The cowpox virus fusion regulator proteins SPI-3 and hemagglutinin interact in infected and uninfected cells

Abstract

The serpin SPI-3 and the hemagglutinin (HA) encoded by cowpox virus (CPV) block cell–cell fusion, and colocalize at the cell surface. wtCPV does not fuse cells, but inactivation of either gene leads to fusion. SPI-3 mAb added to wtCPV-infected cells caused fusion, confirming that SPI-3 protein at the cell surface prevents fusion. The SPI-3 mAb epitope mapped to an 85-amino acid region at the C-terminus. Removal of either 44 residues from the SPI-3 C-terminus or 48 residues following the N-terminal signal sequence resulted in fusion. Interaction between SPI-3 and HA proteins in infected cells was shown by coimmunoprecipitation. SPI-3/HA was not associated with the A27L “fusion” protein. SPI-3 and HA were able to associate in uninfected cells in the absence of other viral proteins. The HA-binding domain in SPI-3 resided in the C-terminal 229 residues, and did not include helix D, which mediates cofactor interaction in many other serpins.