Graduate Thesis Or Dissertation
 

Molecular characterization of the proteinase and RNA-dependent RNA polymerase of Infectious Pancreatic Necrosis Virus, a fish birnavirus

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  • The A segment of infectious pancreatic necrosis virus (IPNV) is expressed as a polyprotein encoding three primary gene products, VP2, NS and VP3, from a large open reading frame. The nucleotide sequence for the A segment of the Sp isolate of IPNV was determined. The NS protein is the putative autocatalytic proteinase responsible for the cleavage of the polyprotein. The functional boundaries of the NS proteinase were mapped by plasmid deletion analysis and examined in an La vitro, translation system. The NS proteolytic activity was determined to lie within the EcoRI and Nsil restriction sites. Characterization of the NS proteinase also was approached by use of proteinase inhibitors and site-directed mutagenesis of the putative catalytic and cleavage sites. Eight proteinase inhibitors, representative of all four proteinase classes, were tested and all failed to inhibit the NS enzyme. Mutagenesis of a putative aspartyl proteinase catalytic motif, DTG, to VTG did not affect proteolytic processing. Additionally, the mutagenesis of the predicted N-terminal cleavage site did not alter processing, however, altered processing was observed when the predicted C-terminal cleavage site was mutated. The major capsid protein, VP2, was mapped with polyclonal and monoclonal antisera. The VP2 gene was digested with Sau3A and subcloned into the pATH expression vector. The trpE-fusion proteins were characterized with polyclonal and monoclonal antisera. Two immunoreactive regions were identified with anti IPNV-Sp sera. A common immunoreactive region, B10, was reactive with antisera to three serotypes of IPNV as well as a neutralizing monoclonal antibody, AS-1. A serotype specific immunoreactive region, A43, also was identified, being recognized only by anti IPNV-Sp sera. The B segment of IPNV encodes the putative RNA-dependent RNA polymerase (RdRp), VP1. The nucleotide sequence for the B segment of the Sp isolate was determined and the deduced amino acid sequences were compared to other polymerases. Concensus sequences associated with GTP-binding proteins and RdRps were identified in the VP1 sequence. However, unlike RdRps associated with single-stranded RNA viruses, the IPNV VP1 proteins lack the Gly-Asp-Asp motif characteristic of this enzyme family. Additionally, the VP1 protein was expressed in a bacterial system and polyclonal antisera was raised against the protein.
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