3ub0
Crystal structure of the nonstructural protein 7 and 8 complex of Feline Coronavirus
Structural highlights
FunctionR1AB_FIPV The replicase polyprotein of coronaviruses is a multifunctional protein: it contains the activities necessary for the transcription of negative stranded RNA, leader RNA, subgenomic mRNAs and progeny virion RNA as well as proteinases responsible for the cleavage of the polyprotein into functional products. The papain-like proteinase 1 (PLP1) and papain-like proteinase 2 (PLP2) are responsible for the cleavages located at the N-terminus of the replicase polyprotein. In addition, PLP2 possesses a deubiquitinating/deISGylating activity and processes both 'Lys-48'- and 'Lys-63'-linked polyubiquitin chains from cellular substrates. PLP2 also antagonizes innate immune induction of type I interferon by blocking the nuclear translocation of host IRF-3 (By similarity). The main proteinase 3CL-PRO is responsible for the majority of cleavages as it cleaves the C-terminus of replicase polyprotein at 11 sites. Recognizes substrates containing the core sequence [ILMVF]-Q-|-[SGACN]. Inhibited by the substrate-analog Cbz-Val-Asn-Ser-Thr-Leu-Gln-CMK. Also contains an ADP-ribose-1-phosphate (ADRP)-binding function (By similarity). The helicase which contains a zinc finger structure displays RNA and DNA duplex-unwinding activities with 5' to 3' polarity. ATPase activity is strongly stimulated by poly(U), poly(dT), poly(C), poly(dA), but not by poly(G) (By similarity). The exoribonuclease acts on both ssRNA and dsRNA in a 3' to 5' direction (By similarity). Nsp7-nsp8 hexadecamer may possibly confer processivity to the polymerase, maybe by binding to dsRNA or by producing primers utilized by the latter (By similarity). Nsp9 is a ssRNA-binding protein (By similarity). NendoU is a Mn(2+)-dependent, uridylate-specific enzyme, which leaves 2'-3'-cyclic phosphates 5' to the cleaved bond (By similarity). Publication Abstract from PubMedNon-structural proteins 7 and 8 of SARS coronavirus 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. Here we present the 2.6-A 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 oligoribomucleotides of up to 6 nucleotides in length when carrying an N-terminal hexahistidine tag. Remarkably, the same protein harbouring 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 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 non-canonical RNA polymerase capable of synthesizing RNA of up to template-length. Non-structural Proteins 7 and 8 of Feline Coronavirus Form a 2:1 Heterotrimer that Exhibits Primer-independent RNA Polymerase Activity.,Xiao Y, Ma Q, Restle T, Shang W, Svergun DI, Ponnusamy R, Sczakiel G, Hilgenfeld R J Virol. 2012 Feb 8. PMID:22318142[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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