The Heliothis armigera single nucleocapsid nucleopolyhedrovirus envelope protein P74 is required for infection of the host midgut

In order to study the function of the envelope protein P74 of Heliothis armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV), a p74-null recombinant baculovirus, rHa-gfΔp74, was constructed by inserting gfp driven by the polyhedrin promoter into the p74 locus of HaSNPV genome. The resulting p74-inactivation occlusion-derived viruses (ODV) failed to infect its natural host larvae per os. However, its inability of oral infectivity was rescued using the purified P74 protein expressed by Bac-to-Bac system in Hz-AM1 cells. Feeding the purified P74 protein along with the p74 deletion mutant virus rHa-gfpΔp74 to H. armigera larvae resulted in the rescue of oral infectivity in a dose dependent manner. The P74 protein was expressed in-frame with GFP to create a P74-GFP chimera for studying the localization of P74, the GFP portion of the chimera facilitating the visualization of the trafficking of P74 in cells. The P74-GFP chimeric proteins localized in the intranuclear ring zone and accumulated into microvesicles. In addition, the specific and saturable binding of P74 protein to its host brush border membrane vesicles (BBMVs) was involved in the invasion of virus. Further investigations (pull-down assay) showed that an about 30 kDa protein in the BBMVs was involved in the specific binding. These results demonstrated that the P74 protein is essential for oral infectivity of ODV and plays a role in midgut attachment and fusion.