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. To obtain a better quantitative understanding of this signalling network we still need to know the absolute numbers of the key molecules. To achieve this goal, we have now used 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 Grb2 or intersectin results in a 1.3- and 2.5-fold reduction in Nck respectively. 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 egress, A36 levels can vary in the viral membrane and the velocity of virus movement depends on Nck.
Competing Interest Statement
The authors have declared no competing interest.
