title: VP3, a structural protein of infectious pancreatic necrosis virus, interacts with RNA-dependent RNA polymerase VP1 and with double-stranded RNA.
abstract: Infectious pancreatic necrosis virus (IPNV) is a bisegmented, double-stranded RNA (dsRNA) virus of the Birnaviridae family that causes widespread disease in salmonids. Its two genomic segments are encapsulated together with the viral RNA-dependent RNA polymerase, VP1, and the assumed internal protein, VP3, in a single-shell capsid composed of VP2. Major aspects of the molecular biology of IPNV, such as particle assembly and interference with host macromolecules, are as yet poorly understood. To understand the infection process, analysis of viral protein interactions is of crucial importance. In this study, we focus on the interaction properties of VP3, the suggested key organizer of particle assembly in birnaviruses. By applying the yeast two-hybrid system in combination with coimmunoprecipitation, VP3 was proven to bind to VP1 and to self-associate strongly. In addition, VP3 was shown to specifically bind to dsRNA in a sequence-independent manner by in vitro pull-down experiments. The binding between VP3 and VP1 was not dependent on the presence of dsRNA. Deletion analyses mapped the VP3 self-interaction domain within the 101 N-terminal amino acids and the VP1 interaction domain within the 62 C-terminal amino acids of VP3. The C-terminal end was also crucial but not sufficient for the dsRNA binding capacity of VP3. For VP1, the 90 C-terminal amino acids constituted the only dispensable part for maintaining VP3-binding ability. Kinetic analysis revealed the presence of VP1-VP3 complexes prior to the formation of mature virions in IPNV-infected CHSE-214 cells, which indicates a role in promoting the assembly process.
authors.affiliation: Department of Marine Biotechnology, Norwegian College of Fishery Sciences, University of Tromsø, N-9037 Tromsø, Norway.
authors.forename: Torunn
authors.initials: T
authors.lastname: Pedersen
authors.forename: Astrid
authors.initials: A
authors.lastname: Skjesol
authors.forename: Jorunn B
authors.initials: JB
authors.lastname: Jørgensen
mesh_terms: D000818:Animals; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D017353:Gene Deletion; D016679:Genome, Viral; D006801:Humans; D047468:Immunoprecipitation; D018061:Infectious pancreatic necrosis virus / Q000378:metabolism*; D007700:Kinetics; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded; D012474:Salmon; D020798:Two-Hybrid System Techniques; D015678:Viral Structural Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D012330:RNA, Double-Stranded; D015678:Viral Structural Proteins; D012321:DNA-Directed RNA Polymerases