Study of RNA-A Initiation Translation of The Infectious Pancreatic Necrosis Virus.

The infectious pancreatic necrosis virus (IPNV) is a salmonid pathogen that causes significant economic losses to the aquaculture industry. IPNV is a non-enveloped virus containing two uncapped and non-polyadenylated double strand RNA genomic segments, RNA-A and RNA-B. The viral protein Vpg is covalently attached to the 5' end of both segments. There is little knowledge about its viral cycle, particularly about the translation of the RNAs. Through experiments using mono and bicistronic reporters, in this work we show that the 120-nucleotide-long 5'-UTR of RNA-A contains an internal ribosome entry site (IRES) that functions efficiently both in vitro and in salmon cells. IRES activity is strongly dependent on temperature. Also, the IRES structure is confined to the 5'UTR and is not affected by the viral coding sequence. This is the first report of IRES activity in a fish virus and can give us tools to generate antivirals to attack the virus without affecting fish directly.

Sequence analysis of infectious pancreatic necrosis virus isolated from Iranian reared rainbow trout (Oncorhynchus mykiss) in 2012.

Infectious pancreatic necrosis virus (IPNV) is the causal agent of a highly contagious disease that affects many species of fish and shellfish. This virus causes economically significant diseases of farmed rainbow trout, Oncorhynchus mykiss (Walbaum), in Iran, which is often associated with the transmission of pathogens from European resources. In this study, moribund rainbow trout fry samples were collected during an outbreak of IPNV in three different fish farms in north and west provinces of Iran in 2012; and we investigated the full genome sequence of Iranian IPNV and compared it with previously identified IPNV sequences. The sequences of different structural and nonstructural-protein genes were compared to those of other aquatic birnaviruses sequenced to date. Our results show that the Iranian isolate falls within genogroup 5, serotype A2 strain SP, having 99% identity with the strain 1146 from Spain. These results suggest that the Iranian isolate may have originated from Europe.

Structure of a VP1-VP3 complex suggests how birnaviruses package the VP1 polymerase.

Infectious pancreatic necrosis virus (IPNV), a member of the family Birnaviridae, infects young salmon, with a severe impact on the commercial sea farming industry. Of the five mature proteins encoded by the IPNV genome, the multifunctional VP3 has an essential role in morphogenesis; interacting with the capsid protein VP2, the viral double-stranded RNA (dsRNA) genome and the RNA-dependent RNA polymerase VP1. Here we investigate one of these VP3 functions and present the crystal structure of the C-terminal 12 residues of VP3 bound to the VP1 polymerase. This interaction, visualized for the first time, reveals the precise molecular determinants used by VP3 to bind the polymerase. Competition binding studies confirm that this region of VP3 is necessary and sufficient for VP1 binding, while biochemical experiments show that VP3 attachment has no effect on polymerase activity. These results indicate how VP3 recruits the polymerase into birnavirus capsids during morphogenesis.

Crystal structure of an Aquabirnavirus particle: insights into antigenic diversity and virulence determinism.

Infectious pancreatic necrosis virus (IPNV), a pathogen of salmon and trout, imposes a severe toll on the aquaculture and sea farming industries. IPNV belongs to the Aquabirnavirus genus in the Birnaviridae family of bisegmented double-stranded RNA viruses. The virions are nonenveloped with a T=13l icosahedral capsid made by the coat protein VP2, the three-dimensional (3D) organization of which is known in detail for the family prototype, the infectious bursal disease virus (IBDV) of poultry. A salient feature of the birnavirus architecture is the presence of 260 trimeric spikes formed by VP2, projecting radially from the capsid. The spikes carry the principal antigenic sites as well as virulence and cell adaptation determinants. We report here the 3.4-A resolution crystal structure of a subviral particle (SVP) of IPNV, containing 20 VP2 trimers organized with icosahedral symmetry. We show that, as expected, the SVPs have a very similar organization to the IBDV counterparts, with VP2 exhibiting the same overall 3D fold. However, the spikes are significantly different, displaying a more compact organization with tighter packing about the molecular 3-fold axis. Amino acids controlling virulence and cell culture adaptation cluster differently at the top of the spike, i.e., in a central bowl in IBDV and at the periphery in IPNV. In contrast, the spike base features an exposed groove, conserved across birnavirus genera, which contains an integrin-binding motif. Thus, in addition to revealing the viral antigenic determinants, the structure suggests that birnaviruses interact with different receptors for attachment and for cell internalization during entry.

Purification, crystallization and preliminary X-ray analysis of truncated and mutant forms of VP4 protease from infectious pancreatic necrosis virus.

In viruses belonging to the Birnaviridae family, virus protein 4 (VP4) is the viral protease responsible for the proteolytic maturation of the polyprotein encoding the major capsid proteins (VP2 and VP3). Infectious pancreatic necrosis virus (IPNV), the prototype of the aquabirnavirus genus, is the causative agent of a contagious disease in fish which has a large economic impact on aquaculture. IPNV VP4 is a 226-residue (24.0 kDa) serine protease that utilizes a Ser/Lys catalytic dyad mechanism (Ser633 and Lys674). Several truncated and mutant forms of VP4 were expressed in a recombinant expression system, purified and screened for crystallization. Two different crystal forms diffract beyond 2.4 A resolution. A triclinic crystal derived from one mutant construct has unit-cell parameters a = 41.7, b = 69.6, c = 191.6 A, alpha = 93.0, beta = 95.1, gamma = 97.7 degrees. A hexagonal crystal with space group P6(1)22/P6(5)22 derived from another mutant construct has unit-cell parameters a = 77.4, b = 77.4, c = 136.9 A.

Structure of birnavirus-like particles determined by combined electron cryomicroscopy and X-ray crystallography.

Birnaviruses possess a capsid with a single protein layer in contrast to most double-stranded RNA viruses infecting multicellular eukaryotes. Using freeze-drying and heavy metal shadowing, the capsids of two birnaviruses, infectious bursal disease virus (IBDV) and infectious pancreatic necrosis virus, as well as of an IBDV virus-like particle (VLP) are shown to follow the same T=13 laevo icosahedral geometry. The structure of the VLP was determined at a resolution of approximately 15 A (1.5 nm) by a combination of electron cryomicroscopy and a recently developed three-dimensional reconstruction method, where the scattering density is expressed in terms of symmetry-adapted functions. This reconstruction methodology is well adapted to the icosahedral symmetry of viruses and only requires a small number of images to analyse. The atomic model of the external capsid protein, VP2, recently determined by X-ray crystallography, fits well into the VLP reconstruction and occupies all the electron densities present in the map. Thus, similarly to the IBDV virion, only VP2 forms the icosahedral layer of the VLP. The other components of both VLP and IBDV particles that play a crucial role in the capsid assembly, VP1, VP3 and the peptides arising from the processing of pVP2, do not follow the icosahedral symmetry, allowing them to be involved in other processes such as RNA packaging.

An anti-apoptosis gene of the Bcl-2 family from Marine Birnavirus inhibiting apoptosis of insect cells infected with baculovirus.

VP5 is a 15-kDa nonstructural protein encoded by a small open reading frame in 5'-terminal of segment A of the Marine Birnavirus (MABV) (strainY-6) genome. Comparisons of the amino acid sequence of the VP5 with other Bcl-2 family member proteins indicated that the VP5 protein contains Bcl-2 homology (BH) domains BH1, BH2, BH3, and BH4, but without the transmembrane region. The VP5 gene from MABV was fused to enhancing green fluorescence protein (eGFP) gene and inserted into the baculovirus genome under the control of polyhedrin gene promoter, and then was highly expressed in insect cells. The expressed VP5 was capable of enhancing insect cell viability, prevented membrane blebbing and delayed DNA internucleosomal cleavage when cells were infected with the recombinant virus. The results suggested that the VP5 of MABV is a novel anti-apoptosis gene, which could regulate the cell apoptosis-off system.

Peptides resulting from the pVP2 C-terminal processing are present in infectious pancreatic necrosis virus particles.

The capsid of birnaviruses contains two proteins, VP2 and VP3, which derive from the processing of a large polyprotein, NH(2)-pVP2-VP4-VP3-COOH. The proteolytic cascade involved in processing the polyprotein, and in the final maturation of pVP2 (the precursor of VP2), has recently been shown to generate VP2 and four structural peptides in infectious bursal disease virus and blotched snakehead virus. The presence of peptides in infectious pancreatic necrosis virus particles was investigated using mass spectrometry and N-terminal sequencing of virus particles. Three peptides deriving from the C terminus of pVP2 (residues 443-486, 487-495 and 496-508 of the polyprotein) and 14 additional peptides produced by further processing of peptides [443-486] and [496-508] were identified. These results indicate that the presence of several virus-encoded peptides in the virions is a hallmark of birnaviruses.

Temporal and subcellular localization of infectious pancreatic necrosis virus structural proteins.

The temporal subcellular localization of the structural proteins VP2 and VP3 of infectious pancreatic necrosis virus was analyzed with monoclonal antibodies conjugated with fluorochromes. Early in the infection both proteins were colocalized in the cytosol, at later times, VP2 was visualized as inclusion bodies around the nuclei of the cells and, sometimes, it was found in elongated tubular structures that might correspond to the type I tubules seen in cells infected with another Birnavirus. As VP2 is a glycosylated protein we determined its subcellular localization compared with that of ER and Golgi probes. These results suggest that VP2 is glycosylated freely in the cytoplasm.

Identification of IPNV-specified components released from productively infected RTG-2 cells following massive cytopathic effect.

Rainbow trout gonad cells (RTG-2) display a dramatic cytopathic effect and lysis following productive infection by infectious pancreatic necrosis virus (IPNV). In this study viruses were efficiently released into the growth medium together with low amounts of the monomeric free form of the structural protein VP3, heterodimers of VP2-VP3, aggregates of pVP2 and viral RNA associated with VP3. Ribonucleoprotein complexes of RNA-VP3 contained RNA equivalent to at the most 25% of full length viral genomes. Infectivity of material released into the growth medium late in infection was only associated with fully assembled viruses and isolated subviral RNA-VP3 complexes were not infectious. Upon purification of IPNV, viral hexa- and pentagonal particles of approx. 15 nm diameter were occasionally co-purified with the virus and then appeared in large quantities. Similar particle-like structures were seen as substructures of purified viruses that were treated with and partially disintegrated by CsCl of virus isodensity concentration.

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 (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 NSt (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 NSt found in infected cells and (ii) a smaller polypeptide NSta. The identity of the virion NSt/NSta was also demonstrated by peptide mapping.

Expression of infectious pancreatic necrosis virus polyprotein and VP1 in insect cells and the detection of the polyprotein in purified virus.

In order to study the molecular biology of infectious pancreatic necrosis virus (IPNV) replication, six different recombinant baculoviruses were constructed. The following four recombinants contained genome segment A-specific sequences; (i) AcPP contained the complete polyprotein coding region and Spodoptera frugiperda (Sf) cells infected by these recombinants synthesized the 106-kDa polyprotein (NH2-preVP2-NS protease-VP3-COOH), which was only partially processed by the protease to yield preVP2 and VP3 and unprocessed polyprotein; (ii) AcPP(S) and AcPP(Ss) represented 3' truncated sequences of the segment A cDNA where the VP3 coding region and that coding for 30 and 98 carboxy terminal amino acids of NS in the two constructs, respectively, were deleted. AcPP(S) demonstrated partial, and that of AcPP(Ss), complete loss of proteolytic activity, demonstrating that the carboxy one-third of the 29-kDa NS protease is necessary for the formation of the active enzyme; and (iii) AcPP(B/B) contained all but the first 180 nt of the pVP2 gene, the complete NS coding region, and the amino end of VP3. Analysis of cells coinfected with AcPP(Ss) and AcPP(B/B) showed either that the protease did not work in trans or that the alteration of the structure of the substrate prevented cleavage. Recombinant baculoviruses AcVP1VL and AcVP1ETL contained IPNV genome segment B cDNA encoding the 94-kDa VP1 which is the viral RNA-dependent RNA polymerase. AcVP1VL contained the whole segment B cDNA, whereas in AcVP1ETL, the 5' non-coding sequences were deleted resulting in the production of large amounts of VP1 when Sf cells were infected with this recombinant. The use of recombinants AcPP and AcVP1ETL as well as monoclonal antibodies and VP1-specific sera allowed the unambiguous identification of the high molecular weight minor polypeptides present in purified IPNV demonstrating the presence of both VP1 and the polyprotein in purified virus preparations.