Abstract
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.
Highlights
Crossing the membrane of host cells, either during entry or escape, is a major obstacle facing prospective viral pathogens during infection
Our results suggest that untethering virus attachment to the cell surface is a rate-limiting step during exocytic release of vaccinia virus
To better understand the role of pathogen-induced actin nucleation in the replication and spread of vaccinia virus (VACV), we tested the effects of blocking the phosphorylation of A36Y112 and A36Y132 on the release of enveloped virus (EEV)
Summary
Crossing the membrane of host cells, either during entry or escape, is a major obstacle facing prospective viral pathogens during infection. Enveloped viruses are released from cells by either the acquisition or loss of an outer membrane and both strategies pose unique challenges to the final separation of pathogen from host. Where viruses gain a membrane, for example influenza virus and human immunodeficiency virus, a tight association must be formed between assembling viral complexes and the internal surface of the cell membrane that is loaded with viral envelope proteins [1,2]. Other viruses, including herpes simplex virus, acquire a double membrane during morphogenesis that is tightly complexed by viral protein interactions across the luminal space [4]. Extracellular CEV remain associated with host cells and this attachment is likely to be mediated by viral envelope proteins [6,7,8]. In support of a role for these luminal domains in virus–host cell adhesion, a number of mutations in these proteins have been
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
