A monoclonal antibody, MAbC3, that reacts with a 14,000-molecular-weight envelope protein (14K protein) of vaccinia virus completely inhibited virus-induced cell fusion during infection. Immunoblot and immunofluorescence studies revealed that the 14K protein was synthesized at about 6 to 7 h postinfection and transported from the cytoplasm to the cell surface. Synthesis and transport of the 14K protein during infection occurred in the presence of rifampin, an inhibitor of virus maturation. Oneand two-dimensional gel electrophoretic analyses demonstrated that the 14K protein forms largely trimers (42K) that are covalently linked by disulfide bonds. The facts that MAbC3 prevents virus uncoating and blocks virus-induced cell fusion but does not prevent virus attachment to cells and the 14K envelope protein forms trimers all suggest that this protein plays major role in virus penetration.
MATERIALS AND METHODSCells and Viruses. FEL cells (a subclone of line 745A) were grown in Dulbecco's modified Eagle's medium (DME medium) containing 10% fetal calf serum and antibiotics. These cells have been previously used to study antiviral and interferon-mediated enzyme activities during infection with vaccinia virus (3). The plaque-purified WR strain of vaccinia virus was grown in HeLa S3 spinner cells at a multiplicity of 0.1 plaque-forming unit (pfu) per cell and purified essentially according to the method of Joklik (4). The particle-to-pfu ratio was about 27 when titrated on monolayer cultures of monkey BSC-40 cells. FEL cells were infected with purified vaccinia virus at 1.0 pfu per cell. On the second and third day ofinfection >90% of the cells had died and remained attached to the dish. Surviving cells were removed by centrifugation and resuspended in fresh medium. After recovery (2-3 weeks) the cells were serially passaged every 4-5 days.Characterization of Intracellular DNA and of Virion DNA.Conditions for extraction of high molecular weight DNA from whole cell lysates, isolation of WR-DNA from purified virus, digestion of DNA with restriction endonucleases, gel electrophoresis, Southern blotting, and hybridization with specific nick-translated DNA probes have been previously described (5, 6). Virus from persistently infected cells was plaque purified by selection of well-isolated plaques on monolayers of BSC-40 cells. Independent virus isolates were propagated in BSC-40 cells and virus was purified as described (7). Where appropriate, these viruses were labeled in the DNA with [32P]orthophosphate or [3H]thymidine (7).
Endoglin is a transmembrane glycoprotein 633 residues in length expressed at the surface of endothelial cells as a disulphide-linked homodimer; the specific cysteine residues involved in endoglin dimerization are unknown. Mutations in the coding region of the endoglin gene are responsible for hereditary haemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder. Many of these mutations, if translated, would lead to truncated forms of the protein. It is therefore of interest to assess the protein expression of different truncated forms of endoglin. Infections in vitro or in vivo with recombinant vaccinia virus, as well as transient transfections with expression vectors, were used to express normal and truncated forms of endoglin. Truncated mutants could be classified into three different groups: (1) those that did not produce stable transcripts; (2) those that produced stable transcripts but did not secrete the protein; and (3) those that secreted a soluble dimeric protein. This is the first time that a recombinant truncated form of endoglin has been found to be expressed in a soluble form. Because a chimaeric construct encoding the N-terminal sequence of platelet/endothelial cell adhesion molecule (PECAM-1) antigen fused to residues Ile281-Ala658 of endoglin also yielded a dimeric surface protein, these results suggest that cysteine residues contained within the fragment Cys330-Cys412 are involved in disulphide bond formation. Infection with vaccinia recombinants encoding an HHT1 mutation did not affect the expression of the normal endoglin, and did not reveal an association of the recombinant soluble form with the transmembrane endoglin, supporting a haploinsufficiency model for HHT1.
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