Volume 288, number 1,2, 201-205 FEES 10058 August 1991 0 1991 Federation of European Biochemical Societies 00145793/91/53.50 ADONIS 001457939100768X Putative papain-related thiol prsteases of positive-strand RNA viruses Identification of rubi- and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, a- and coronaviruses Alexander E. Gorbalenyal, Eugene V. Koonin2 and Michael M.-C. Lai3 1 Institute of Poliomyelitis and Viral Encephalitides, USSR Academy of Medical Sciences, 142782 Moscow Region, USSR, aInstitute of MicroBiology, USSR Academy of Sciences, II 7811 Moscow, USSR and 3Howard Hughes Medical Institute and Department of Microbioiogy. University of Southern California School of Medicine, Los Angeles, CA 900.93, USA Received 13 June 1991 A computer-assisted comparative analysis of the amino acid sequences of (putative) thiol proteases encoded by the genomcs of scvcral diverse groups of positive-stranded RNA viruses and distantly related to the family of cellular papain-like proteases is presented. A high level of similarity was detected between the leader protcasc of foot-and-mouth-disease virus and the protease of murine hepatitis coronavirus which cleaves the N-terminal 28 protein from the polyprotcin. Statistically significant alignment of a portion of the rubella virus polyprotein with cellular papain-like proteascs was obtained, lasding to tentative identification of the papain-like protcase as the enzyme mediating processing of the non-structural proteins of this virus. Specific grouplyg between the sequences of the proteases of cr-viruses, and poty- and bymoviruses was revealed. It was noted that papain- like protcases of positive-str;qded RNA viruws are much more variable both in their sequences and in genomic locations than chymotrypsin-related proteases found in the same vim: class. A novel conarvcd domain of unknown function has also been identified which flanks the papain-like pro. teases of er-. rubi- and coronaviruses. Papain-like protease; RNA virus; Polyprotein processing; Sequence motif; Catalytic center 1. INTRODUCTION Polyprotein processing is the strategy employed by a number of groups of positive-stranded RNA viruses for genome expression (for review see l). Processing of membrane proteins of enveloped viruses is usually mediated by cellular proteases, whereas processing of non-membrane proteins by virus-encoded proteases. A Correspondence ud CPl and CP2, Dictyostelium discoideum cysteine proteinase I and 2; actin. Actinidia chinensis actinidin; papain and omega, Carica papaya pro- teinase I and 111, respectively; aleur, Hordeum vtdgare aleurain; Cat.H and L, Rattus norvegicus cathepsins H and L; Cat.B, Homo supiens cathepsin B; SH-EP, Vigno mungo cysteine endopeptidase; bromel, Ananas comosus bromelain; derpt. Detrnatophagoides pteronyssinus major mite fecal allergen; calp, Mus musculus calpain; SFV, Semliki forest virus; SNBV, Sindbis virus; VEBV, Venezuelan equine encephalomyelitis virus; ONNV, ONyong-Nyong virus; RRV, Ross River virus; MidV. Middelburg virus (alphaviruses); PVY, potato virus Y; PPV. plum pox virus; TEV, tobacco etch virus; TVMV, tobacco vein mottling virus (potyviruses); BaYMV, barley yellow mosaic virus (bymovirus); MHV, murine hepatitis virus: 1BV. avian bronchitis virus (coronaviruses); FMDV AlO, foot-and-mouth- disease virus A10 strain (aphthovirus); RuV, rubella virus (rubivirus); HC, helper component; M-pro, main protease; L-pro, leader, or accessory protease (see text); SPL, Streptococcus-like protease; CI, cylindrical inclusion (potyvirus protein). large superfamily of virus-encoded proteases related to chymotrypsin-like cellular serine proteases has been described 12-51. Some of these viral proteases have the substitution of Cys for the principal catalytic Ser, not found in cellular enzymes, comprising a unique group of cysteine proteases. Only very recently, the existence of classical cys- teine proteases related to papain-like cellular enzymes has been claimed for several positive-stranded RN;i viruses. The essential Cys and His residues were iden- tified in the potyvirus CI 6, cr-virus nsP2 7,8 and murine coronavirus leader (L-pro) (9, and Baker, et al., submitted) proteases by site-directed mutagenesis. The relative positions of these residues in the respective proteins and their amino acid contexts resemble those of the catalytic residues in the papain-like proteases 6-g. Two other putative papain-like proteases were revealed in the polyproteins of coronaviruses by com- parative sequence analysis. The putative main. pro- tease (M-pro) is related to MHV L-pro and is conserved in both IBV and MHV polyproteins lo, and the putative SPL protease shares similarity to the protease from Streptococcus and is present in IBV polyprotein only ll. Two proteases of picornaviruses, L-pro of FMDV and VPO, have not been characterized with respect to the type of the catalytic residues l. In addition, the