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ABSTRACT Venturia canescensis a parasitoid wasp that harbors a domesticated endogenous virus (DEV) and parasitizes host insects likeEphestia kuehniella. TheV. canescensDEV evolved from an alphanudivirus and produces virus-like particles (VLPs) in females that protect wasp eggs from a host immune defense called encapsulation. In contrast, very few DEV genes required for VLP formation and function have been identified. In this study, we characterized fiveV. canescensDEV genes of unknown function that all nudiviruses encode. Three of these genes are single copy (OrNVorf18-like,OrNVorf61-like, andOrNVorf76-like), whileOrNVorf41-likehas expanded into a six-member family andOrNVorf47-likehas expanded into a three-member family. Sequence analysis indicated all of these genes retain essential motifs present in nudivirus homologs, while transmission electron microscopy (TEM) studies characterized the timing of VLP formation during the wasp pupal stage. RNA interference (RNAi) assays identifiedOrNVorf18-like,OrNVorf61-like,OrNVorf41-like-1,andOrNVorf41-like-2as genes that are required for normal VLP formation. Knockdown ofOrNVorf47-likefamily members did not affect VLP formation but did disable binding of VLPs toV. canescenseggs and protection against encapsulation. Disabled formation of VLPs in response to RNAi knockdown ofOrNVorf18-like,OrNVorf61-like,OrNVorf41-like-1,andOrNVorf41-like-2also resulted in wasp eggs being encapsulated. In contrast, knockdown ofOrNVorf76-likehad no effect on VLP assembly, egg binding, or encapsulation. Altogether, reported results significantly advance our understanding ofV. canescensVLP (VcVLP) formation and function. IMPORTANCEUnderstanding howV. canescenscoopted an alphanudivirus to produce VcVLPs is of interest to the study of virus evolution. Our results show that three nudivirus core genes have essential functions in VcVLP formation, while one is essential for the novel function of binding to wasp eggs and protection from encapsulation, which is the most important immune defense of insects against parasitoids. 

ABSTRACT Systemic viral infection of insects typically begins with the primary infection of midgut epithelial cells (enterocytes) and subsequent transit of the progeny virus in an apical-to-basal orientation into the hemocoel. For insect-vectored viruses, an oppositely oriented process (basal-to-apical transit) occurs upon secondary infection of salivary glands and is necessary for virus transmission to non-insect hosts. To examine this inversely oriented virus transit in these polarized tissues, we assessed the intracellular trafficking of two model viral envelope proteins (baculovirus GP64 and vesicular stomatitis virus G) in the midgut and salivary gland cells of the model insect,Drosophila melanogaster. Using fly lines that inducibly express either GP64 or VSV G, we found that each protein, expressed alone, was trafficked basally in midgut enterocytes. In salivary gland cells, VSV G was trafficked apically in most but not all cells, whereas GP64 was consistently trafficked basally. We demonstrated that a YxxØ motif present in both proteins was critical for basal trafficking in midgut enterocytes but dispensable for trafficking in salivary gland cells. Using RNAi, we found that clathrin adaptor protein complexes AP-1 and AP-3, as well as seven Rab GTPases, were involved in polarized VSV G trafficking in midgut enterocytes. Our results indicate that these viral envelope proteins encode the requisite information and require no other viral factors for appropriately polarized trafficking. In addition, they exploit tissue-specific differences in protein trafficking pathways to facilitate virus egress in the appropriate orientation for establishing systemic infections and vectoring infection to other hosts. IMPORTANCEViruses that use insects as hosts must navigate specific routes through different insect tissues to complete their life cycles. The routes may differ substantially depending on the life cycle of the virus. Both insect pathogenic viruses and insect-vectored viruses must navigate through the polarized cells of the midgut epithelium to establish a systemic infection. In addition, insect-vectored viruses must also navigate through the polarized salivary gland epithelium for transmission. Thus, insect-vectored viruses appear to traffic in opposite directions in these two tissues. In this study, we asked whether two viral envelope proteins (VSV G and baculovirus GP64) alone encode the signals necessary for the polarized trafficking associated with their respective life cycles. UsingDrosophilaas a model to examine tissue-specific polarized trafficking of these viral envelope proteins, we identified one of the virus-encoded signals and several host proteins associated with regulating the polarized trafficking in the midgut epithelium.