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

文献类型：期刊论文

作者Xiao, Yibei2,4; Ma, Qingjun2; Restle, Tobias3; Shang, Weifeng5; Svergun, Dmitri I.5; Ponnusamy, Rajesh2; Sczakiel, Georg3; Hilgenfeld, Rolf1,2,6
刊名JOURNAL OF VIROLOGY
出版日期2012-04
卷号86期号:8页码:4444-4454
ISSN号0022-538X
DOI10.1128/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.
WOS关键词SARS-CORONAVIRUS ; SOLUTION SCATTERING ; HEPATITIS-VIRUS ; DEPENDENT RNA ; TRANSCRIPTION ; REPLICATION ; INSIGHTS ; NSP7
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研究方向Virology
语种英语
WOS记录号WOS:000302185400038
出版者AMER SOC MICROBIOLOGY
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.

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来源：上海药物研究所

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