The vaccinia virus fusion inhibitor proteins SPI-3 (K2) and HA (A56) expressed by infected cells reduce the entry of superinfecting virus

The orthopoxvirus SPI-3 (K2) and A56 (hemagglutinin, HA) proteins interact and together prevent cell–cell fusion. SPI-3/A56 has been proposed to prevent the superinfection of previously infected cells by reducing virus–cell fusion. Binding of mature virions of vaccinia virus (VV) to VV-infected cells was unaffected by SPI-3 or A56 on the surface of infected cells. Entry of VV into infected cells was assessed using VV-P_T7-luc carrying the luciferase reporter under T7 control. Cells infected with VV or cowpox virus (CPV) expressing T7 RNA polymerase and lacking SPI-3 and/or A56 were superinfected with VV-P_T7-luc, and luciferase activity was measured. Inactivation of SPI-3 or A56 from the pre-infecting virus resulted in greater luciferase expression from the superinfecting VV-P_T7-luc. Antibody against SPI-3 present during infection with wild-type CPV-T7 increased luciferase expression from superinfecting VV-P_T7-luc. The SPI-3/A56 complex on the infected cell surface therefore appears to reduce the entry of virions into infected cells.