Vaccinia virus (VACV) produces two distinct enveloped virions, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), but the entry mechanism of neither virion is understood. Here, the binding and entry of IMV particles have been investigated. The cell receptors for IMV are unknown, but it was proposed that IMV can bind to glycosaminoglycans (GAGs) on the cell surface and three IMV surface proteins have been implicated in this. In this study, the effect of soluble GAGs on IMV infectivity was reinvestigated and it was demonstrated that GAGs affected IMV infectivity partially in some cells, but not at all in others. Therefore, binding of IMV to GAGs is cell type-specific and not essential for IMV entry. By using electron microscopy, it is demonstrated that IMV from strains Western Reserve and modified virus Ankara enter cells by fusion with the plasma membrane. After an IMV particle bound to the cell, the IMV membrane fused with the plasma membrane and released the virus core into the cytoplasm. IMV surface antigen became incorporated into the plasma membrane and was not left outside the cell, as claimed in previous studies. Continuity between the IMV membrane and the plasma membrane was confirmed by tilt-series analysis to orientate membranes perpendicularly to the beam of the electron microscope. This analysis shows unequivocally that IMV is surrounded by a single lipid membrane and enters by fusion at the cell surface.

Hitherto, all enveloped viruses were thought to shed their lipid membrane during entry into cells by membrane fusion. The extracellular form of Vaccinia virus has two lipid envelopes surrounding the virus core, and consequently a single fusion event will not deliver a naked core into the cell. Here we report a previously underscribed mechanism in which the outer viral membrane is disrupted by a ligand-induced nonfusogenic reaction, followed by the fusion of the inner viral membrane with the plasma membrane and penetration of the virus core into the cytoplasm. The dissolution of the outer envelope depends on interactions with cellular polyanionic molecules and requires the virus glycoproteins A34 and B5. This discovery represents a remarkable example of how viruses manipulate biological membranes, solves the topological problem of how a double-enveloped virus enters cells, reveals a new effect of polyanions on viruses, and provides a therapeutic approach for treatment of poxvirus infections, such as smallpox.antiviral therapy ͉ extracellular enveloped virus ͉ membrane dissolution ͉ Vaccinia virus ͉ virus entry H itherto, membrane fusion was the only known mechanism by which enveloped viruses overcome the lipid barrier to enter and replicate in cells (1, 2). Here we show that the extracellular enveloped virus (EEV) of Vaccinia virus (VACV) sheds its outer lipid membrane by a ligand-dependent nonfusogenic mechanism.VACV replication produces several distinct virions: the intracellular mature virus (IMV), intracellular enveloped virus, cellassociated enveloped virus (CEV), and EEV (3, 4). IMV is surrounded by one lipid membrane (5-9) and is physically robust to aid virus transmission between hosts. CEV and EEV are IMV particles wrapped with an additional membrane derived from the trans-Golgi network (10) or endosomes (11) and are responsible for virus dissemination within the host (4). This extra lipid envelope (EEV membrane) and the associated virus and host membrane proteins serve to protect the IMV particle within from immune surveillance and may contribute to a broader cell tropism of the virus (4). Recently, we provided unequivocal electron micrographs showing that IMV enters by fusion with the plasma membrane (8), consistent with previous reports (6,12,13), and the recent genetic evidence for entry by fusion (14-17). Once the naked core has entered the cytoplasm, it moves deeper into the cell on microtubules (18). However, for VACV EEV, the additional EEV membrane presents an unexplained topological problem for entry, because fusion of the EEV outer envelope with the plasma membrane or the membrane of an intracellular vesicle will release only an IMV, instead of a naked core, into the cytosol. For a recent review of VACV entry, see ref.19.Here we studied the entry of EEV by immuno-EM and demonstrated that the EEV outer membrane is disrupted at the point of cell contact after binding. This enables the IMV within to enter the cell by fusion with the plasma membrane. The ligands required for membrane rupture were ...

Infection with Vaccinia virus (VV) produces several distinct virions called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). In this report, we have investigated how incoming virus cores derived from IMV are transported within the cell. To do this, recombinant VVs (vA5L-EGFP-N and vA5L-EGFP-C) were generated in which the A5L virus core protein was fused with the enhanced green fluorescent protein (EGFP) at the N or C terminus. These viruses were viable, induced formation of actin tails and had a plaque size similar to wild-type. Immunoblotting showed the A5L-EGFP fusion protein was present in IMV particles and immunoelectron microscopy showed that the fusion protein was incorporated into VV cores. IMV made by vA5L-EGFP-N were used to follow the location and movement of cores after infection of PtK 2 cells. Confocal microscopy showed that virus cores were stained with anti-core antibody only after they had entered the cell and, once intracellular, were negative for the IMV surface protein D8L. These cores co-localized with microtubules and moved in a stop-start manner with an average speed of 51?8 (±3?9) mm min "1 , consistent with microtubular movement. Treatment of cells with nocodazole or colchicine inhibited core movement, but addition of cytochalasin D did not. These data show that VV cores derived from IMV use microtubules for intracellular transport after entry.

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.