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

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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.

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  1. In summary, our quantitative approach in determining precise numbers of molecules at CEV has unexpectedly revealed that A36 levels in the viral membrane are variable. It has also demonstrated that the velocity of virus movement depends on Nck rather than N-WASP which activates Arp2/3 complex dependent actin polymerization.

    Very cool & elegant work!!!

  2. The striking difference between these two mutant viruses is the level of Nck recruited to the CEV, with the numbers being 61% lower for the A36△NPF mutant compared to the wild-type virus. Nck and N-WASP are both essential for Vaccinia induced actin polymerization, but how their relative levels impact on the rate of actin-based motility is not established.

    Is the entire CEV-Actin machinery known? While Nck is altered, there is probably a lot more happening, right?

  3. Loss of Grb2 or intersectin recruitment leads to even lower levels of N-WASP (245±26 and 276±66 respectively).

    Could this be confirmed with a Grb2 or Intersectin mutant? Right now this claim is based on the A36 mutants, correct? But the A36 mutations could have many effects beyond loss of Grb2/Intersectin recruitment, right?

  4. Interestingly, however, in human cells, ~200 copies of the Arp2/3 complex are recruited during clathrin-mediated endocytic events (Akamatsu et al. 2020).

    Are the cell lines in Akamatsu et al 2020 able to be infected by the virus? It'd be neat to one day have this direct molecule count to confirm the 2 NPFs per Arp2/3 complex in vaccinia motility!

  5. verall, there appears to be a 4:2:1 ratio for the A36:Nck:N-WASP signalling network in MEFs.

    Very cool! Do you think there are 2 Nck molecules binding to each A36 protein, or that half of the A36 proteins aren't binding Nck?