Structural and functional analyses of infectious pancreatic necrosis virus (IPNV)

Abstract: Infectious pancreatic necrosis virus (IPNV) is a naked virus of an uncomplicated structure. In the interior part of the virion, the two segments of double-stranded RNA are located together with the minor structural protein VP1. This protein is detected in its free form as well as linked to the genome. A polymerase activity has been assigned for VP1. The localisation of the viral protein VP3 has also been proposed to be internal. We have shown that VP3 is quantitatively associated with the entire viral genome and VP3/RNA cores are released from disintegrated virions and visualized as entangled filaments with an estimated diameter of 5.0-6.5 nm. The highly basic carboxy terminal end of polypeptide VP3 may have a neutralizing effect on the RNA and facilitate the packaging of the genome into the capsid. VP3/RNA complexes were also shown to be released from infected cells during lysis, however, the nucleic acids of these components consist of incomplete RNA segments and are non-infectious. The lack of infectivity of these components was assessed by an improved TCID50 end-point titration method evaluated by an immuno dot blot assay developed by us. The nucleoprotein core of IPNV is surrounded by a single-shelled capsid built by subunits of one protein, VP2. Our studies, involving several different methods, have demonstrated that this is a glycoprotein. The fact that low quantities of carbohydrates actually are present on VP2, may offer an interesting issue concerning future studies of the virus-receptor interactions and the questions of tropism. During the cycle of replication, we have also detected VP2 in granulas in the perinuclear region; these granulas increase in size and are found as inclusion bodies at the end of multiplication, before the cellular cytoskeleton is disrupted. These inclusion bodies, most likely correspond to pseudocrystalline aggregates of assembled virions as revealed in the electron microscope. Our improved virus purification technique, using anion exchange chromatography, together with the end-point titration method, are valuable tools in the research of IPNV, and would offer some assistance in pursuing the future tasks. Also the fact that preparations of purified IPNV indeed will retain maximum infectivity levels upon freezing in the presence of a suitable cryoprotective agent is advantageous for all structural and biological studies.