Further Requirements for Cleavage by the Murine Coronavirus 3C-like Proteinase: Identification of a Cleavage Site within ORF1b Josefina D. Pin o n, Henry Teng, and Susan R. Weiss1 Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076 Received May 25, 1999; returned to author for revision July 1, 1999; accepted August 4, 1999 The coronavirus mouse hepatitis virus strain A59 (MHV-A59) encodes a 3C-like proteinase (3CLpro) that is proposed to be responsible for the majority of the processing events that take place within the replicase polyproteins pp1a and pp1ab. In this study we demonstrate that the Q9392S940 peptide bond, located between the polymerase and Zn-finger regions of pp1ab (the POL2Zn site), is processed by the 3CLpro, albeit inefficiently. Mutagenesis of the POL2Zn site, as well as the previously identified HD123C site in the 1a region of pp1a and pp1ab, demonstrated that the amino acid residues at the P2 and P1 positions of the cleavage site, occupied by L and Q, respectively, were important determinants of 3CLpro substrate specificity. Finally, a direct comparison of the 3CLpro-mediated cleavages at the HD123C and POL2Zn sites was made by determining the rate constants using synthetic peptides. The results show that while a larger polypeptide substrate carrying the HD123C site was processed more efficiently than a polypeptide substrate carrying the POL2Zn site, cleavage of the synthetic peptide substrates containing these two cleavage sites occurred at similar efficiencies. This indicates that the overall conformation of a large polyprotein substrate is important in the accessibility of the cleavage site to the proteinase. 1999 Academic Press INTRODUCTION The family Coronaviridae is composed of a group of viruses that cause a variety of diseases in different animal hosts. The murine coronavirus, mouse hepatitis virus (MHV), causes a range of diseases in mouse, including enteritis, hepatitis, encephalitis, and a demy- elinating disease (Holmes and Lai, 1996; Houtman and Fleming, 1996; Lai, 1990). Coronaviruses, along with the arteriviruses, are classified under the newly established order Nidovirales based on the similarities in their ge- nome organization and replication strategy (Cavanagh, 1997; de Vries et al., 1997). The name Nidovirales origi- nates from the Latin word nidus, meaning “nest,” and refers to the 39 nested set of subgenomic mRNAs that is produced during viral infection (de Vries et al., 1997). As with all positive-strand RNA viruses, entry of the viral genome into the cytoplasm is followed by the translation of the positive-strand RNA genome, resulting in the ex- pression of viral proteins. The coronavirus genome is organized into seven genes that are separated by stop codons and intergenic sequences (Holmes and Lai, 1996; Lai, 1990). Thus, translation of the viral genome results only in the expression of gene 1 proteins. The rest of the viral genome is expressed through subgenomic mRNAs that are transcribed by the viral RNA-dependent RNA polymerase encoded in gene 1. Replication of the viral genome also requires the replicase proteins en- coded in gene 1. Thus, for both viral replication and subgenomic mRNA transcription to take place, the ex- pression of gene 1 products is essential. The replicase gene (gene 1) (Fig. 1) of coronaviruses, spanning 2022 kb, is organized into two overlapping open reading frames, ORF1a and ORF1b (Bonilla et al., 1994; Lee et al., 1991). The expression of the downstream ORF1b is mediated by a ribosomal frameshift event that is aided by the formation of a pseudoknot structure within the overlapping region (Bredenbeek et al., 1990; Brierley et al., 1987; Herold and Siddell, 1993). Thus, two polypeptides, pp1a and pp1ab, are expressed from gene 1, with the translation of pp1ab being only 2540% as efficient as that of pp1a in in vitro studies (Bredenbeek et al., 1990; Brierley et al., 1987). Through a series of intri- cate cotranslational and posttranslational processing events, these polyproteins are converted into a func- tional complex that in turn is responsible for both genomic RNA replication and subgenomic mRNA tran- scription (de Vries et al., 1997). Responsible for these processing events are at least two or three viral protein- ases encoded within the ORF1a region of gene 1 (Fig. 1). Two of these proteinase domains, by sequence analysis, share similarities with the cellular proteinase papain. A third proteinase, resembling the poliovirus 3C protein- ase, has also been identified (Gorbalenya et al., 1989; Lee et al., 1991). The coronavirus 3C-like proteinase 1To whom correspondence and reprint requests should be ad- dressed at 203A Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104-6076. Fax: (215) 573 4858. E-mail: weisssrmail.med. upenn.edu. Virology 263, 471484 (1999) Article ID viro.1999.9954, available online at on 0042-6822/99 $30.00 Copyright 1999 by Academic Press