Baculovirus midgut entry: the role of per os infectivity factors (PIFs)

Baculoviruses infect insect midgut epithelial cells in the form of occlusion-derived virus (ODVs) particles..For Autographa californica multiple nucleopolyhedrovirus (AcMNPV) at least seven conserved ODV membrane proteins are essential for oral infectivity. These proteins are called per os infectivity factors (PIFs). We identified a multi-molecular complex composed of PIF1, PIF2, PIF3, and PIF4. Co-immunoprecipitation (CoIP) combined with proteomic analysis was used to identify other components of the AcMNPV PIF complex. Differentially denaturing SDS-PAGE further confirmed that PIF4 forms a stable complex with PIF1, PIF2, and PIF3. Deletion of the genes for either PIF1, PIF2, PIF3, or PIF4 impaired complex formation, but absence of P74 or PIF5 did not, in line with the fact that P74 was more loosely associated with the complex and PIF 5 was not found in the complex. CO-IP indicated that PIF-6 is also associated with the PIF complex, as well as three other proteins P95, AC5 and AC!08. To monitor the entry of virus into primary midgut epithelium cells in detail we constructed a baculovirus with EGFP fused to the VP939 nucleocapsid protein. This allowed us to study the ability to bind and enter host cells ex vivo using confocal fluorescence microscopy. When applied to wild type AcMNPV and viruses with mutations in P74 (PIF0), PIF1 or PIF2, it was shown that P74 is required for ODV binding. Viruses with PIF1 and PIF2 deletions still bound to midgut epithelial cells but were not able to enter. The live imaging of ODV entry into midgut cells complements the genetic and biochemical evidence for the role of PIFs in the oral infection process.