1. Cellular mechanisms of alpha herpesvirus egress: live cell fluorescence microscopy of pseudorabies virus exocytosis
- Author
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Ian B. Hogue, Jens B. Bosse, Jiun-Ruey Hu, Lynn W. Enquist, and Stephan Y. Thiberge
- Subjects
Viral Diseases ,viruses ,Nucleocapsids ,Virions ,Molecular Cell Biology ,Capsids ,Medicine and Health Sciences ,lcsh:QH301-705.5 ,Virus Release ,Vesicle ,Vector Construction ,Herpesvirus 1, Suid ,Secretory Vesicle ,3. Good health ,Cell biology ,Infectious Diseases ,Veterinary Diseases ,Viral Envelope ,Research Article ,lcsh:Immunologic diseases. Allergy ,Immunology ,Sexually Transmitted Diseases ,Viral Structure ,DNA construction ,Biology ,Microbiology ,Exocytosis ,Cell Line ,Gene Delivery ,RAB6A ,Virology ,Gene Expression and Vector Techniques ,Genetics ,Viral Components ,Humans ,Molecular Biology ,Secretory pathway ,Molecular Biology Assays and Analysis Techniques ,Biology and life sciences ,Lipid bilayer fusion ,Herpes Simplex ,Epithelial Cells ,Cell Biology ,Recombinant Gene Expression Techniques ,Veterinary Virology ,Molecular biology techniques ,Microscopy, Fluorescence ,lcsh:Biology (General) ,rab GTP-Binding Proteins ,Viral exocytosis ,Veterinary Science ,Parasitology ,Rab ,Carrier Proteins ,lcsh:RC581-607 - 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., Author Summary Pseudorabies virus, an alpha herpesvirus, is an important veterinary pathogen, and related to human varicella-zoster virus and herpes simplex viruses. 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. In particular, how this process unfolds over time is not easily observed using previous methods. In this study, we developed a new method to observe this egress process. Using this method, we described how virus particles move on their way out: individual virus particles travel to the cell surface, directly to the exit site, where they pause for several seconds before crossing out of the cell. We identified several cellular proteins that are involved in this process. After exiting, virus particles remained stuck to the outer cell surface. Finally, we draw connections between our observations and other recent studies to propose an integrated model of how alpha herpesvirus particles exit from infected cells.
- Published
- 2014