Abstract
Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV) particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence (TIRF) microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement analysis to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically associated with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5β, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biological and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress.
Highlights
Pseudorabies virus (PRV; suid herpesvirus 1) is a veterinary pathogen, widely used as a neuroanatomical tracing tool, and related to the human alpha herpesviruses varicella-zoster virus (VZV) and herpes simplex virus 1 and 2 (HSV-1 & -2)
New alpha herpesvirus particles are assembled inside an infected cell, and must exit from the infected cell by taking advantage of cellular mechanisms
How these virus particles are transported inside the infected cell and secreted at the cell surface is not understood in great detail
Summary
Pseudorabies virus (PRV; suid herpesvirus 1) is a veterinary pathogen, widely used as a neuroanatomical tracing tool, and related to the human alpha herpesviruses varicella-zoster virus (VZV) and herpes simplex virus 1 and 2 (HSV-1 & -2). Viral membrane proteins are produced in the secretory pathway and traffic to the site of secondary envelopment, thought to be trans-Golgi [2,3,4] and/or endosomal membranes [5,6]. Virus particles acquire their envelopes by budding into these membranes, producing an enveloped virion inside an intracellular vesicle. This virion transport vesicle traffics to the plasma membrane, where the virion exits the infected cell by exocytosis. While this general description of viral egress is widely accepted, the specific mechanisms involved are not well studied
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