Entry is a rate-limiting step for viral infection in a Drosophila melanogaster model of pathogenesis

Abstract

The identification of host factors that control susceptibility to infection has been hampered by a lack of amenable genetic systems. We established an in vivo model to determine the host factors that control pathogenesis and identified viral entry as a rate-limiting step for infection. We infected Drosophila melanogaster cells and adults with drosophila C virus and found that the clathrin-mediated endocytotic pathway is essential for both infection and pathogenesis. Heterozygosity for mutations in genes involved in endocytosis is sufficient to protect flies from pathogenicity, indicating the exquisite sensitivity and dependency of the virus on this pathway. Thus, this virus model provides a sensitive and efficient approach for identifying components required for pathogenesis.

Figure 1

DCV is infectious in vitro. (a) Flow cytometry of DL2 cells stained with propidium iodide at 4 d after infection (unshaded) or after being left uninfected (shaded). Cellular debris were electronically ‘gated out’ of the analysis. The percentage of dead cells before and after infection is calculated from the electronically gated regions shown (bracketed line). The shift from 3% to 80% dead cells indicates substantial cytopathic effect. (b) Immunoblot probed with anti-DCV of lysates from adult flies left uninfected (Un), 48 h after buffer injection (Buff) or at 48 h after infection (Inf). Infected DL2 cells (DL2 inf) and purified virions (Pure virus) are also shown. Lysates were quantified and normalized before loading, excluding the purified virions.

Figure 2

DCV traffics through a vesicular compartment and requires a functional endocytic apparatus in vitro. (a) Time course of infection monitored by immunofluorescence. Cells are uninfected (A), infected for 3 h at 4 °C to allow surface binding but no endocytosis (B), infected for 3 h at 4 °C followed by 3 h at 25 °C to release the block to endocytosis and monitor viral trafficking (C), and infected for 3 h at 4 °C followed by 7.5 h at 25 °C to monitor the initiation of new viral replication (D). Panels E–H are the same time course and conditions as in A–D except valinomycin was added 15 min before t = 0. Green, anti-DCV; red, Alexa Fluor-568–phalloidin. (b,c) Immunofluorescence assay of DL2 cells infected with DCV at 24 h after infection shows that cells treated with bafilomycin A1 (Baf), valinomycin (Val) or cycloheximide (Cyclo), have much less immunostaining than untreated cells (Untreated; no drugs). All nuclei are labeled in red (Hoescht 33342) and infected cells, in green (anti-DCV). This is quantified in c for three sites per well in three independent experiments. Data are presented relative to the mean fractional infection seen in untreated cells (0.52 ± 0.16).

Figure 3

Clathrin-mediated endocytosis is required for DCV infection. (a,b) The clathrin heavy chain (chc4/+), α-Adaptin (α-Ada3/α-Ada4), synaptotagmin (sytT77/+) and abnormal wing discs (awd^K–pn/awd^K–pn) mutants are more resistant to DCV infection than are wild-type flies. In contrast, other mutations that perturb vesicular trafficking, tested as homozygous (or¹, g², rb¹, dor⁸, dor¹, lt¹, hk¹, com^ST53, com¹⁹¹, ca¹, pn¹, ltd¹, p^p and p^snb) or heterozygous (lap¹, hsc70–4⁰³⁵⁵⁰, csp⁰³⁹⁸, syb^k07703, nsyb^I18 and syx^L266) mutations, were not substantially different from wild-type (a representative experiment is shown with a subset of mutants). Groups of >25 flies were injected with DCV and monitored daily for mortality. (c) Immunoblot analysis shows that the delayed mortality correlates with the production of viral antigens. Wild-type, α-Ada3/α-Ada4 or chc4/+ flies were left uninjected (0) or were challenged with DCV, and protein lysates were generated (time points after infection, above lanes), normalized and probed with anti-DCV. (d) Immunofluorescence analysis of DCV-infected wild-type or α-Ada3/α-Ada4 fat body and egg chambers shows vastly reduced immunostaining in the infected mutant tissues. Green, anti-DCV; red, Alexa Fluor-568–phalloidin.

Figure 4

Autonomous requirement for dynamin in viral entry. Immunofluorescence analysis of fat body and egg chambers of wild-type flies or mosaic flies (yolk-Gal4/UAS-shi^dn) shows that loss of dynamin activity in the fat body is sufficient to block the production of viral antigens but not in the egg chamber of the same fly. Green, anti-DCV; red, Alexa Fluor-568–phalloidin.