The level of Nck rather than N-WASP determines the rate of actin-based motility of Vaccinia

ABSTRACT Vaccinia virus exiting from host cells activates Src/Abl kinases to phosphorylate A36, an integral membrane viral protein. Phosphorylated A36 binds the adaptors Nck and Grb2 which recruit N-WASP to activate Arp2/3-driven actin polymerisation to promote viral spread. A36 also recruits intersectin, which enhances actin polymerization via AP-2/clathrin and Cdc42. How many viral and host molecules does vaccinia hijack to induce actin polymerization? To advance our quantitative understanding of this process, we now determined absolute numbers of the essential molecules in the vaccinia signalling network using fluorescent molecule counting approaches in live cells. There are 1156±120 A36 molecules on virus particles inducing actin polymerization in HeLa cells. This number, however, is over 2000 in mouse embryonic fibroblasts (MEFs), suggesting that A36 levels on the virion are not fixed. In MEFs, viruses recruit 1032±200 Nck and 434±10 N-WASP molecules, suggesting a ratio of 4:2:1 for the A36:Nck:N-WASP signalling network. Loss of A36 binding to either secondary adaptors Grb2 or intersectin results in a 1.3- and 2.5-fold reduction in Nck respectively. Curiously, despite recruiting comparable numbers of the Arp2/3 activator, N-WASP (245±26 and 276±66), these mutant viruses move at different speeds that inversely correlate with the number of Nck molecules. Our analysis has uncovered two unexpected new aspects of Vaccinia virus egress, numbers of the viral protein A36 can vary in the virion membrane and the velocity of virus movement depends on the levels of the adaptor protein Nck.