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

Ian Hogue, Jens B. Bosse, Jiun Ruey Hu, Stephan Y. Thiberge, Lynn W. Enquist

Research output: Contribution to journalArticle

36 Citations (Scopus)

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.

Original languageEnglish (US)
JournalPLoS Pathogens
Volume10
Issue number12
DOIs
StatePublished - Jan 1 2014
Externally publishedYes

Fingerprint

Suid Herpesvirus 1
Herpesviridae
Exocytosis
Fluorescence Microscopy
Virion
Cell Membrane
Secretory Pathway
Secretory Vesicles
rab GTP-Binding Proteins
Viruses
Viral Structures
Membrane Fusion
Molecular Dynamics Simulation
Microtubules
Epithelial Cells

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Molecular Biology
  • Genetics
  • Virology

Cite this

Cellular Mechanisms of Alpha Herpesvirus Egress : Live Cell Fluorescence Microscopy of Pseudorabies Virus Exocytosis. / Hogue, Ian; Bosse, Jens B.; Hu, Jiun Ruey; Thiberge, Stephan Y.; Enquist, Lynn W.

In: PLoS Pathogens, Vol. 10, No. 12, 01.01.2014.

Research output: Contribution to journalArticle

Hogue, Ian ; Bosse, Jens B. ; Hu, Jiun Ruey ; Thiberge, Stephan Y. ; Enquist, Lynn W. / Cellular Mechanisms of Alpha Herpesvirus Egress : Live Cell Fluorescence Microscopy of Pseudorabies Virus Exocytosis. In: PLoS Pathogens. 2014 ; Vol. 10, No. 12.
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