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. / Graduation date: 1993
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/36739 |
| Date | 30 September 1992 |
| Creators | Mason, Carla L. |
| Contributors | Leong, Jo-Ann C. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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