Novel Role for ESCRT-III Component CHMP4C in the Integrity of the Endocytic Network Utilized for Herpes Simplex Virus Envelopment

Enveloped viruses exploit cellular trafficking pathways for their morphogenesis, providing potential scope for the development of new antiviral therapies. We have previously shown that herpes simplex virus 1 (HSV1) utilizes recycling endocytic membranes as the source of its envelope, in a process involving four Rab GTPases. To identify novel factors involved in HSV1 envelopment, we have screened a small interfering RNA (siRNA) library targeting over 80 human trafficking proteins, including coat proteins, adaptor proteins, fusion factors, fission factors, and Rab effectors. The depletion of 11 factors reduced virus yields by 20- to 100-fold, including three early secretory pathway proteins, four late secretory pathway proteins, and four endocytic pathway proteins, three of which are membrane fission factors. Five of the 11 targets were chosen for further analysis in virus infection, where it was found that the absence of only 1, the fission factor CHMP4C, but not the CHMP4A or CHMP4B paralogues, reduced virus production at the final stage of morphogenesis. Ultrastructural and confocal microscopy of CHMP4C-depleted, HSV1-infected cells showed an accumulation of endocytic membranes; extensive tubulation of recycling, transferrin receptor-positive endosomes indicative of aberrant fission; and a failure in virus envelopment. No effect on the late endocytic pathway was detected, while exogenous CHMP4C was shown to localize to recycling endosomes. Taken together, these data reveal a novel role for the CHMP4C fission factor in the integrity of the recycling endosomal network, which has been unveiled through the dependence of HSV1 on these membranes for the acquisition of their envelopes.