Abstract
Vaccinia virus (VACV) spreads across cell monolayers fourfold faster than predicted from its replication kinetics. Early after infection, infected cells repulse some superinfecting extracellular enveloped virus (EEV) particles by the formation of actin tails from the cell surface, thereby causing accelerated spread to uninfected cells. This strategy requires the expression of two viral proteins, A33 and A36, on the surface of infected cells and upon contact with EEV this complex induces actin polymerization. Here we have studied this phenomenon further and investigated whether A33 and A36 expression in cell lines causes an increase in VACV plaque size, whether these proteins are able to block superinfection by EEV, and which protein(s) on the EEV surface are required to initiate the formation of actin tails from infected cells. Data presented show that VACV plaque size was not increased by expression of A33 and A36, and these proteins did not block entry of the majority of EEV binding to these cells. In contrast, expression of proteins A56 and K2 inhibited entry of both EEV and intracellular mature virus. Lastly, VACV protein B5 was required on EEV to induce the formation of actin tails at the surface of cells expressing A33 and A36, and B5 short consensus repeat 4 is critical for this induction.
Highlights
Vaccinia virus (VACV) is a member of the genus Orthopoxvirus; it replicates in the cytoplasm (Moss, 2007) and produces multiple distinct virions (Smith et al, 2002; Condit et al, 2006)
Protein expression was confirmed by immunoblotting with anti-A36 or anti-A33 antibodies (Fig. 1a). Expression levels in these cell lines was comparable to that achieved in the HeLa cells used previously (Doceul et al, 2010) and was slightly less than obtained in cells infected with VACV in the presence of cytosine arabinoside
In this paper we have investigated additional features of this phenomenon and addressed the following questions: (i) does the constitutive expression of A33–A36 in cell lines make VACV spread faster than it does in normal cells?; (ii) does this complex prevent the cells being infected by the majority of enveloped virus (EEV) particles?; and (iii) which components of the EEV particle are needed for the interaction with the A33–A36 complex to induce actin polymerization?
Summary
Vaccinia virus (VACV) is a member of the genus Orthopoxvirus; it replicates in the cytoplasm (Moss, 2007) and produces multiple distinct virions (Smith et al, 2002; Condit et al, 2006). Form a cell-associated enveloped virus (CEV) that remains attached to the cell surface. Viral proteins A36 (Parkinson & Smith, 1994; van Eijl et al, 2000) and F12 (Zhang et al, 2000; van Eijl et al, 2002) are associated with IEV and CEV membranes and are absent from IMV and EEV. Proteins A33 (Roper et al, 1996), A34 (Duncan & Smith, 1992), B5 (Engelstad et al, 1992; Isaacs et al, 1992), F13 (Blasco & Moss, 1991) and A56 (Payne & Norrby, 1976) are associated with IEV, CEV and EEV. CEV promotes cell-to-cell spread of virus by the induction of actin tails from the cell surface beneath newly synthesized virions, and EEV promotes the dissemination of virus in cultured cells and in vivo (Smith et al, 2003)
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