Pseudorabies Virus Entry and Egress Visualized by Fluorescence Microscopy and a pH-Sensitive Reporter

Abstract

Conflicting reports have emerged as to whether entry of alpha-herpesvirus particles occurs by receptor-mediated membrane fusion or pH-dependent endocytosis, and whether the method of entry differs by cell type. At the other end of the virus replication cycle, it is unclear whether egress of alpha-herpesvirus particles occurs at terminal synapses, varicosities, or inter-varicosity sites of neuronal axons. We aimed to explore the pathway of virus entry and the sites of virus egress through live cell imaging of virus particles tagged with a pH-sensitive fluorescent protein, pHluorin. Here, we characterized PRV483, a novel recombinant of PRV Becker encoding both a fusion between capsid protein VP26 and monomeric red fluorescent protein, as well as a fusion between envelope glycoprotein M and pHluorin. These studies mark the first time, to our knowledge, that a pH-sensitive fluorescent protein has been used to investigate virus particle egress. We imaged rat superior cervical ganglion neurons infected with PRV483, starting around 10 minutes post infection to observe virus entry, and 10 hours post infection to observe virus egress. In entry experiments, doubly-labeled puncta were observed, albeit rarely, to undergo directional movement, consistent with microtubule-based transport, after attachment to the axon, suggesting that endocytosis may be a mode of entry used by some portion of virus particles. In egress experiments, red puncta were observed to accumulate at varicosities and become dual-labeled, suggesting that virus egress occurs at varicosities. The findings from this exploratory study question the existing consensus on herpesvirus entry pathway in neurons and provide clues to herpesvirus sites of egress from neurons.