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From Proteopedia
The structure of PEDV NSP9
Structural highlights
FunctionR1AB_PEDV7 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).[PROSITE-ProRule:PRU00772] 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. Nsp7-nsp8 hexadecamer may possibly confer processivity to the polymerase, maybe by binding to dsRNA or by producing primers utilized by the latter. Nsp9 is a ssRNA-binding protein. NendoU is a Mn(2+)-dependent, uridylate-specific enzyme, which leaves 2'-3'-cyclic phosphates 5' to the cleaved bond. Publication Abstract from PubMedCoronaviruses pose serious health threats to humans and other animals. Understanding the mechanisms of their replication has important implications for global health and economic stability. Nonstructural protein 9 (nsp9) is an essential RNA binding protein for coronavirus replication. However, the mechanisms of the dimerization and nucleic acid binding of nsp9 remain elusive. Here, we report four crystal structures, including wild-type porcine delta coronavirus (PDCoV) nsp9, PDCoV nsp9-DeltaN7 (N-terminal 7 amino acids deleted), wild-type porcine epidemic diarrhea virus (PEDV) nsp9, and PEDV nsp9-C59A mutant. These structures reveal the diverse dimerization forms of coronavirus nsp9. We first found that the N-finger of nsp9 from PDCoV plays a critical role in dimerization. Meanwhile, PEDV nsp9 is distinguished by the presence of a disulfide bond in the dimer interface. Interestingly, size exclusion chromatography and analytical ultracentrifugation analyses indicate that the PDCoV nsp9-DeltaN7 and PEDV nsp9-C59A mutants are monomeric in solution. In addition, electrophoretic mobility shift assays and microscale thermophoresis analysis indicate that the monomeric forms of PDCoV nsp9 and PEDV nsp9 still have nucleic acid binding affinity, although it is lower than that of the wild type. Our results show that the diverse dimerization forms of coronavirus nsp9 proteins enhance their nucleic acid binding affinity.IMPORTANCE Coronaviruses cause widespread respiratory, gastrointestinal, and central nervous system diseases in humans and other animals, threatening human health and causing economic loss. Coronavirus nsp9, a member of the replication complex, is an important RNA binding subunit in the RNA-synthesizing machinery of all coronaviruses. However, the mechanisms of the dimerization and nucleic acid binding of nsp9 remain elusive. In this study we determined the nsp9 crystal structures of PDCoV and PEDV. We first found that the N-finger of nsp9 from PDCoV plays a critical role in dimerization. Meanwhile, PEDV nsp9 is distinguished by the presence of a disulfide bond in the dimer interface. This study provides a structural and functional basis for understanding the mechanism of dimerization and shows that the diverse dimerization modes of coronavirus nsp9 proteins enhance their nucleic acid binding affinity. Importantly, these findings may provide a new insight for antiviral drug development. Dimerization of Coronavirus nsp9 with Diverse Modes Enhances Its Nucleic Acid Binding Affinity.,Zeng Z, Deng F, Shi K, Ye G, Wang G, Fang L, Xiao S, Fu Z, Peng G J Virol. 2018 Aug 16;92(17). pii: JVI.00692-18. doi: 10.1128/JVI.00692-18. Print , 2018 Sep 1. PMID:29925659[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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