Nonstructural Proteins 7 and 8 of Feline Coronavirus Form a 2:1 Heterotrimer That Exhibits Primer-Independent RNA Polymerase Activity

doi:10.1128/JVI.06635-11

	Nonstructural Proteins 7 and 8 of Feline Coronavirus Form a 2:1 Heterotrimer That Exhibits Primer-Independent RNA Polymerase Activity
	Xiao, Yibei 2,4; Ma, Qingjun 2; Restle, Tobias 3; Shang, Weifeng 5; Svergun, Dmitri I.5; Ponnusamy, Rajesh 2; Sczakiel, Georg 3; Hilgenfeld, Rolf 1,2,6
刊名	JOURNAL OF VIROLOGY
	2012-04
卷号	86 期号:8 页码:4444-4454
ISSN号	0022-538X
DOI	10.1128/JVI.06635-11
文献子类	Article
英文摘要	Nonstructural proteins 7 and 8 of severe acute respiratory syndrome coronavirus (SARS-CoV) have previously been shown by X-ray crystallography to form an 8:8 hexadecamer. In addition, it has been demonstrated that N-terminally His(6)-tagged SARS-CoV Nsp8 is a primase able to synthesize RNA oligonucleotides with a length of up to 6 nucleotides. We present here the 2.6-angstrom crystal structure of the feline coronavirus (FCoV) Nsp7:Nsp8 complex, which is a 2:1 heterotrimer containing two copies of the alpha-helical Nsp7 with conformational differences between them, and one copy of Nsp8 that consists of an alpha/beta domain and a long-alpha-helix domain. The same stoichiometry is found for the Nsp7:Nsp8 complex in solution, as demonstrated by chemical cross-linking, size exclusion chromatography, and small-angle X-ray scattering. Furthermore, we show that FCoV Nsp8, like its SARS-CoV counterpart, is able to synthesize short oligoribonucleotides of up to 6 nucleotides in length when carrying an N-terminal His(6) tag. Remarkably, the same protein harboring the sequence GPLG instead of the His(6) tag at its N terminus exhibits a substantially increased, primer-independent RNA polymerase activity. Upon addition of Nsp7, the RNA polymerase activity is further enhanced so that RNA up to template length (67 nucleotides) can be synthesized. Further, we show that the unprocessed intermediate polyprotein Nsp7-10 of human coronavirus (HCoV) 229E is also capable of synthesizing oligoribonucleotides up to a chain length of six. These results indicate that in case of FCoV as well as of HCoV 229E, the formation of a hexadecameric Nsp7:Nsp8 complex is not necessary for RNA polymerase activity. Further, the FCoV Nsp7:Nsp8 complex functions as a noncanonical RNA polymerase capable of synthesizing RNA of up to template length.
资助项目	European Commission[LSHG-CT-2004-511960] ; European Commission[HEALTH-F3-2010-260644] ; DFG Cluster of Excellence[EXC306] ; Fonds der Chemischen Industrie[00000000] ; Chinese Academy of Sciences (CAS)[2010T1S6] ; BMBF[05K10YEA]
WOS关键词	SARS-CORONAVIRUS ; SOLUTION SCATTERING ; HEPATITIS-VIRUS ; DEPENDENT RNA ; TRANSCRIPTION ; REPLICATION ; INSIGHTS ; NSP7
WOS研究方向	Virology
语种	英语
出版者	AMER SOC MICROBIOLOGY
WOS记录号	WOS:000302185400038
内容类型	期刊论文
源URL	[http://119.78.100.183/handle/2S10ELR8/278135]
专题	中国科学院上海药物研究所
通讯作者	Hilgenfeld, Rolf
作者单位	1.Chinese Acad Sci, Shanghai Inst Mat Med, Shanghai 200031, Peoples R China 2.Univ Lubeck, Ctr Struct & Cell Biol Med, Inst Biochem, Lubeck, Germany; 3.Univ Lubeck, Ctr Struct & Cell Biol Med, Inst Mol Med, Lubeck, Germany; 4.Univ Lubeck, Grad Sch Comp Med & Life Sci, Lubeck, Germany; 5.DESY, EMBL Hamburg Outstat, D-2000 Hamburg, Germany; 6.DESY, Lab Struct Biol Infect & Inflammat, D-2000 Hamburg, Germany;
推荐引用方式 GB/T 7714	Xiao, Yibei,Ma, Qingjun,Restle, Tobias,et al. Nonstructural Proteins 7 and 8 of Feline Coronavirus Form a 2:1 Heterotrimer That Exhibits Primer-Independent RNA Polymerase Activity[J]. JOURNAL OF VIROLOGY,2012,86(8):4444-4454.
APA	Xiao, Yibei.,Ma, Qingjun.,Restle, Tobias.,Shang, Weifeng.,Svergun, Dmitri I..,...&Hilgenfeld, Rolf.(2012).Nonstructural Proteins 7 and 8 of Feline Coronavirus Form a 2:1 Heterotrimer That Exhibits Primer-Independent RNA Polymerase Activity.JOURNAL OF VIROLOGY,86(8),4444-4454.
MLA	Xiao, Yibei,et al."Nonstructural Proteins 7 and 8 of Feline Coronavirus Form a 2:1 Heterotrimer That Exhibits Primer-Independent RNA Polymerase Activity".JOURNAL OF VIROLOGY 86.8(2012):4444-4454.