The amount of Nck rather than N-WASP correlates with the rate of actin-based motility of Vaccinia virus

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 polymerization 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 such a virus-hijacked signaling network engage? To advance our quantitative understanding of this model signaling network, we determined the absolute numbers of the key molecules using fluorescent molecule-counting approaches in live cells. There are 1,156 ± 120 A36 molecules on virus particles inducing actin polymerization in HeLa cells. This number, however, is over 2,000 in mouse embryonic fibroblasts (MEFs), suggesting that A36 levels on the virion are not fixed. In MEFs, viruses recruit 1,032 ± 200 Nck and 434 ± 10 N-WASP molecules, suggesting a ratio of 4:2:1 for the A36:Nck:N-WASP signaling network. Loss of A36 binding to either secondary factor 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 rate of virus movement depends on the adaptor protein Nck. IMPORTANCE Vaccinia virus is a large double-stranded DNA virus and a close relative of Mpox and Variola virus, the causative agent of smallpox. During infection, Vaccinia hijacks its host’s transport systems and promotes its spread into neighboring cells by recruiting a signaling network that stimulates actin polymerization. Over the years, Vaccinia has provided a powerful model to understand how signaling networks regulate actin polymerization. Nevertheless, we still lack important quantitative information about the system, including the precise number of viral and host molecules required to induce actin polymerization. Using quantitative fluorescence microscopy techniques, we have determined the number of viral and host signaling proteins accumulating on virions during their egress. Our analysis has uncovered two unexpected new aspects of this process: the number of viral proteins in the virion is not fixed and the velocity of virus movement depends on the level of a single adaptor within the signaling network.