Baculovirus per os infectivity: a complex matter

Bob Boogaard

Research output: Thesisinternal PhD, WU


Insect larvae are infected by baculoviruses when they consume plant material that is contaminated with occlusion bodies (OBs). OBs are polyhedral shaped particles of crystallized protein (polyhedrin or granulin), in which the occlusion derived viruses (ODVs) are embedded. Upon ingestion of the OBs, the ODVs are released in the alkaline lumen of the midgut and infect the midgut epithelial cells. To be infectious for these cells under alkaline conditions, the ODV envelope contains a set of proteins, which are called per os infectivity factors or PIFs. Eight different PIFs had been identified at the start of this research, of which six were known to participate in the formation of a large complex: the ODV entry complex. Presently, ten different PIFs are known, of which nine form the entry complex. This complex has a stable core that is formed by PIF1 to 4, which even resists treatment with denaturing and reducing agents (SDS and β-mercaptoethanol), and five additional PIFs (PIF0 and PIF6 to 9), which are associated to the complex more loosely. All these individual components are essential for complex formation. PIF5 is the only PIF-protein that is not a constituent of the entry complex.  

Our initial studies with pif deletion mutant viruses were hampered by the disappearance of not only the targeted, but also non-targeted PIFs, when the OBs were produced in insect larvae, while the various PIFs were all detectable for  the wild type virus. However, those remaining PIFs were found again in these mutant viruses when the larval derived OBs were treated with heat prior to ODV isolation, or when the OBs were produced in cultured insect cells. These observations showed that proteases from the host, which had been reported to be co-occluded in the OBs, are able to degrade PIF proteins when complex formation is disrupted, as was the case in the various pif deletion mutant viruses. These observations provided a first clue on why the PIFs form a complex, which might be important to resist proteolytic degradation by proteases in the gut of the host.

Previous research in our laboratory had identified viral protein AC108 as a PIF1 interaction partner in a co-immunoprecipitation study, indicating that this protein might be involved in oral infectivity as well. The research presented in this thesis demonstrated that mutation of the ac108 gene from the viral genome completely abolished the oral infectivity of ODVs, which was recovered when this gene was repaired. Further protein analyses revealed that AC108 is a loosely associated but nevertheless an essential component of the ODV entry complex. This protein was therefore designated as PIF9.

The biological function of PIF9 was further investigated by the generation of fluorescently labelled ODVs of the pif9 mutant. These virus particles were then combined with isolated midgut cells of Spodoptera exigua larvae and monitored by confocal microscopy in a time lapse experiment. It was observed that in absence of PIF9, the ODVs still bind to the midgut cell brush border, but that the nucleocapsids failed to enter the cell, in contrast to fluorescent ODVs that have all PIFs. Fluorescent ODVs were also generated for a pif3 deletion mutant as it had been shown previously by others that this (complex deficient) mutant is able to bind and fuse with the host cell plasma membrane as the wild type virus, but nevertheless fails to establish a midgut infection. Our analysis with the confocal microscope showed that this mutant displays the same phenotype as the pif9 deletion mutant virus: ODV binding, but no entry of the nucleocapsids. We therefore proposed a two-step fusion process, in which first the outer leaflets of the lipid bilayers fuse and subsequently the inner leaflets. PIF3 might be important to complete the fusion process by aiding fusion of the inner leaflets of the lipid bilayers.  

PIF1 and 2 had been reported to be important for ODV binding and were later also shown to be crucial for the formation of the ODV entry complex. Furthermore, ODV binding had been shown to be one of the viral host range determinants and as most baculoviruses have a narrow host range, we hypothesized that the more variable C-terminal parts of PIF1 and 2 are important for host interaction, while the highly conserved N-terminal parts are needed for complex formation. However, limited C-terminal truncation of either PIF1 or 2 abolished formation of the entry complex as PIF0 and 8 failed to bind the stable core in the truncation mutants. The lost ability to form the entry complex in the truncation mutants was accompanied by a severely hampered oral infectivity in S. exigua larvae. Some larvae incidentally got infected by one of the truncation mutants despite abolished complex formation. It was therefore speculated that although the ODV entry complex is the main determinant for the infectivity of ODVs, additional factors are present in the ODV that is able to incidentally cause infections in absence of the entry complex.

To determine the context in which the PIFs function, the interaction partners were mapped of three loosely associated components of the entry complex (PIF6, 8 and 9) and the single solitary PIF, PIF5. These analyses revealed that the PIFs interact with a plethora of viral proteins with a wide variety of functions, from nucleocapsid assembly to OB formation. PIF5 also interacted with two components of the entry complex, PIF0 and 1. This indicated that PIF0 and 1 are not only present in the ODV envelope as part of the entry complex, but also occur outside the complex where these proteins interact with PIF5. This study shows that the PIFs not only interact with each other to form the entry complex, but are part of a network of protein interactions in ODVs. The biological significance of these interactions for midgut infection remains enigmatic.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • van Oers, Monique, Promotor
  • van Lent, J.W.M., Co-promotor
Award date15 Jan 2020
Place of PublicationWageningen
Print ISBNs9789463952132
Publication statusPublished - 15 Jan 2020


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