Infectious bursal disease virus polyprotein processing does not involve cellular proteases

Abstract

The larger genome segment, segment A, of infectious bursal disease virus (IBDV) encodes VP2, VP3 and VP4 as a precursor polyprotein. The viral protease, VP4, is responsible for self-processing of the polyprotein, 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 transcription-translation system and in the Sindbis virus expression system (with BHK-21 and Vero cell cultures) were used to study processing of the polyprotein. In both expression systems, we identified three main gene products with molecular masses of 48, 34, and 30.5 kDa corresponding to VPX, VP3, and VP4, respectively, as found in IBDV-infected Vero cell cultures, although the amount of each product was variable. A translational time course of the polyprotein gene and analyses of products specified by various sub-clones of the full-length cDNA were used to distinguish primary processing products of translation from secondary products generated by proteolytic processing during in vitro coupled transcription-translation expression. The VPX, VP3 and VP4, which are the primary processing products, first appeared after 20 min of incubation and their production was maximum by 75 min of the coupled transcription-translation reaction. Cycloheximide chases demonstrated that there is no secondary processing of VPX (or VP3 and VP4). Thus VP2, the major 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 secondary 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.

Identification and Molecular Characterization of the RNA Polymerase-Binding Motif of Infectious Bursal Disease Virus Inner Capsid Protein VP3

“…VPX, VP2, and VP3 are the major structural proteins. VP4 is responsible for the proteolytic maturation of the polyprotein, and it is not incorporated into the virion (2,9,12). VP3 contains a highly hydrophilic C-terminal region rich in prolines and charged amino acids (see Fig.…”

Infectious Bursal Disease Virus Capsid Protein VP3 Interacts both with VP1, the RNA-Dependent RNA Polymerase, and with Viral Double-Stranded RNA

“…The viral protease VP4, through a catalytic site belonging to the Lon protease family, is responsible for this self-processing of the polyprotein (5, 27, 35). During virus maturation, the precursor pVP2 is further processed into mature VP2 (40 kDa), probably as a result of a site-specific cleavage of pVP2 by VP4 protease activity (25,27).VP2 and VP3 are the major viral structural proteins. They form the proteinaceous capsid, the structure of which was shown by cryoelectron microscopy and image reconstruction to exhibit a Tϭ13 lattice (10, 13).…”

“…The viral protease VP4, through a catalytic site belonging to the Lon protease family, is responsible for this self-processing of the polyprotein (5,27,35). During virus maturation, the precursor pVP2 is further processed into mature VP2 (40 kDa), probably as a result of a site-specific cleavage of pVP2 by VP4 protease activity (25,27).…”

Rescue of Very Virulent and Mosaic Infectious Bursal Disease Virus from Cloned cDNA: VP2 Is Not the Sole Determinant of the Very Virulent Phenotype

“…1B) which is autocatalytically cleaved to yield the viral proteins pVP2 (also known as VPX; 48 kDa), VP4 (29 kDa), and VP3 (33 kDa). During in vivo virus maturation pVP2 is processed into VP2 (41 to 38 kDa), probably resulting from site-specific cleavage of the pVP2 by a host cell-encoded protease (19). VP2 and VP3 are the two proteins that constitute the shell of the virion.…”