Vaccinia virus protein complex F12/E2 interacts with kinesin light chain isoform 2 to engage the kinesin-1 motor complex.

During vaccinia virus morphogenesis, intracellular mature virus (IMV) particles are wrapped by a double lipid bilayer to form triple enveloped virions called intracellular enveloped virus (IEV). IEV are then transported to the cell surface where the outer IEV membrane fuses with the cell membrane to expose a double enveloped virion outside the cell. The F12, E2 and A36 proteins are involved in transport of IEVs to the cell surface. Deletion of the F12L or E2L genes causes a severe inhibition of IEV transport and a tiny plaque size. Deletion of the A36R gene leads to a smaller reduction in plaque size and less severe inhibition of IEV egress. The A36 protein is present in the outer membrane of IEVs, and over-expressed fragments of this protein interact with kinesin light chain (KLC). However, no interaction of F12 or E2 with the kinesin complex has been reported hitherto. Here the F12/E2 complex is shown to associate with kinesin-1 through an interaction of E2 with the C-terminal tail of KLC isoform 2, which varies considerably between different KLC isoforms. siRNA-mediated knockdown of KLC isoform 1 increased IEV transport to the cell surface and virus plaque size, suggesting interaction with KLC isoform 1 is somehow inhibitory of IEV transport. In contrast, knockdown of KLC isoform 2 did not affect IEV egress or plaque formation, indicating redundancy in virion egress pathways. Lastly, the enhancement of plaque size resulting from loss of KLC isoform 1 was abrogated by removal of KLC isoforms 1 and 2 simultaneously. These observations suggest redundancy in the mechanisms used for IEV egress, with involvement of KLC isoforms 1 and 2, and provide evidence of interaction of F12/E2 complex with the kinesin-1 complex.This work was supported by grant G1000207 from the Medical Research Council, UK and grant 090315 from The Wellcome Trust. GLS is a Wellcome Trust Principal research Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final published version. It first appeared at http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004723

Acidic residues in the membrane-proximal stalk region of vaccinia virus protein B5 are required for glycosaminoglycan-mediated disruption of the extracellular enveloped virus outer membrane

The extracellular enveloped virus (EEV) form of vaccinia virus (VACV) is surrounded by two lipid envelopes. This presents a topological problem for virus entry into cells, because a classical fusion event would only release a virion surrounded by a single envelope into the cell. Recently, we described a mechanism in which the EEV outer membrane is disrupted following interaction with glycosaminoglycans (GAGs) on the cell surface and thus allowing fusion of the inner membrane with the plasma membrane and penetration of a naked core into the cytosol. Here we show that both the B5 and A34 viral glycoproteins are required for this process. A34 is required to recruit B5 into the EEV membrane and B5 acts as a molecular switch to control EEV membrane rupture upon exposure to GAGs. Analysis of VACV strains expressing mutated B5 proteins demonstrated that the acidic stalk region between the transmembrane anchor sequence and the fourth short consensus repeat of B5 are critical for GAG-induced membrane rupture. Furthermore, the interaction between B5 and A34 can be disrupted by the addition of polyanions (GAGs) and polycations, but only the former induce membrane rupture. Based on these data we propose a revised model for EEV entry