Proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites

Abstract

Proteolytic processing of the polyprotein encoded by mRNA 1 is an essential step in coronavirus RNA replication and gene expression. We have previously reported that an open reading frame (ORF) 1a-specific proteinase of the picornavirus 3C proteinase group is involved in processing of the coronavirus infectious bronchitis virus (IBV) 1a/1b polyprotein, leading to the formation of a mature viral protein of 100 kDa. We report here the identification of a novel 10-kDa polypeptide and the involvement of the 3C-like proteinase in processing of the ORF 1a polyprotein to produce the 10-kDa protein species. By using a region-specific antiserum, V47, raised against a bacterial-viral fusion protein containing IBV sequence encoded between nucleotides 11488 and 12600, the 10-kDa polypeptide was detected in lysates from both IBV-infected and plasmid DNA-transfected Vero cells. Coexpression, deletion, and mutagenesis studies showed that this novel polypeptide was encoded by ORF 1a from nucleotide 11545 to 11878 and was cleaved from the 1a polyprotein by the 3C-like proteinase domain. Evidence presented suggested that a previously predicted Q-S (Q 3783 S 3784) dipeptide bond encoded by ORF 1a between nucleotides 11875 and 11880 was responsible for the release of the C terminus of the 10-kDa polypeptide and that a novel Q-N (Q 3672 N 3673) dipeptide bond encoded between nucleotides 11542 and 11547 was responsible for the release of the N terminus of the 10-kDa polypeptide.

Complete genomic sequence of turkey coronavirus

“…NSP5 was similar by 52%, 44%, and 92% at the nucleotide sequence level and by 39%, 44%, and 93% at the amino acids level to HCoV-229E, BCoV, and IBV, respectively. This region was believed to play a critical role for ORF1b processing since the deletion of NSP5 in IBV resulted in the unprocessing of 1b protein (Liu et al, 1997). NSP3, NSP4 and NSP6 were predicted to carry a hydrophobic transmembrane domain which may play an important role in the transcription/replication process as recently discussed for other coronaviruses (Sawicki et al, 2007).…”

Towards construction of viral vectors based on avian coronavirus infectious bronchitis virus for gene delivery and vaccine development

“…The 5 twothird region of mRNA1 comprises two large ORFs, 1a and 1b, and encodes two polyproteins. The two polyproteins are cleaved proteolytically by two virus-encoded proteinases, the papain-like and 3C-like proteinases, into 15 functional proteins (Nsp2-Nsp16) (Liu et al, 1995(Liu et al, , 1997Ng and Liu, 1998Xu et al, 2001). Compared to other coronaviruses, Nsp1 is absent in IBV but Nsp2 is considerably larger (Lim and Liu, 1998a,b;Lim et al, 2000;Liu et al, 1998).…”

Prediction and biochemical analysis of putative cleavage sites of the 3C-like protease of Middle East respiratory syndrome coronavirus

“…Comparing with the previous researches on the prediction and identification of 3CLpro cleavage sites, the scanning condition IV showed its advantages. For example, the two nonexistent putative cleavage sites predicted within nsp6 of IBV (Gorbalenya et al, 1989;Liu et al, 1997;Ng and Liu, 1998) were avoided in our prediction method (data not shown). Notably, the noncanonical cleavage site at the end of MHV nsp7 identified by Deming et al could be predicted using scanning condition IV.…”

“…Some cleavage sites have been identified and confirmed by previous studies, including three cleavage sites of PLpros of human coronavirus 229E (HCoV 229E), mouse hepatitis virus (MHV), SARS-CoV, MERS-CoV and infectious bronchitis virus (IBV), whose cleavages release the first 3 non-structural proteins (Bonilla et al, 1995;Kilianski et al, 2013;Lim and Liu, 1998;Ziebuhr et al, 2007). The canonical cleavage sites of 3CLpros, the sites between the recognized nsps, have also been characterized, including all sites of MHV, IBV, SARS-CoV and a fraction of sites of HCoV 229E which release the non-structural proteins from nsp4 to nsp16 (Deming et al, 2007;Grotzinger et al, 1996;Liu et al, 1994Liu et al, , 1997Lu et al, 1995). For 3CLpro of MERS-CoV, two cleavage sites releasing nsp4 to nsp6 have been identified (Kilianski et al, 2013).…”

“…Gorbalenya et al (1989) made the first systematical prediction on IBV pp1a/1ab according to the substrate specificity of 3C protease of picornaviruses. However, two of their predicted cleavage sites within nsp6 of IBV were proved uncleavable (Liu et al, 1997;Ng and Liu, 2000). Gao et al (2003) developed a software (ZCURVE CoV) to predict the nsps as well as gene-encoded ORFs of coronaviruses more accurately based on previous studies of 3CLpros cleavage sites of IBV, MHV and HCoV 229E.…”