Turkey Coronavirus Non-Structure Protein NSP15 – An Endoribonuclease

Abstract

Turkey coronavirus (TCoV) polyprotein was predicted to be cleaved into 15 non-structural proteins (nsp2 to nsp16), but none of these nsps have been characterized. TCoV nsp15 consists of 338 residues and shares 40% sequence similarity to U-specific Nidovirales endoribonuclease (NendoU) of severe acute respiratory syndrome coronavirus. Objective: The purpose of the present study was to characterize TCoV nsp15. Methods: The TCoV nsp15 gene was cloned into pTriEX1 and expressed as a C-terminal His-tagged recombinant protein in BL21 (DE3). The recombinant nsp15 was purified by Ni-NTA resin. Synthetic RNA substrates were used to determine the substrate specificity of the TCoV nsp15. RNA zymography was used to determine the active form of the nsp15. Results: The TCoV nsp15 did not cleave DNA but degraded total cellular RNA. The TCoV nsp15 cleaved single-stranded (ss) RNA at the uridylate site. The TCoV nsp15 cleaved hairpin RNA, pRNA, and double-stranded RNA (dsRNA) of infectious bursal disease virus very slowly, implying that dsRNA is not a good substrate for the TCoV nsp15. No divalent metal ion was required for in vitro enzymatic activity of the TCoV nsp15. The active form of the TCoV nsp15 was a homohexamer and disulfide bond was essential for the enzymatic activity. Conclusion: The TCoV nsp15 is a NendoU but has some characteristics different from other NendoU.

Comparative in vivo analysis of the nsp15 endoribonuclease of murine, porcine and severe acute respiratory syndrome coronaviruses

“…As a result, intermediate precursors likely exist and interactions with other viral nsps may interfere with its self-interaction. In this regard, it is important to note that although bacteria-expressed nsp15 forms hexamers and exhibits endonuclease activity in vitro (Cao et al, 2008;Guarino et al, 2005), these biochemical properties have yet to be demonstrated in virus-infected or ectopically expressed cells.…”

“…Indeed, comparative sequence analysis has predicted that coronaviral nsp15 may have endoribonuclease activity similar to Xenopus endoU (XendoU) that cleaves U on single-stranded RNA molecule and requires Mn 2+ as a cofactor (Bhardwaj et al, 2004;Gioia et al, 2005;Laneve et al, 2003). The nidoviral endoU (NendoU) activity was first demonstrated biochemically in vitro for SARS-CoV nsp15 expressed from bacteria (Bhardwaj et al, 2004), and subsequently confirmed in bacteria-expressed nsp15s from MHV (Kang et al, 2007;Xu et al, 2006), human CoV-229E (Ivanov et al, 2004), turkey CoV (Cao et al, 2008), and in nsp11 of equine arteritis virus, a cousin of coronavirus within the order of Nidovirales (Nedialkova et al, 2009). Unlike XendoU which acts as a monomer, NendoU appears to maintain its activity as a hexamer in vitro (Guarino et al, 2005;Joseph et al, 2007;Ricagno et al, 2006;Xu et al, 2006).…”

Structure and Cleavage Specificity of the Chymotrypsin-Like Serine Protease (3CLSP/nsp4) of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

“…Disulfide bonds are rare in proteins that function in the cytosol, but for instance in coronavirus nonstructural proteins, several cases have been observed. [42][43][44] We presume that the side chain of Cys111 can adopt a different conformation and form a disulfide bond with Cys115, perhaps after substrate binding to facilitate the proteolysis. This should be addressed in the future with a substrate-enzyme complex structure.…”

Nidovirus ribonucleases: Structures and functions in viral replication

“…42 Endoribonuclease activity has been confirmed for nsp15 from the coronaviruses SARS-CoV, HCoV-229E, MHV-A59, Avian infectious bronchitis virus (IBV) and Turkey coronavirus and for nsp11 from the arteriviruses Equine arteritis virus (EAV) and PRRSV. 41,42,[75][76][77] Similar to XendoU, NendoUs are endoribonucleases that cleave 3' of pyrimidines, preferring uridine over cytidine and releasing products with 2',3'-cyclic phosphate and 5'-OH ends. A marked preference for cleavage after unpaired over paired pyrimidines has been demonstrated comparing the efficiency of cleavage of ssRNA versus dsRNA and using an RNA with a known secondary structure.…”