Cellular Mechanisms of Alpha Herpesvirus Egress: Live Cell Fluorescence Microscopy of Pseudorabies Virus Exocytosis

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.

Mildly Acidic pH Triggers an Irreversible Conformational Change in the Fusion Domain of Herpes Simplex Virus 1 Glycoprotein B and Inactivation of Viral Entry

“…1). From the time of assembly and egress of a newly synthesized virion to its entry into a new target cell, a single enveloped HSV particle may encounter multiple acidic-pH environments (2,31). We next assessed the effect of multiple low-pH exposures on HSV-1 infectivity.…”

The diffusive way out: Herpesviruses remodel the host nucleus, enabling capsids to access the inner nuclear membrane

“…Finally, the vesicle containing the mature enveloped particle fuses with the plasma membrane, releasing the mature virion in an exocytosis-like event. [1][2][3] In the last 20 y or so it became very clear that both during entry as well as exit, herpesvirus particles use microtubule-associated, motor-dependent transport to cross the cytoplasmic space. [4][5][6] However much less is known of how newly formed and packaged capsids cross the nuclear space to the reach the inner nuclear membrane for egress.…”

Repair of spinal cord injury with neuronal relays: From fetal grafts to neural stem cells

“…Transsynaptic viral labeling such as pseudorabies virus (PRV) (Lee et al, 2014) and wheat germ agglutinin (WGA) expressing vectors (Fujimoto et al, 2012) can be used to trace the entire circuit, but alternate routes of viral transmission should be considered when interpreting data from incomplete injuries. Although the available data supports the hypothesis that both PRV and WGA are transferred at active synaptic connections, many of those studies have been conducted in vitro (Hogue et al, 2014) or intact CNS (Ohashi et al, 2011) rather than a relay model of SCI repair. The release of vesicles from growth cones (Sabo and McAllister, 2003; Tojima et al, 2007), or the establishment and retraction of functional synapses within a relay could lead to labeling of neurons without permanent synaptic connections.…”