Turkey coronavirus non-structure protein NSP15--an endoribonuclease.
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.


CONCLUSIONS
The TCoV nsp15 is a NendoU but has some characteristics different from other NendoU.
Cao
Jianzhong
J
Department of Comparative Pathobiology, Purdue University, West Lafayette, IN, USA.
Wu
Ching-Ching
CC
Lin
Tsang Long
TL
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004752:Coronavirus, Turkey / Q000201:enzymology*; D004247:DNA / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D020536:Enzyme Activators / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D055503:Protein Multimerization; D012313:RNA / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
D020536:Enzyme Activators; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D012313:RNA; D004247:DNA; D004722:Endoribonucleases