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Characterization of novel components of the baculovirus per os infectivity factor complex.

ABSTRACT: Baculovirus occlusion-derived virus (ODV) infects insect midgut cells under alkaline conditions, a process mediated by highly conserved per os infectivity factors (PIFs), P74 (PIF0), PIF1, PIF2, PIF3, PIF4, and PIF5 (ODV-E56). Previously, a multimolecular complex composed of PIF1, PIF2, PIF3, and P74 was identified which was proposed to play an essential role during ODV entry. Recently, more proteins have been identified that play important roles in ODV oral infectivity, including PIF4, PIF5, and SF58, which might work in concert with previously known PIFs to facilitate ODV infection. In order to understand the ODV entry mechanism, the identification of all components of the PIF complex is crucial. Hence, the aim of this study was to identify additional components of the PIF complex. Coimmunoprecipitation (CoIP) combined with proteomic analysis was used to identify the components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex. PIF4 and P95 (AC83) were identified as components of the PIF complex while PIF5 was not, and this was confirmed with blue native PAGE and a second CoIP. Deletion of the pif4 gene impaired complex formation, but deletion of pif5 did not. Differentially denaturing SDS-PAGE further revealed that PIF4 forms a stable complex with PIF1, PIF2, and PIF3. P95 and P74 are more loosely associated with this complex. Three other proteins, AC5, AC68, and AC108 (homologue of SF58), were also found by the proteomic analysis to be associated with the PIF complex. Finally the functional significance of the PIF protein interactions is discussed.

SUBMITTER: Peng K

PROVIDER: S-EPMC3347349 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Characterization of novel components of the baculovirus per os infectivity factor complex.

Peng Ke K van Lent Jan W M JW Boeren Sjef S Fang Minggang M Theilmann David A DA Erlandson Martin A MA Vlak Just M JM van Oers Monique M MM

Journal of virology 20120229 9

Baculovirus occlusion-derived virus (ODV) infects insect midgut cells under alkaline conditions, a process mediated by highly conserved per os infectivity factors (PIFs), P74 (PIF0), PIF1, PIF2, PIF3, PIF4, and PIF5 (ODV-E56). Previously, a multimolecular complex composed of PIF1, PIF2, PIF3, and P74 was identified which was proposed to play an essential role during ODV entry. Recently, more proteins have been identified that play important roles in ODV oral infectivity, including PIF4, PIF5, an ...[more]

PMID: 22379094

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Project description:Baculovirus entry into insect midgut cells is dependent on a multiprotein complex of per os infectivity factors (PIFs) on the envelopes of occlusion-derived virions (ODVs). The structure and assembly of the PIF complex are 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 blotting was conducted on three different baculoviruses. The results showed that the PIF complex has a molecular mass of ∼500 kDa and consists of nine PIFs, including a newly discovered member (PIF9). To decipher the assembly process, each pif gene was knocked out from the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome individually by use of synthetic baculovirus technology, and the impact on PIF complex formation was investigated. Deletion of pif8 resulted in the formation of an ∼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, our data identified a core complex of ∼230 kDa, consisting of PIF1, -2, and -3. 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 subsequently incorporated into the envelopes of ODVs. Only the full complex, not the subcomplex, is resistant to proteolytic attack, indicating the essentiality of correct complex assembly for oral infection.IMPORTANCE Entry of baculovirus into host insects is mediated by a per os infectivity factor (PIF) complex on the envelopes of occlusion-derived viruses (ODVs). Knowledge of the composition and structure of the PIF complex is fundamental to understanding its mode of action. By using multiple approaches, we determined the complete list of proteins (nine) in the PIF complex. In contrast to previous knowledge in the field, the core complex is revised to ∼230 kDa and consists of PIF1 to -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 ODVs. Only the full complex is resistant to proteolytic degradation in the 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.

Project description:Baculovirus occlusion-derived virus (ODV) initiates infection of lepidopteran larval hosts by binding to the midgut epithelia, which is mediated by per os infectivity factors (PIFs). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes seven PIF proteins, of which PIF1 to PIF4 form a core complex in ODV envelopes to which PIF0 and PIF6 loosely associate. Deletion of any pif gene results in ODV being unable to bind or enter midgut cells. AC83 also associates with the PIF complex, and this study further analyzed its role in oral infectivity to determine if it is a PIF protein. It had been proposed that AC83 possesses a chitin binding domain that enables transit through the peritrophic matrix; however, no chitin binding activity has ever been demonstrated. AC83 has been reported to be found only in the ODV envelopes, but in contrast, the Orgyia pseudotsugata MNPV AC83 homolog is associated with both ODV nucleocapsids and envelopes. In addition, unlike known pif genes, deletion of ac83 eliminates nucleocapsid formation. We propose a new model for AC83 function and show AC83 is associated with both ODV nucleocapsids and envelopes. We also further define the domain required for nucleocapsid assembly. The cysteine-rich region of AC83 is also shown not to be a chitin binding domain but a zinc finger domain required for the recruitment or assembly of the PIF complex to ODV envelopes. As such, AC83 has all the properties of a PIF protein and should be considered PIF8. In addition, pif7 (ac110) is reported as the 38th baculovirus core gene.IMPORTANCE ODV is essential for the per os infectivity of the baculovirus AcMNPV. To initiate infection, ODV binds to microvilli of lepidopteran midgut cells, a process which requires a group of seven virion envelope proteins called PIFs. In this study, we reexamined the function of AC83, a protein that copurifies with the ODV PIFs, to determine its role in the oral infection process. A zinc finger domain was identified and a new model for AC83 function was proposed. In contrast to previous studies, AC83 was found to be physically located in both the envelope and nucleocapsid of ODV. By deletion analysis, the AC83 domain required for nucleocapsid assembly was more finely delineated. We show that AC83 is required for PIF complex formation and conclude that it is a true per os infectivity factor and should be called PIF8.

Project description:The relatively low infectivity of baculoviruses to their host larvae limits their use as insecticidal agents on a larger scale. In the present study, a novel strategy was developed to efficiently embed foreign proteins into Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion bodies (OBs) to achieve stable expression of foreign proteins and to improve viral infectivity. A recombinant AcMNPV bacmid was constructed by expressing the 150-amino-acid (aa) N-terminal segment of polyhedrin under the control of the p10 promoter and the remaining C-terminal 95-aa segment under the control of the polyhedrin promoter. The recombinant virus formed OBs in Spodoptera frugiperda 9 cells, in which the occlusion-derived viruses were embedded in a manner similar to that for wild-type AcMNPV. Next, the 95-aa polyhedrin C terminus was fused to enhanced green fluorescent protein, and the recombinant AcMNPV formed fluorescent green OBs and was stably passaged in vitro and in vivo The AcMNPV recombinants were further modified by fusing truncated Agrotis segetum granulovirus enhancin or truncated Cydia pomonella granulovirus ORF13 (GP37) to the C-terminal 95 aa of polyhedrin, and both recombinants were able to form normal OBs. Bioactivity assays indicated that the median lethal concentrations of these two AcMNPV recombinants were 3- to 5-fold lower than that of the control virus. These results suggest that embedding enhancing factors in baculovirus OBs by use of this novel technique may promote efficient and stable foreign protein expression and significantly improve baculovirus infectivity.IMPORTANCE Baculoviruses have been used as bioinsecticides for over 40 years, but their relatively low infectivity to their host larvae limits their use on a larger scale. It has been reported that it is possible to improve baculovirus infectivity by packaging enhancing factors within baculovirus occlusion bodies (OBs); however, so far, the packaging efficiency has been low. In this article, we describe a novel strategy for efficiently embedding foreign proteins into AcMNPV OBs by expressing N- and C-terminal (dimidiate) polyhedrin fragments (150 and 95 amino acids, respectively) as fusions to foreign proteins under the control of the p10 and polyhedrin promoters, respectively. When this strategy was used to embed an enhancing factor (enhancin or GP37) into the baculovirus OBs, 3- to 5-fold increases in baculoviral infectivity were observed. This novel strategy has the potential to create an efficient protein expression system and a highly efficient virus-based system for insecticide production in the future.

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Characterization of novel components of the baculovirus per os infectivity factor complex.

Publications

Characterization of novel components of the baculovirus per os infectivity factor complex.

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