Characterization of a vaccinia virus-encoded 42-kilodalton class I membrane glycoprotein component of the extracellular virus envelope

Abstract

Using a reverse genetic approach, we have demonstrated that the product of the B5R open reading frame (ORF), which has homology with members of the family of complement control proteins, is a membrane glycoprotein present in the extracellular enveloped (EEV) form of vaccinia virus but absent from the intracellular naked (INV) form. An antibody (C'-B5R) raised to a 15-amino-acid peptide from the translated B5R ORF reacted with a 42-kDa protein (gp42) found in vaccinia virus-infected cells and cesium chloride-banded EEV but not INV. Under nonreducing conditions, an 85-kDa component, possibly representing a hetero- or homodimeric form of gp42, was detected by both immunoprecipitation and Western immunoblot analysis. Metabolic labeling with [3H]glucosamine and [3H]palmitate revealed that the B5R product is glycosylated and acylated. The C-terminal transmembrane domain of the protein was identified by constructing a recombinant vaccinia virus that overexpressed a truncated, secreted form of the B5R ORF product. By N-terminal sequence analysis of this secreted protein, the site of signal peptide cleavage of gp42 was determined. A previously described monoclonal antibody (MAb 20) raised to EEV, which immunoprecipitated a protein with biochemical characteristics similar to those of wild-type gp42, reacted with the recombinant, secreted product of the B5R ORF. Immunofluorescence of wild-type vaccinia virus-infected cells by using either MAb 20 or C'-B5R revealed that the protein is expressed on the cell surface and within the cytoplasm. Immunogold labeling of EEV and INV with MAb 20 demonstrated that the protein was found exclusively on the EEV membrane.

The antiviral activity of antibodies in vitro and in vivo

“…Orthopoxviruses use both these strategies. First, the EEV, which is responsible for cell-to-cell and long-range spread of 226 PARREN AND BURTON orthopoxvirus infection (Blasco and Moss, 1992), expresses a viral protein on its membrane, B5R, which has homology to complement control proteins and may provide protection against complement-mediated virolysis of EEV (Engelstad et al, 1992;Isaacs et al, 1992;Vanderplasschen et al, 1998). Vaccinia virus, cowpox, and variola virus secrete complement control proteins that inhibit complement activation via inhibition of C3 convertase activity (Kotwal et al, 1990;Rosengard et al, 1999;Tortorella et al, 2000).…”

The formin FHOD1 and the small GTPase Rac1 promote vaccinia virus actin–based motility

“…The viral proteins B5 and A36 are essential for vaccinia actin-based motility (Sanderson et al, 1998;Wolffe et al, 1998;Katz et al, 2003). B5 and A36 are located in the inner and outer membrane of IEVs, respectively (Engelstad et al, 1992;Isaacs et al, 1992;Parkinson and Smith, 1994;Röttger et al, 1999). The fusion of the outer membrane of IEVs to the plasma membrane exposes B5 at the surface of CEVs (Engelstad et al, 1992;Isaacs et al, 1992) and positions A36 in the plasma membrane, underneath CEVs (van Eijl et al, 2000).…”

Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread

“…The EEV envelope contains several viral proteins, including A56R [21,22], F13L [23,24], B5R [13,25], A36R [26], A34R [27,28], and A33R [29]. Among those, B5 [30] and A33 [31] proteins are known neutralization or viral spread inhibition targets associated with the EEV membrane and/or infected cells.…”