INFECTIOUS BURSAL DISEASE VIRUS POLYPROTEIN PROCESSING DOES NOT INVOLVE CELLULAR PROTEASES

The larger genome segment, segment A, of infectious bursal disease vir us (IBDV) encodes VP2, VP3 and VP4 as a precursor polyprotein. The vir al protease, VP4, is responsible for self-processing of the polyprotei n, however, there are additional secondary precursor products such as VPX whose further processing has not been defined. Expression of IBDV cDNAs in vitro with rabbit reticulocyte lysates in a coupled transcrip tion-translation system and in the Sindbis virus expression system (wi th BHK-21 and Vero cell cultures) were used to study processing of the polyprotein. In both expression systems, we identified three main gen e products with molecular masses of 48, 34, and 30.5 kDa corresponding to VPX, VP3, and VP4, respectively, as found in IBDV-infected Vero ce ll cultures, although the amount of each product was variable. A trans lational time course of the polyprotein gene and analyses of products specified by various sub-clones of the full-length cDNA were used to d istinguish primary processing products of translation from secondary p roducts generated by proteolytic processing during in vitro coupled tr anscription-translation expression. The VPX, VP3 and VP4, which are th e primary processing products, first appeared after 20 min of incubati on and their production was maximum by 75 min of the coupled transcrip tion-translation reaction. Cycloheximide chases demonstrated that ther e is no secondary processing of VPX (or VP3 and VP4). Thus VP2, the ma jor capsid protein in virions, was not detected either in translation products of rabbit reticulocyte lysates or in lysates of Sindbis virus recombinant-infected cell cultures indicating the absence of secondar y processing of VPX to VP2 during foreign expression of the segment A cDNA. We conclude that VPX maturation to VP2 does not involve cellular proteases.