Baculovirus per os Infectivity Factor Complex: Components and Assembly

ABSTRACT

Baculovirus entry into insect midgut cells is dependent on a multiprotein complex of per os infectivity factors (PIFs) on the envelope of occlusion derived virions (ODVs). The structure and assembly of the PIF complex is largely unknown. To reveal the complete members of the complex, a combination of blue native polyacrylamide gel electrophoresis, liquid chromatography-tandem mass spectrometry, and Western blots were conducted on three different baculoviruses. The results showed that the PIF complex is ∼500-kDa in size consisting of nine PIFs including a newly discovered member (PIF9). To decipher the assembly process, each pif was knockout from the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome individually using synthetic baculovirus technology and the impact on PIF complex formation was investigated. Deletion of pif8 resulted in the formation of a ∼400 kDa subcomplex. Deletion of pif0, 4, 6, 7, or 9 resulted in a subcomplex of ∼230 kDa, but deletion of pif1, 2 or 3 abolished formation of any complex. Taken together, a core-complex of ∼230-kDa consisting of PIF1, 2 and 3 was identified. This revised the previous knowledge that the core-complex was about 170 kDa and contained PIF1 to 4. Analysis of the PIF complex in cellular fractions suggested that it is assembled in the cytoplasm before being transported to the nucleus and subsequent incorporation into the envelope of ODV. Only the full complex, and not the subcomplexes, is resistant to proteolytic attack, indicating the essentiality of correct complex assembly for oral infection.

IMPORTANCE Entry of baculovirus in host insects is mediated by a per os infectivity factor (PIF) complex on the envelope of occlusion-derived virus (ODV). Knowledge of the composition and structure of the PIF complex is fundamental to understanding its mode of action. By using multiple approaches, we provide here a complete list of proteins (nine) in the PIF complex. Different from previous knowledge in the field, the core-complex is revised to ∼230 kDa in size and consisting of PIF1-3 but not PIF4. Interestingly, our results suggest that the PIF complex is formed in the cytoplasm prior to its transport to the nucleus and subsequent incorporation into ODV. Only the full complex is resistant to proteolytic degradation in insect midgut implying the critical role of the entire complex. These findings provide the baseline for future studies on the ODV entry mechanism mediated by the multiprotein complex.