A36-dependent Actin Filament Nucleation Promotes Release of Vaccinia Virus

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.

The role of signalling and the cytoskeleton during Vaccinia Virus egress

“…Consistent with this, treatment of mice with the Abl-family kinase inhibitor Gleevec/STI-571/Imatinib reduces viral spread and promotes their survival from an otherwise lethal infection (Reeves et al, 2005;Reeves et al, 2011). The activity of Abl and Arg clearly enhances A36-dependent release of Vaccinia from infected cells (Horsington et al, 2013). In addition, both kinases also promote viral release independently of their ability to phosphorylate A36 (Horsington et al, 2013).…”

“…More than 30 years ago it was demonstrated that inhibition of actin polymerization with cytochalasin D inhibits release of vaccinia from infected cells (Payne and Kristensson, 1982). More recently, it was found that A36 phosphorylation and actin polymerization promotes virus release by driving CEV out of invaginations in the plasma membrane (Horsington et al, 2013). The activity of Abl and Arg but not Src family kinases also promotes the release of CEV from infected cells (Reeves et al, 2005).…”

“…The activity of Abl and Arg clearly enhances A36-dependent release of Vaccinia from infected cells (Horsington et al, 2013). In addition, both kinases also promote viral release independently of their ability to phosphorylate A36 (Horsington et al, 2013). The identity of this additional Abl and Arg substrate that presumably promotes actin-dependent viral release remains to be established.…”

Functional Comparison of Herpes Simplex Virus 1 (HSV-1) and HSV-2 ICP27 Homologs Reveals a Role for ICP27 in Virion Release

“…6D) phenotypes were more extreme than those of K2F1, again suggesting a linkage between these traits. We also note that, in the vaccinia virus system, similar 2-to 4-fold reductions in viral release efficiency can have similar negative effects on the formation of comet-like plaques (61). The deletions in the dAc and d2-3 mutants encompass ICP27 residues 20 to 63 and 64 to 108, respectively.…”

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

“…Fluorescent tags are expressed from the viral genome, eliminating the need for transfection, enabling primary cells derived from infected animals or nontransfectable cells to be easily analyzed. Initially, fluorescent VACVs were used for simple subcellular tracking of virus movement 30 , but more recent approaches have expanded their utility to include FRET studies 31 , FRAP at single virus particles 32 , promoter reporters 33 , intravital imaging 34 , and structural studies [35][36][37] . All these techniques could be within easier and closer reach coupled with this method of creating recombinant fluorescent viruses.…”