Polydnaviruses associated with ichneumonid parasitoid wasps (Ichnoviruses) encode large numbers of genes, often in multigene families. The Ichnovirus Vinnexin gene family, which is expressed in parasitized lepidopteran larvae, encodes homologues of Innexins, the structural components of insect gap junctions. Here, we have examined intracellular behaviours of the Campoletis sonorensis Ichnovirus (CsIV) Vinnexins, alone and in combination with a host Innexin orthologue, Innexin2 (Inx2). QRT‐PCR verified that transcription of CsIV vinnexins occurs contemporaneously with inx2, implying co‐occurrence of Vinnexin and Inx2 proteins. Confocal microscopy demonstrated that epitope‐tagged VinnexinG (VnxG) and VinnexinQ2 (VnxQ2) exhibit similar subcellular localization as Spodoptera frugiperda Inx2 (Sf‐Inx2). Surface biotinylation assays verified that all three proteins localize to the cell surface, and cytochalasin B and nocodazole that they rely on actin and microtubule cytoskeletal networks for localization. Immunomicroscopy following co‐transfection of constructs indicates extensive co‐localization of Vinnexins with each other and Sf‐Inx2, and live‐cell imaging of mCherry‐labelled Inx2 supports that Vinnexins may affect Sf‐Inx2 distribution in a Vinnexin‐specific fashion. Our findings support that the Vinnexins may disrupt host cell physiology in a protein‐specific manner through altering gap junctional intercellular channel communication, as well as indirectly by affecting multicellular junction characteristics.

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