bioRxiv2024
Systemic viral infection of insects typically begins with primary infection of midgut epithelial cells (enterocytes) and subsequent transit of virus in an apical-to-basal orientation through the polarized enterocytes into the hemocoel. In the case of insect-vectored viruses, a similar yet oppositely oriented process (basal-to-apical virus 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 glycoprotein, VSV G) in the midgut and salivary gland cells of the model insect, Drosophila melanogaster. Using transgenic Drosophila fly lines that inducibly express either GP64 or VSV G, we found that both proteins were trafficked basally in midgut enterocytes. In salivary gland cells, VSV G was trafficked to apical membranes in most but not all cells, whereas GP64 was trafficked consistently to basal membranes. We further examined the mechanism of polarized trafficking in midgut and salivary gland epithelia and found that a cytoplasmic YxxO motif in both VSV G and GP64 proteins is critical for basal trafficking of each envelope protein in midgut enterocytes, but dispensable for their trafficking in salivary gland epithelial cells. Using RNAi, we found that clathrin adapter protein complexes AP1 and AP3, as well as several Rab GTPases (Rab1, 4, 8, 10, 23, 30, and - 35), 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. Author SummaryViruses that use insects as hosts must navigate specific routes through the insects tissues to complete their life cycles. The routes may differ substantially depending on the life cycle of the virus. Some insect pathogenic viruses, such as baculoviruses, establish a systemic infection and this represents an endpoint in the infection cycle in the insect. In contrast, many insect-vectored viruses establish a systemic infection in the insect, but must also deliver infectious virus to the insects non-insect host. In both cases, the virus must first navigate through the midgut epithelium to establish a systemic infection, but insect-vectored viruses must also navigate through the salivary gland epithelium. Both midgut and salivary gland cells are polarized, and insect-vectored viruses appear to traffic in opposite directions in these two tissues. In this study, we asked whether two viral envelope proteins alone encode the signals necessary for polarized trafficking associated with their respective life cycles. Using two representative viral envelope proteins (VSV G and baculovirus GP64) and Drosophila as a model insect to examine tissue-specific polarized trafficking of 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.