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1、Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase Yu Chena, Hui Caia, Jian Pana, Nian Xianga, Po Tiena, Tero Aholab, and Deyin Guoa,1 aState Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan Univer
2、sity, Wuhan 430072, Peoples Republic of China; and bInstitute of Biotechnology, University of Helsinki, 00014, Helsinki, Finland Edited by Paul Ahlquist, University of Wisconsin, Madison, WI, and approved December 30, 2008 (received for review September 4, 2008) The N7-methylguanosine (m7G) cap is t
3、he defi ning structural feature of eukaryotic mRNAs. Most eukaryotic viruses that repli- cate in the cytoplasm, including coronaviruses, have evolved strat- egies to cap their RNAs. In this report, we used a yeast genetic system to functionally screen for the cap-forming enzymes en- coded by severe
4、acute respiratory syndrome (SARS) coronavirus and identifi ed the nonstructural protein (nsp) 14 of SARS corona- virus as a (guanine-N7)-methyltransferase (N7-MTase) in vivo in yeast cells and in vitro using purifi ed enzymes and RNA substrates. Interestingly, coronavirus nsp14 was previously charac
5、terized as a 3?-to-5? exoribonuclease, and by mutational analysis, we mapped the N7-MTase domain to the carboxy-terminal part of nsp14 that shows features conserved with cellular N7-MTase in structure- based sequence alignment. The exoribonuclease active site was dispensable but the exoribonuclease
6、domain was required for N7-MTase activity. Such combination of the 2 functional domains in coronavirus nsp14 suggests that it may represent a novel form of RNA-processing enzymes. Mutational analysis in a replicon system showed that the N7-MTase activity was important for SARS virusreplication/trans
7、criptionandcanthusbeusedasanattractive drug target to develop antivirals for control of coronaviruses including the deadly SARS virus. Furthermore, the observation that the N7-MTase of RNA life could function in lieu of that in DNA life provides interesting evolutionary insight and practical possibi
8、lities in antiviral drug screening. RNA capping ? exoribonuclease ? yeast ? alphavirus ? fl avivirus E ukaryotic mRNAs possess a 5?-terminal cap structure, in which an N7-methyl-guanine moiety is linked to the first transcribed nucleotide by a 5?-5? triphosphate bridge. Cap formation requires 3 sequ
9、ential reactions catalyzed by RNA triphosphatase (TPase), guanylyltransferase (GTase), and (gua- nine-N7)-methyltransferase (N7-MTase) (1). Most cytoplasmi- cally replicating eukaryotic viruses that do not have access to the cellular capping apparatus located in the nucleus have evolved strategies t
10、o cap their RNAs. Although the final cap structures of viral and cellular mRNAs are similar, some RNA viruses adopt unique biochemical mechanisms to build the cap structure (24). Coronaviruses are the etiological agents of respiratory and enteric diseases in humans and livestock, exemplified by the
11、life-threatening severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) (5). The large genomic RNA of coronaviruses contains 2 large ORFs, 1a and 1b, encoding for viral replicase/transcriptase at the 5? terminus and a varied number of ORFs encoding structural and virus- speci
12、fic accessory proteins at the 3? terminus. The large polypep- tides translated from ORFs 1a and 1b are processed into 16 nonstructural proteins (nsps) by 23 viral proteinases (6). Strik- ingly, the coronavirus genome is predicted to encode several RNA processing enzymes that are not present in small
13、 RNA viruses, including nsp14 exoribonuclease (ExoN) (7, 8) and nsp15 endoribonuclease (9). Although the RNAs of coronaviruses and related toroviruses appear to carry a 5?-cap structure (10, 11), the enzymes involved in RNA capping are poorly characterized. Coronavirus nsp13 has been shown to exhibi
14、t an RNA TPase activity in vitro (12) but a direct role of nsp13 in RNA capping still awaits experi- mental evidence. Coronavirus nsp16 was predicted to be an S-adenosyl-L-methionine (SAM)-dependent RNA 2?-O MTase (6, 13), and, recently, this activity was confirmed for nsp16 of Feline coronavirus (F
15、CoV) (14). However, the N7-MTase and GTase essential for RNA cap formation are currently unknown for coronaviruses. Although a bioinformatic analysis predicted that the SARS-CoV unique domain (SUD) within nsp3 (nsp3- SUD) may possess N7-MTase activity (15), this assumption is weakened by the fact th
16、at most coronaviruses do not encode this domain (6). Therefore, identification and characterization of N7-MTase and GTase would be of paramount importance to elucidation of the RNA capping mechanisms of coronaviruses. Here we used a yeast genetic system to screen for the 3 essential capping enzymes (TPase, GTase, and N7-MTase) possibly encoded by coronaviruses. In previous studies, it has been shown that the capping functions of yeast cells can be replaced by the cap-forming enzymes of mammals a
