Peter Dobos

The molecular biology of infectious pancreatic necrosis virus (IPNV)

Abstract IPNV is a medium-sized, unenveloped bisegmented dsRNA-containing virus in the family Birnaviridae. Genome segment A (3097 bp) contains two overlapping open-reading frames (ORFs). A large ORF encodes a 106 kDa polyprotein (NH2-pVP2-NS protease-VP3-COOH) which is cotranslationally cleaved by the protease to generate the major capsid proteins VP2 and VP3, and a second, small ORF which overlaps the amino end of the large ORF but in a different reading frame, and encodes a 17 kDa arginine-rich minor polypeptide. Genome segment B (2784 bp) encodes a minor internal capsid polypeptide VP1 (94 kDa), which based on its size, low copy number and the presence of several conserved domains associated with RNA-dependent RNA polymerases (RdRp) of other RNA viruses, is the putative virion-associated RdRp. VP1 is present in the virion in two forms: as a free polypeptide and as a genome-linked protein (VPg) covalently attached to the 5′ ends of both genome segments. During in vitro RNA transcription, VP1 serves as a primer and remains attached to the 5′ end of the RNA thereby becoming a VPg. Transcription follows a semi-conservative, strand-displacement mechanism. In infected cells two genome-length 24S viral mRNAs lacking 3′ poly A tracts are synthesized that can hybridize to the two denatured genome segments. In vivo protein synthesis involves both polyprotein processing and internal initiation of translation at some of the in-phase methionine codons. The virus-coded protease functions only in cis and its insensitivity to a number of proteinase inhibitors suggests that it may be a novel viral protease. The putative cleavage sites on the polyprotein have been mapped to within a few amino acids but the exact boundary between pVP2 NS and NS VP3 has not been established. A universal, group-specific epitope has been mapped to near the amino terminus of VP2, whereas a serotype-specific epitope was found to be located in the middle of the polypeptide.

Sequence analysis of infectious pancreatic necrosis virus genome segment B and its encoded VP1 protein: A putative RNA-dependent RNA polymerase lacking the Gly-Asp-Asp motif

Abstract The genome segment B sequence of infectious pancreatic necrosis virus was determined for both the Jasper and Sp serotypes. The sequences are 2784 and 2630 by long, respectively, and contain a single large open reading frame encoding the VP1 protein, the putative RNA-dependent RNA polymerase (RdRp) of IPNV. The proteins exhibit an 88% homology with each other, but only 41% with infectious bursal disease virus (IBDV) VP1, another member of the Birnaviridae. Despite the low overall homology between the IPNV and IBDV VP1 proteins, homologous regions were detected within the central portion of the proteins. The carboxy-proximal regions of the VP1, which contain very low amino acid homology, displayed evidence of conservation in structural features such as a hydrophilic, highly basic domain. Consensus sequences associated with GTP-binding proteins and RdRps were also detected in VP1. However, unlike the RdRps associated with single-stranded plus RNA viruses, the birnavirus RdRp lacks the Gly-Asp-Asp motif characteristic of this enzyme family.

Protein synthesis in cells infected by Autographa californica nuclear polyhedrosis virus (Ac-NPV): The effect of cytosine arabinoside

Twenty-two virion polypeptides (VP) were detected reproducibly when the occluded form (OV) of [35S]methionine-labeled Ac-NPV, purified from polyhedral inclusion bodies (PIB), was analyzed by polyacrylamide gel electrophoresis and autoradiography. Ten of the VPs and the polyhedral protein (PP) were phosphorylated and 6 VPs were glycoproteins. Purified, nonoccluded virus (NOV) revealed 25 polypeptides. During a single cycle of virus replication, the synthesis of 33 infected cell polypeptides (1CP) was detected in different relative proportions at different times. They were arbitrarily designated as early (0-12hr), middle (12-18 hr), and late 18-24 hr) polypeptides. Most of the middle and late proteins seemed to be viral structural proteins. Rapid post-translational cleavage of IPCs was not observed; however, pulse-chase experiments revealed post-translational modifications of at least four polypeptides. Inhibition of DNA synthesis at the time of infection did not prevent the synthesis of ICPs, VPs, or the formation of PIBs. Progeny PIB from both control and cytosine arabinoside (Ara C)-treated cells that were infected with radiochemically pure [3H]thymidine-labeled NOV contained parental virus DNA. Electron micrographs of thin sections showed that, whereas PIBs from control cultures contained complete virions, those from Ara (C-treated cells contained both full and empty virus.

Identification of the proteins encoded by each genome segment of infectious pancreatic necrosis virus

Abstract Infectious pancreatic necrosis virus (IPNV) contains two segments of double-stranded RNA. Temperature-sensitive (ts) mutants were isolated from two serotypes (1 and 2) of IPNV after treatment with nitrosoguanidine. Crosses between is mutants showed that two recombination groups (named I and II) were present within each serotype. Crosses between serotypes yielded viable ts + recombinant viruses that were shown by gel electrophoresis to contain one RNA segment from each parent. Analysis of the proteins of each hybrid showed that the large RNA coded for the major capsid protein, the internal virion protein, and a nonstructural protein, and the small RNA coded for the 100,000-dalton protein.

Effect of ribavirin on the replication of infectious pancreatic necrosis virus in fish cell cultures.

Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) at concentrations of 10 microgram/ml or more, inhibited the replication of infectious pancreatic necrosis virus (IPNV) in both Chinook salmon embryo (CHSE-214) and rainbow trout gonad (RTG-2) cells. The drug was most effective when added just before or within 8 h p.i. Incorporation studies with radioactive precursors demonstrated that ribavirin suppressed cellular DNA and RNA synthesis within 2 to 3 h after addition of the drug. The inhibition of nucleic acid synthesis and the antiviral activity was gradually reversed within 3 to 5 days after removal of the drug from the infected cells. Polyacrylamide slab-gel electrophoresis combined with fluorography revealed that: (i) 0.5 microgram/ml actinomycin D sufficiently inhibited host cell RNA synthesis thereby enabling the study of virus-specific RNA synthesis in infected cells and (ii) ribavirin inhibited the synthesis of all three virus RNA forms: the transcription intermediate, virus mRNA and progeny dsRNA.

Cloning and expression of NDV hemagglutinin-neuraminidase cDNA in a baculovirus expression vector system

The hemagglutinin-neuraminidase (HN) gene of the Hitchner B1 strain of Newcastle disease virus (NDV) was cloned as a cDNA and inserted into a baculovirus expression vector. The recombinant HN (recHN) expressed in Spodoptera frugiperda cells had both hemagglutinating and neuraminidase activities both of which were inhibited by polyclonal anti-NDV sera or a monoclonal antibody (MAb) against HN. Infected insect cells could hemadsorb chicken red blood cells suggesting that the recHN is properly glycosylated and transported to the cell surface. A 67-kDa recHN precursor and a 74-kDa, presumably mature, recHN from infected cells were detected by Western blot analysis and were found to comigrate with similar proteins from NDV-infected chick embryo fibroblast cells. The kinetics of synthesis of recHN was similar to that for polyhedrin and some HN appeared in the extracellular medium. HN was copurified with extracellular virus (ECV) from the extracellular medium and was used to immunize chickens. The anti recHN serum was specific to NDV in both ELISA and Western blot analysis.

Synthesis of Drosophila X virus proteins in cultured Drosophila cells.

The genome of Drosophila X virus (DXV) is made up of two segments of dsRNA with molecular weights of 2.3 X 10(6) (A) and 2.2 X 10(6) (B). Agarose gel electrophoresis of RNA fragments produced by S1 nuclease digestion of partially denatured, purified segment A and B indicated that the two genome segments have different nucleotide sequences. The dose-response curve of virus infectivity was linear, indicating that the two genome segments reside in the same particle. The virions contained five polypeptides (VPs) that fell into three molecular weight size classes; large, 110K; medium, 49K and 45K; and small, 34K and 27K. Virus infection of Drosophila cells induced the synthesis of five infected cell polypeptides (ICPs); 110K, 67K, 49K, 34K, and 27K. Pulse-chase experiments and peptide mapping revealed that four of these (110K, 67K, 34K, and 27K) were primary gene products and that ICP 49 was generated by post-translational cleavage of ICP 67. The major capsid protein, VP 45, was cleaved from ICP 49; however, this cleavage was incomplete because both polypeptides were present in purified virus. The results suggest that the strategy for protein synthesis of DXV differs from that of other dsRNA viruses.

Antigenic and Genomic Differences of Two Jasper Strains of Infectious Pancreatic Necrosis Virus

Strains of infectious pancreatic necrosis virus (IPNV-Jasper) obtained from two different laboratories were compared serologically with polyclonal and monoclonal antibodies. Nucleotide sequence and restriction endonuclease patterns of 359-bp fragment of genome segment A cDNA were also compared. Substantial differences were found in both analyses that will support the fact that the two Jasper strains are not identical.