1887

Abstract

Baculovirus occlusion-derived virions (ODVs) contain a number of infectivity factors essential for the initiation of infection in larval midgut cells. Deletion of any of these factors neutralizes infectivity by the route. We have observed that P74 of the group I alphabaculovirus multiple nucleopolyhedrovirus (AcMNPV) is N-terminally cleaved when a soluble form of the protein was incubated with insect midgut tissues under alkaline conditions and that cleavage was prevented by soybean trypsin inhibitor (SBTI). Presently, biological assays were carried out that suggest SBTI inhibits and trypsin enhances baculovirus infectivity. We developed a method to rescue infectivity of a P74 null virus involving co-transfection of viral DNA with a plasmid that transiently expresses . We used this plasmid rescue method to functionally characterize P74. A series of site-directed mutants were generated at the N terminus to evaluate if trypsin cleavage sites were necessary for function. Mutagenesis of R195, R196 and R199 compromised infectivity and rendered P74 resistant to midgut trypsin.

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2008-10-01
2020-01-27
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vol. , part 10, pp. 2388–2397

Primers and two PCR-based strategies used to make site-directed mutants in the gene

Mortality (dead/total) of 2nd instar larvae 7 days post feeding OBs in presence of SBTI or trypsin

Mortality (dead/total) of 2nd instar larvae 7 days post feeding of OBs produced during expression of site-directed mutant constructs (mutations in P74 region R114–R199)

Mortality (dead/total) of 2nd instar larvae 7 days post feeding of OBs produced during expression of different site-directed mutant constructs (mutations in P74 region R195–R199)

Source virus full names and P74 protein homologue protein accession numbers (GenBank)



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