D3224: Viral protein trafficking in polarized epithelial cells of Drosophila melanogaster

Arbovirus transmission to vertebrate hosts requires sequential infection of polarized midgut enterocytes (ECs) and salivary gland (SG) epithelial cells of vector insects. Virus exit from ECs into the hemocoel requires viral envelope protein trafficking to the basal surface of ECs whereas virus exit from SG cells into the lumen requires trafficking of these viral envelope proteins to the apical surface. It is unclear how the same viral envelope protein is differentially trafficked, in a polarized manner, within cells of these two tissues. To begin to address this issue, we identified several host genes important for trafficking of the baculovirus (AcMNPV) GP64 envelope glycoprotein to the surface of cultured Drosophila melanogaster cells. We generated transgenic Drosophila flies to study the in vivo trafficking of GP64 and the vesicular stomatitis virus G (VSV G) envelope glycoproteins in polarized midgut ECs and SG cells. For this we used Drosophila that conditionally expresses VSV G or GP64, in the absence of infection or any other viral proteins, and found each viral protein is trafficked basally in ECs. We developed a method for assessing viral protein localization in ECs quantitatively by immunofluorescence. Using this approach, we examined several clathrin adaptor proteins and found AP-2mu to be important for basal localization of VSV G in enterocytes. Both VSV G and GP64 possess a tyrosine-based sorting motif (YxxΦ) in their short cytoplasmic tails. We also found that alanine substitutions in this motif (YxxΦ to AxxA) resulted in loss of basal trafficking of both VSV G and GP64 in ECs in vivo. These studies will provide a better understanding of viral transit through tissues of insect hosts and vectors, and should lead to the development of novel approaches to reduce vector competence for important viral diseases.