Inactivation Analysis of HcNPV Cysteine Protease Gene and Chitinase Gene.

The fragment of Hyphantria cunea nuclear polyhedrosis virus (HcNPV), which contains cysteine protease gene(CP) and chitinase gene (ChiA),was inserted into plasmid PCRII to construct transfer vector pHcCVdel. Recombinant transfer vector pHcCvpolh was constructed by inserting the fragment of HcNPV polyhedrin gene (polh) into the EcoRI site of the pHcCVdel. By contransfection of the pHcCvpolh and HcNPV-PTTH(+) DNA, which contained Bombyx mori prothoracicotropic hormone gene (PTTH) into SPIM cells, the region from +76 downstream of ChiA gene initiation codon to +20 downstream of CP gene initiation codon was substituted by the polh gene, and the recombinant virus HcNPVpolh(+)CP(-)ChiA(-)PTTH(+) was produced. The recombinant virus, in which CP gene and ChiA gene were inactive, could express PTTH gene and form polyhedra. SPIM cells infected with the recombinant virus revealed that CP and ChiA gene were not essential for viral replication and had no significant effect on polyhedron formation, but the infected cells survived 2 days more than those infected with HcNPV and HcNPVPTTH(+). Therefore, the expression time of foreign gene may be prolonged when CP gene and ChiA gene were inactivated.