 14962394
references.citation: J Gen Virol. 2003 Sep;84(Pt 9):2305-2315
references.pmid: 12917450
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13190-5
references.pmid: 14585926
references.citation: J Virol. 1996 Mar;70(3):1923-30
references.pmid: 8627718
references.citation: J Virol. 2007 Jan;81(1):20-9
references.pmid: 16928755
references.citation: J Virol. 2004 Nov;78(22):12218-24
references.pmid: 15507608
references.citation: Virology. 2009 Jan 20;383(2):328-37
references.pmid: 19022466
references.citation: J Virol. 2009 Jul;83(13):6957-62
references.pmid: 19386712
references.citation: Biochemistry. 2004 Apr 20;43(15):4568-74
references.pmid: 15078103
references.citation: PLoS Pathog. 2005 Dec;1(4):e39
references.pmid: 16341254
references.citation: J Virol. 2008 Sep;82(17):8647-55
references.pmid: 18562531
references.citation: Virology. 2008 Sep 30;379(2):175-80
references.pmid: 18678384
references.citation: J Virol. 2007 Jul;81(13):7086-98
references.pmid: 17428870
references.citation: Proc Natl Acad Sci U S A. 2004 Aug 24;101(34):12694-9
references.pmid: 15304651
references.citation: J Gen Virol. 2002 Mar;83(Pt 3):595-599
references.pmid: 11842254
references.citation: J Virol. 2004 Jun;78(11):5619-32
references.pmid: 15140959
references.citation: J Virol. 1999 Mar;73(3):2027-37
references.pmid: 9971783
references.citation: Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122-6
references.pmid: 3095828
references.citation: J Biol Chem. 2008 Feb 8;283(6):3655-3664
references.pmid: 18045871
references.citation: Proc Natl Acad Sci U S A. 2004 Mar 16;101(11):3792-6
references.pmid: 15007178
references.citation: J Mol Biol. 2003 Aug 29;331(5):991-1004
references.pmid: 12927536
references.citation: J Virol. 2006 Apr;80(8):4157-67
references.pmid: 16571831
references.citation: Nucleic Acids Res. 2006;34(17):4816-25
references.pmid: 16971454
references.citation: J Virol. 2004 Jul;78(14):7833-8
references.pmid: 15220459
references.citation: Proc Natl Acad Sci U S A. 2006 Aug 8;103(32):11892-7
references.pmid: 16882730
references.citation: Virology. 1998 Jul 5;246(2):288-97
references.pmid: 9657947
references.citation: J Virol. 2002 Jun;76(12):6257-67
references.pmid: 12021359
references.citation: J Biol Chem. 2002 Oct 18;277(42):39960-6
references.pmid: 12163505
references.citation: J Virol. 2007 Oct;81(19):10280-91
references.pmid: 17634238
references.citation: Nat Struct Mol Biol. 2005 Nov;12(11):980-6
references.pmid: 16228002
references.citation: J Virol. 2006 Aug;80(16):7902-8
references.pmid: 16873247
references.citation: J Mol Biol. 2005 Nov 11;353(5):1106-17
references.pmid: 16216269
references.citation: Proc Natl Acad Sci U S A. 2004 Jul 6;101(27):10012-7
references.pmid: 15226499
references.citation: J Gen Virol. 2000 Apr;81(Pt 4):853-79
references.pmid: 10725411
references.citation: J Biol Chem. 1996 Mar 1;271(9):4864-71
references.pmid: 8617757
references.citation: Virology. 1995 Apr 1;208(1):1-8
references.pmid: 11831690
references.citation: J Virol. 2003 Jan;77(2):1415-26
references.pmid: 12502857
references.citation: Biochem Biophys Res Commun. 2005 Apr 15;329(3):934-40
references.pmid: 15752746
references.citation: Nucleic Acids Res. 1989 Jun 26;17(12):4847-61
references.pmid: 2526320
references.citation: J Med Chem. 2006 Aug 10;49(16):4971-80
references.pmid: 16884309
references.citation: J Virol. 2007 Jun;81(12):6356-68
references.pmid: 17392363
references.citation: Curr Top Microbiol Immunol. 2005;287:57-94
references.pmid: 15609509
references.citation: J Virol. 2007 Nov;81(22):12135-44
references.pmid: 17804504
references.citation: Proc Natl Acad Sci U S A. 2006 Mar 28;103(13):5108-13
references.pmid: 16549795
references.citation: Microbiol Immunol. 2005;49(10):899-908
references.pmid: 16237267
references.citation: J Virol. 2007 Feb;81(4):1574-85
references.pmid: 17121802
references.citation: FEBS Lett. 2003 Oct 23;553(3):451-6
references.pmid: 14572668
references.citation: Biochemistry. 2007 Jul 31;46(30):8744-52
references.pmid: 17605471
references.citation: EMBO J. 2006 Oct 18;25(20):4933-42
references.pmid: 17024178
references.citation: J Virol. 1997 Mar;71(3):1814-20
references.pmid: 9032311
references.citation: FEMS Microbiol Rev. 2006 Sep;30(5):760-73
references.pmid: 16911043
references.citation: Virology. 2007 Feb 5;358(1):136-47
references.pmid: 16979681
references.citation: Virology. 2001 Sep 30;288(2):212-22
references.pmid: 11601893
references.citation: Virology. 1998 Mar 15;242(2):288-302
references.pmid: 9514967
references.citation: Virology. 1998 Apr 10;243(2):388-95
references.pmid: 9568037
references.citation: J Virol. 2006 Aug;80(16):7909-17
references.pmid: 16873248
references.citation: Science. 2003 Jun 13;300(5626):1763-7
references.pmid: 12746549
references.citation: J Virol. 2006 Aug;80(16):7894-901
references.pmid: 16873246
references.citation: J Virol. 2007 Dec;81(24):13587-97
references.pmid: 17898055
references.citation: EMBO J. 2002 Jul 1;21(13):3213-24
references.pmid: 12093723
references.citation: J Virol. 1994 Sep;68(9):5772-80
references.pmid: 8057459
references.citation: J Virol. 2008 Aug;82(16):8071-84
references.pmid: 18417574
references.citation: Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3484-9
references.pmid: 19208801
references.citation: J Virol. 2001 Jul;75(14):6676-81
references.pmid: 11413334
references.citation: Virology. 1995 Jun 1;209(2):420-7
references.pmid: 7778277
references.citation: PLoS Biol. 2005 Oct;3(10):e324
references.pmid: 16128623
references.citation: Cell. 2003 Jun 13;113(6):701-2
references.pmid: 12809601
references.citation: Virology. 2000 Jun 20;272(1):27-39
references.pmid: 10873746
references.citation: J Virol. 2008 Mar;82(5):2515-27
references.pmid: 18094151
references.citation: J Mol Biol. 2006 Aug 11;361(2):243-56
references.pmid: 16828802
references.citation: J Virol. 2007 Jun;81(12):6700-8
references.pmid: 17409150
references.citation: J Virol. 2008 Dec;82(24):12392-405
references.pmid: 18842706
references.citation: J Virol. 2006 Nov;80(21):10900-6
references.pmid: 16928748