CTMI (2005) 287:57-94 ? Springer-Verlag 2005 The Coronavirus Replicase J. Ziebuhr Institute of Virology and Immunology, University of W?rzburg, Versbacher Str. 7, 97078 W?rzburg, Germany j.ziebuhrmail.uni-wuerzburg.de 1Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58 2Organization and Expression of the Replicase Gene . . . . . . . . . . .59 3Replicase Polyproteins. . . . . . . . . . . . . . . . . . . . . . . . . . . .61 3.1Functional Domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61 3.2Proteolytic Processing by Viral Cysteine Proteinases . . . . . . . . . . .64 3.2.1Accessory Proteinases . . . . . . . . . . . . . . . . . . . . . . . . . . . .66 3.2.2Main Proteinase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69 3.3Helicase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76 3.4RNA-Dependent RNA Polymerase . . . . . . . . . . . . . . . . . . . . .78 4Subcellular Localization of the Coronavirus Replicase . . . . . . . . . .79 5Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . .83 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83 Abstract Coronavirus genome replication and transcription take place at cytoplasmic membranes and involve coordinated processes of both continuous and discontinu- ous RNA synthesis that are mediated by the viral replicase, a huge protein complex encoded by the 20-kb replicase gene. The replicase complex is believed to be com- prised of up to 16 viral subunits and a number of cellular proteins. Besides RNA-de- pendent RNA polymerase, RNA helicase, and protease activities, which are common to RNA viruses, the coronavirus replicase was recently predicted to employ a variety of RNA processing enzymes that are not (or extremely rarely) found in other RNA viruses and include putative sequence-specific endoribonuclease, 30-to-50exoribonu- clease, 20-O-ribose methyltransferase, ADP ribose 100-phosphatase and, in a subset of group 2 coronaviruses, cyclic phosphodiesterase activities. This chapter reviews (1) the organization of the coronavirus replicase gene, (2) the proteolytic processing of the replicase by viral proteases, (3) the available functional and structural informa- tion on individual subunits of the replicase, such as proteases, RNA helicase, and the RNA-dependent RNA polymerase, and (4) the subcellular localization of coronavirus proteins involved in RNA synthesis. Although many molecular details of the corona- virus life cycle remain to be investigated, the available information suggests that these viruses and their distant nidovirus relatives employ a unique collection of en- zymatic activities and other protein functions to synthesize a set of 50-leader-con- taining subgenomic mRNAs and to replicate the largest RNAvirus genomes current- ly known. 1 Introduction Plus-strand (+) RNA viruses exhibit an enormous genetic diversity that also applies to their RNA synthesis machinery. The RNA-depen- dent RNA polymerase (RdRp) is the only enzyme to be absolutely con- served, whereas other replicative and accessory protein domains vary considerably, in terms of both number and arrangement in the polypro- tein (Koonin and Dolja 1993). Despite this diversity, phylogenetic rela- tionships have been identified and used to group +RNA viruses into large superfamilies (or classes) (Goldbach 1987; Strauss and Strauss 1988; Koonin and Dolja 1993). As few as three superfamilies, the pico- rnavirus-like, flavivirus-like and alphavirus-like viruses, were proposed to accommodate the vast majority of +RNA viruses infecting animals, plants, and microorganisms (Koonin and Dolja 1993). Interestingly, coronaviruses were among the few exceptions that did not easily fit into one of the established superfamilies; and the sequence analysis and characterization of arteri-, toro-, and roniviruses suggested that coron- aviruses and their relatives may indeed exemplify a viral life form that, in several fundamental aspects, differs from that of other +RNA viruses (Gorbalenya et al. 1989c; Snijder et al. 1990a; den Boon et al. 1991; Sni- jder and Horzinek 1993; de Vries et al. 1997; Lai and Cavanagh 1997; Snijder and Meulenberg 1998; Cowley et al. 2000). Thus coronaviruses (and all their relatives) (1) produce a nested set of 30-coterminal mRNAs (Lai et al. 1983; Spaan et al. 1983), (2) use ribosomal frameshifting into the 1 frame to express their key replicative functions (Brierley et al. 1987, 1989), (3) have a unique set of conserved functional domains that are arranged in the viral polyproteins in the following order: chymo- trypsin-like proteinase, RdRp, helicase, and endoribonuclease (from N- to C-terminus) (Gorbalenya et al. 1989c; Gorbalenya 2001; Snijder et al. 2003), and (4) use RdRp and helicase activities that, based on the conservation of signature motifs, have been classified as belonging to the RdRp and helicase superfamilies 1, respectively (Ko