• BMB Reports
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• v.35 no.6
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• pp.562-567
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• 2002

In order to find the cellular interaction factors of the Heliothis armigera nuclear polyhedrosis virus capsid protein VP39, a Heliothis armigera cell cDNA library was constructed. Then VP39 was used as bait. The host actin gene was isolated from the cDNA library with the yeast two-hybrid system. This demonstrated that VP39 could interact with its host actin in yeast. In order to corroborate this interaction in vivo, the vp39 gene was fused with the green fluorescent protein gene in plasmid pEGFP39. The fusion protein was expressed in the Hz-AM1 cells under the control of the Autographa californica multiple nucleopolyhedrovirus immediate early gene promoter. The host actin was labeled specifically by the red fluorescence substance, tetramethy rhodamine isothicyanete-phalloidin. Observation under a fluorescence microscopy showed that VP39, which was indicated by green fluorescence, began to appear in the cells 6 h after being transfected with pEGFP39. Red actin cables were also formed in the cytoplasm at the same time. Actin was aggregated in the nucleus 9 h after the transfection. The green and red fluorescence always appeared in the same location of the cells, which demonstrated that VP39 could combine with the host actin. Such a combination would result in the actin skeleton rearrangement.

• BMB Reports
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• v.38 no.1
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• pp.41-48
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• 2005

In this study we developed the transposon-mediated shuttle vector 'Hanpvid', which composed of HaNPV (Heliothis armigera nuclear polyhedrosis virus) genomic DNA and a transposon cassette from Bacmid of Bac-to-Bac system. Hanpvid replicates in E. coli in the same way as Bacmid and retains infective function in cotton bollworm cells (Hz-AM1). Using Hanpvid we constructed a recombinant virus, which could infect Hz-AM1 cells and generate recombinant HaNPV (rHa-Bar) containing the barnase gene, a ribonuclease gene from Bacillus amyloliquefaciens. Since the expression vector carrying barnase gene cannot replicate in the absence of barstar, a specific inhibitor of barnase, we constructed a new cotton bollworm cell line (AM1-NB) using the marker rescue method. In AM1-NB barstar was integrated into the cellular chromosome to sustain the replication of rHa-Bar. To screen out recombinant HaNPV for potential use as biopesticide, Hz-AM1 and AM1-NB cell lines were infected with rHa-Bar, respectively. The results obtained indicate that Viral progenies in AM1-NB were 23 and 160 times greater than those in Hz-AM1 48 h and 72 h after infection, respectively. With additional insertion of the polyhedron gene from AcNPV (Autographa californica nuclear polyhedrosis virus) into the Hanpvid genome, rHa-Bar regained the polyhedron phenotype and its pest-killing rate greatly improved. Toxic analysis showed that the lethal dosages ($LD_{50}$) and the lethal time(s) ($LT_{50}$) of rHa-Bar were reduced by 20% and 30%, respectively, compared to wt-HaNPV in the third instar larvae of cotton bollworm. This study shows that in AM1-NB barnase can be effectively produced and used as pest-killing agent for the biological control of cotton pests.