[Show abstract][Hide abstract] ABSTRACT: Vaccinia virus has a broad range of infectivity in many cell lines and animals. Although it is known that the vaccinia mature virus binds to cell surface glycosaminoglycans and extracellular matrix proteins, whether additional cellular receptors are required for virus entry remains unclear. Our previous studies showed that the vaccinia mature virus enters through lipid rafts, suggesting the involvement of raft-associated cellular proteins. Here we demonstrate that one lipid raft-associated protein, integrin β1, is important for vaccinia mature virus entry into HeLa cells. Vaccinia virus associates with integrin β1 in lipid rafts on the cell surface, and the knockdown of integrin β1 in HeLa cells reduces vaccinia mature virus entry. Additionally, vaccinia mature virus infection is reduced in a mouse cell line, GD25, that is deficient in integrin β1 expression. Vaccinia mature virus infection triggers the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling, and the treatment of cells with inhibitors to block P13K activation reduces virus entry in an integrin β1-dependent manner, suggesting that integrin β1-mediates PI3K/Akt activation induced by vaccinia virus and that this signaling pathway is essential for virus endocytosis. The inhibition of integrin β1-mediated cell adhesion results in a reduction of vaccinia virus entry and the disruption of focal adhesion and PI3K/Akt activation. In summary, our results show that the binding of vaccinia mature virus to cells mimics the outside-in activation process of integrin functions to facilitate vaccinia virus entry into HeLa cells.
Journal of Virology 04/2012; 86(12):6677-87. DOI:10.1128/JVI.06860-11 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mature vaccinia virus enters cells through either fluid-phase endocytosis/macropinocytosis or plasma membrane fusion. This may explain the wide range of host cell susceptibilities to vaccinia virus entry; however, it is not known how vaccinia virus chooses between these two pathways and which viral envelope proteins determine such processes. By screening several recombinant viruses and different strains, we found that mature virions containing the vaccinia virus A25 and A26 proteins entered HeLa cells preferentially through a bafilomycin-sensitive entry pathway, whereas virions lacking these two proteins entered through a bafilomycin-resistant pathway. To investigate whether the A25 and A26 proteins contribute to entry pathway specificity, two mutant vaccinia viruses, WRDeltaA25L and WRDeltaA26L, were subsequently generated from the wild-type WR strain. In contrast to the WR strain, both the WRDeltaA25L and WRDeltaA26L viruses became resistant to bafilomycin, suggesting that the removal of the A25 and A26 proteins bypassed the low-pH endosomal requirement for mature virion entry. Indeed, WRDeltaA25L and WRDeltaA26L virus infections of HeLa, CHO-K1, and L cells immediately triggered cell-to-cell fusion at a neutral pH at 1 to 2 h postinfection (p.i.), providing direct evidence that viral fusion machinery is readily activated after the removal of the A25 and A26 proteins to allow virus entry through the plasma membrane. In summary, our data support a model that on vaccinia mature virions, the viral A25 and A26 proteins are low-pH-sensitive fusion suppressors whose inactivation during the endocytic route results in viral and cell membrane fusion. Our results also suggest that during virion morphogenesis, the incorporation of the A25 and A26 proteins into mature virions may help restrain viral fusion activity until the time of infections.
Journal of Virology 09/2010; 84(17):8422-32. DOI:10.1128/JVI.00599-10 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The vaccinia virus WR53.5L/F14.5L gene encodes a small conserved protein that was not detected previously. However, additional
proteomic analyses of different vaccinia virus isolates and strains revealed that the WR53.5 protein was incorporated into
intracellular mature virus (IMV). The WR53.5 protein contains a putative N-terminal transmembrane region and a short C-terminal
region. Protease digestion removed the C terminus of WR53.5 protein from IMV particles, suggesting a similar topology to that
of the IMV type II transmembrane protein. We generated a recombinant vaccinia virus, vi53.5L, that expressed WR53.5 protein
under isopropyl-β-d-thiogalactopyranoside (IPTG) regulation and found that the vaccinia virus life cycle proceeded normally with or without IPTG,
suggesting that WR53.5 protein is not essential for vaccinia virus growth in cell cultures. Interestingly, the C-terminal
region of WR53.5 protein was exposed on the cell surface of infected cells and mediated calcium-independent cell adhesion.
Finally, viruses with inactivated WR53.5L gene expression exhibited reduced virulence in mice when animals were inoculated
intranasally, demonstrating that WR53.5 protein was required for virus virulence in vivo. In summary, we identified a new
vaccinia IMV envelope protein, WR53.5, that mediates cell adhesion and is important for virus virulence in vivo.
Journal of Virology 09/2008; 82(20):10079-87. DOI:10.1128/JVI.00816-08 · 4.44 Impact Factor