Identification of pif-2, a third conserved baculovirus gene required for per os infection of insects

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 (pi. 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 Baculoviridae.