A36-dependent Actin Filament Nucleation Promotes Release of Vaccinia Virus

PLoS Pathogens

Russell J. Diefenbach¹  Cynthia B. Whitchurch²  Timothy P. Newsome³  Delfine Cheng⁴  Filip Braet⁴  Helena Lynn⁵  Jacquelyn Horsington⁵  Guna Karupiah⁶  Lynne Turnbull⁶ 

[1] Australian Centre for Microscopy & Microanalysis, University of Sydney, Sydney, New South Wales, Australia;Centre for Virus Research, Westmead Millennium Institute, University of Sydney, Westmead, New South Wales, Australia;John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory, Australia;School of Medical Sciences (Discipline of Anatomy and Histology), The Bosch Institute, The University of Sydney, New South Wales, Australia;School of Molecular Bioscience, University of Sydney, Sydney, New South Wales, Australia;The ithree institute, University of Technology Sydney, Sydney, New South Wales, Australia

关键词: Cell membranes;    Actins;    Nucleation;    Membrane proteins;    Pathogen motility;    Vaccinia virus;    Cell motility;    Actin filaments;   

DOI  :  10.1371/journal.ppat.1003239

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36YdF virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36YdF infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36YdF extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5P189S mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force of actin nucleation or by mutations in luminal proteins that weaken these interactions.

【 授权许可】

CC BY