A major research obstacle in the study of the Norwalk-like human caliciviruses has been our inability to grow them in cell culture. A major goal of this project is to develop systems for study of the basic molecular biology and replication of these fastidious human pathogens. We have developed the molecular characterization of cultivatable feline calicivirus (FCV) as a model for study of the human caliciviruses associated with epidemic gastroenteritis. The FCV RNA genome shares several important structural features with the human calicivirus genome but in contrast to the human strains, FCV grows efficiently in cell culture (feline kidney). A full-length cDNA clone of the RNA genome of the URB FCV strain was constructed in our laboratory and placed downstream of the T7 RNA polymerase promoter. RNA transcripts generated in vitro from this clone were infectious when introduced into feline kidney cells. This infectious clone has served as an important model in the development of such a system for the fastidious human caliciviruses. Previous studies of the proteins synthesized in feline kidney cells following infection with FCV had shown the presence of nonstructural proteins with molecular weights of approximately 96, 75, 39, 36 and 27 kDa. It was proposed that several of the proteins were cleavage products (intermediate-sized and mature forms) from a larger polyprotein. The presence in infected cells of ?intermediate? viral proteins suggests that regulatory mechanisms are involved in processing of the nonstructural polyprotein and correspondingly, in viral replication. Last year, we established that the ?3C-like? proteinase encoded in the FCV first open reading frame (ORF1) was responsible for cleavage of the capsid protein precursor translated from the subgenomic RNA message. The major goal of our work this year was to map the cleavage sites in the FCV nonstructural polyprotein in order to identify the boundaries of the mature FCV nonstructural proteins. In vitro translation of the FCV ORF1 cloned into the pTM1 plasmid led to the production of an active protease. Instead of the predicted polyprotein with an estimated size of 195 kDa, we observed at least four major protein bands with sizes ranging from approximately 30 to 80 kDa and several minor bands including a protein of approximately 14 kDa that were immunoprecipitated with cat FCV-infection serum. These four major proteins corresponded in size to four bands detected in FCV-infected CRFK cells in a Western blot reacted with the cat FCV infection serum. The similarities between products of this autocatalytic cleavage and proteins in FCV-infected cells suggested that the protease sequences encoded in ORF1 were sufficient to mediate cleavage of the ORF1 polyprotein in the absence of cellular factors and that the majority of proteins synthesized in vitro were identical in size to mature forms of the viral nonstructural proteins in infected cells. The similarity between the cleavage products observed in vitro and in infected cells allowed us to use the FCV ORF1 clone as a tool to identify the polyprotein cleavage. Antisera were developed in guinea pigs that were specific for discrete regions spanning the entire ORF1. In addition, the radiolabeled in vitro translation products were analyzed by direct microsequence analysis. A preliminary map of the FCV ORF1 polyprotein was determined. The map contains a total of six cleavage sites, that are cleaved in vitro to different extents. The map shares certain similarities with Southampton virus (SV), a ?Norwalk-like? virus, and rabbit hemorrhagic disease virus (RHDV) genomes. For example, all three viruses produce a predicted cleaved protein containing the ?2C? region that is similar in size (37-41 kDa). In addition, a small N-terminal cleavage product is apparently produced from the FCV and RHDV polyproteins. An interesting difference between FCV and RHDV is that the predicted mature 3C protease differs in size. In FCV, the 3C cleavage fragment is approximately 30 kDa; in RHDV, it is 15 kDa. An interesting difference between FCV and RHDV when compared with SV is that fewer cleavage products are observed in in vitro processing studies of the SV polyprotein. It is not yet clear whether the inefficient processing of the SV and Norwalk virus polyprotein is related to the inability to grow these viruses in available cell cultures.
