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Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies.

ABSTRACT: 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.

SUBMITTER: Yang S

PROVIDER: S-EPMC5494622 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies.

Yang Shili S Zhao Lijuan L Ma Ruipeng R Fang Wei W Hu Jia J Lei Chengfeng C Sun Xiulian X

Applied and environmental microbiology 20170630 14

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 <i>Autographa californica multiple nucleopolyhedrovirus</i> (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 ...[more]

PMID: 28500037

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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.

Dataset Information

Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies.

Publications

Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies.

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