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.
Magyar
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Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest.
Dobos
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D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000276:immunology; D002213:Capsid / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D036022:Capsid Proteins; D016679:Genome, Viral; D018061:Infectious pancreatic necrosis virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009036:Moths / Q000166:cytology; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D011499:Protein Processing, Post-Translational; D012313:RNA; D011994:Recombinant Proteins / Q000096:biosynthesis; D014764:Viral Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D014771:Virion / Q000737:chemistry
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D036022:Capsid Proteins; D011498:Protein Precursors; C039919:RNA, recombinant; D011994:Recombinant Proteins; D014764:Viral Proteins; D012313:RNA