Evidence for the Detection of the Infectious Pancreatic Necrosis Virus Polyprotein and the 17-kDa Polypeptide in Infected Cells and of the NS Protease in Purified Virus

Abstract The larger genome segment A of infectious pancreatic necrosis virus (IPNV) contains two open reading frames (ORFs): (i) the large ORF encodes a 106-kDa polyprotein (PP) (NH2-pVP2-NS-VP3-COOH) which is cotranslationally cleaved by the NS protease to generate the major capsid polypeptides VP2 and VP3; (ii) the second small ORF, which overlaps the 5′ end of the PP ORF but in a different reading frame, encodes a 17-kDa arginine-rich polypeptide. Hitherto, neither the PP nor the 17-kDa polypeptide have been identified in infected cells, and the NS (nonstructural) polypeptide was thought not to be part of the virion. The smaller genome segment B of IPNV encodes a 94-kDa minor polypeptide VP1. Using recombinant baculoviruses expressing VP1 and PP as markers, the PP could be unambiguously identified in Western blots of infected fish-cell lysates and in purified IPNV. Anti-17-kDa and anti-NS serum was produced by injecting rabbits with bacterially expressed fusion proteins containing these polypeptides. Labeled 17-kDa polypeptide was immune-precipitated from infected cell lysates using the anti-17-kDa serum, whereas the NS and NS, (a truncated form of NS) polypeptides were identified in infected cells by immune precipitation and Western blotting using the anti-NS serum. Western blots of purified virus revealed two forms of truncated NS: (i) the NS, found in infected cells and (ii) a smaller polypeptide NS ta . The identity of the virion NSt/NSta was also demonstrated by peptide mapping.