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Updated: Sep 23, 2026
Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay
Published on: February 22, 2018
Identification of pif-2, a third conserved baculovirus gene required for per os infection of insects
Gorben P Pijlman1, Andrea J P Pruijssers1, Just M Vlak1
1Wageningen University, Laboratory of Virology, Binnenhaven 11, 6709 PD Wageningen, The Netherlands.
Abstract:
Infection of cultured insect cells with Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) resulted in the generation of mutants with major genomic deletions. Some of the mutants lacked the ability to infect S. exigua larvae per os. The gene(s) responsible for this phenotype in SeMNPV was mapped within a contiguous sequence encoding ORFs 29-35. In this paper we have shown that SeMNPV ORFs 15-35 (including genes encoding cathepsin, chitinase, GP37, PTPT-2, EGT, PKIP-1 and ARIF-1) are not essential for virus replication in cell culture or by in vivo intrahaemocoelic injection. By site-specific deletion mutagenesis of a full-length infectious clone of SeMNPV (bacmid) using ET recombination in E. coli, a series of SeMNPV bacmid mutants with increasing deletions in ORFs 15-35 was generated. Analyses of these mutants indicated that a deletion of SeMNPV ORF35 (Se35) resulted in loss of oral infectivity of polyhedral occlusion bodies. Reinsertion of ORF35 in SeMNPV bacmids lacking Se35 rescued oral infectivity. We propose the name pif-2 for Se35 and its baculovirus homologues (e.g. Autographa californica MNPV ORF22), by analogy to a different gene recently characterized in Spodoptera littoralis NPV, which was designated per os infectivity factor (pif). Similar to the p74 gene, which encodes an essential structural protein of the occlusion-derived virus envelope, pif and pif-2 belong to a group of 30 genes that are conserved among the Baculoviradae.
Insights
Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) ORF35 deletion mutants lost oral infectivity. Reinserting ORF35 restored infectivity, identifying it as a per os infectivity factor (pif-2).
Area of Science:
- Virology
- Insect Pathology
- Molecular Biology
Background:
- Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) infection can generate mutants with genomic deletions.
- Some SeMNPV mutants exhibit a loss of oral infectivity in Spodoptera exigua larvae.
- Previous studies mapped the gene(s) responsible for this oral infectivity defect to a region encoding ORFs 29-35.
Purpose of the Study:
- To identify the specific gene(s) within SeMNPV ORFs 15-35 responsible for oral infectivity.
- To characterize the role of SeMNPV ORF35 in the infection process of Spodoptera exigua larvae.
- To propose nomenclature for the identified gene and its homologues.
Main Methods:
- Site-specific deletion mutagenesis of a full-length SeMNPV bacmid clone using ET recombination in E. coli.
- Generation of SeMNPV bacmid mutants with progressive deletions within ORFs 15-35.
- Analysis of mutant infectivity in Spodoptera exigua larvae and rescue experiments by reinserting specific ORFs.
Main Results:
- SeMNPV ORFs 15-35 are not essential for virus replication in cell culture or via intrahaemocoelic injection.
- Deletion of SeMNPV ORF35 (Se35) specifically resulted in the loss of polyhedral occlusion body oral infectivity.
- Reinsertion of ORF35 into Se35 deletion mutants rescued oral infectivity, confirming its essential role.
Conclusions:
- SeMNPV ORF35, proposed as pif-2, is essential for oral infectivity of Spodoptera exigua larvae.
- pif-2 is a novel per os infectivity factor, analogous to previously identified pif genes in other NPVs.
- pif-2 belongs to a conserved group of 30 baculovirus genes, including the structural protein gene p74.
