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

The larger genome segment A of infectious pancreatic necrosis virus (I PNV) contains two open reading frames (ORFs): (i) the large ORF encode s a 106-kDa polyprotein (PP) (NH2-pVP2-NS-VP3-COOH) which is cotransla tionally cleaved by the NS protease to generate the major capsid polyp eptides 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 a rginine-rich polypeptide. Hitherto, neither the PP nor the 17-kDa poly peptide have been identified in infected cells, and the NS (nonstructu ral) polypeptide was thought not to be part of the virion. The smaller genome segment B of IPNV encodes a 94-kDa minor polypeptide VP1. Usin g recombinant baculoviruses expressing VP1 and PP as markers, the PP c ould be unambiguously identified in Western blots of infected fish-cel l lysates and in purified IPNV. Anti-17-kDa and anti-NS serum was prod uced by injecting rabbits with bacterially expressed fusion proteins c ontaining these polypeptides. Labeled 17-kDa polypeptide was immune-pr ecipitated from infected cell lysates using the anti-17-kDa serum, whe reas the NS and NSt (a truncated form of NS) polypeptides were identif ied in infected cells by immune precipitation and Western blotting usi ng the anti-NS serum. Western blots of purified virus revealed two for ms of truncated Ns: (i) the Ns(t) found in infected cells and (ii) a s maller polypeptide NSta. The identity of the virion NSt/NSta was also demonstrated by peptide mapping. (C) 1994 Academic Press, Inc.