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.

2) Wording is a bit tricky when to referring to where the various proteins are localized.Technically, none of these are "on" CEV but lie underneath them.
3) The authors should explain how "inclusion of an actin marker ensures that only CEV are analyzed" (line 117).4) Viruses A36-Y132 and A36∆NPF are not described in the text nor the materials and methods.Exactly how they were created should be described (or cited).5) The sentence on line 228 in incomplete.6) Looking at the diagram in Fig. 1, the assumption (line 246) that the area under the CEV (plasma membrane) is equal to the size of the outer IEV membrane is inaccurate.Once the IEV fuses with the outer membrane, much of the IEV membrane (lipids and proteins) would likely diffuse away and only the part that is directly under the CEV virion would contain proteins interacting with the CEV.This assumption is also made on line 293.
Reviewer #2 (Comments for the Author): This manuscript by Basant and Way continues this group's longstanding dissection of actin-based motility in the Vaccinia virus system.Here, they rigorously quantify the number of A36, Nck, and N-WASP molecules recruited to cell-associated enveloped virus particles (CEV) associated with actin comets in infected cells.They do this both in human HeLa cells and mouse embryonic fibroblasts (MEF), which must be used to quantify Nck and N-WASP levels.Somewhat surprisingly, they find that A36 levels associated with CEV are higher in MEF cells than HeLa, perhaps due to the slower kinetics of infection in murine cells (allowing more time for A36 to accumulate).Quantification in MEF cells demonstrated that A36:Nck:N-WASP ratios were approximately 4:2:1 using wt virus.Mutation of the Grb2 SH2 binding site in A36 modestly decreased Nck binding, while mutation of the intersectin binding site more strongly decreased Nck binding; both mutations strongly decreased N-WASP binding to CEV.Finally, and perhaps most surprisingly, using these mutants the speed of actin motility on CEV was shown to be inversely proportional to the level of Nck binding.
Overall, the experiments are technically sound and carefully interpreted, and thus provide valuable new information for understanding the design principles that underlie localized actin polymerization.Solid data on numbers of molecules will likely be valuable in quantitative modeling studies as well.

Specific points:
1.One potential technical issue with the quantification of Nck and N-WASP is that it is impossible to measure more than one of the three molecules (A36, Nck, N-WASP) in the same cells--not only is only one tag used for quantification, but Nck and N-WASP must be quantified in knockout cells reconstituted with tagged Nck and N-WASP.This to some extent weakens the ability to correlate amounts of the three different molecules and their ratios.In principle, reconstitution with tagged Nck and N-WASP could affect other components associating with CEV.I am not sure this can be experimentally addressed, but the issue should at least be discussed briefly in the text.
2. Similarly, it would be useful to quantify the number of mutant A36 molecules (Y132F and deltaNPY) associated with CEV, to ensure that differential expression of the mutants is not what is driving differences observed in Nck and N-WASP recruitment.
3. A36-Nck-Grb2-NWASP complexes (perhaps also including WIP, intersectin, etc.) involve the type of relatively weak multivalent interactions that have been shown to lead to formation of biomolecular condensates.Indeed, the first in vitro studies of such condensates used constructs that mimic multivalent Nck/N-WASP interactions.It might be interesting to discuss potential effects of condensate formation on dwell-time of components, and how this might affect the extent of actin polymerization and its speed.I know this group has previously looked at apparent off-rates for various components in the Vaccinia system, which may be relevant.
4. The authors are careful to provide multiple validations for extrapolating outside the existing standard curve to quantify A36 levels (Fig. 2).However, the quantification of B5 and F13 are much further outside the standard curve.The authors should at minimum comment on the reasons they feel this extrapolation is justified.5. P. 7, line 199: The statement of changes in ratios of A36 to Nck to N-WASP should perhaps be phrased differently; currently it says differences in NPF mutants are "significantly reduced" while differences in Y132F mutants are "reduced."If the intent is to discuss statistical significance, my guess is only Nck binding to the NPF mutant would be significant.5. Discussion, p. 9: The authors speculate that for the deltaNPF mutant, the level of Nck binding is in a "sweet spot" to maximize activation of Arp2/3 by N-WASP.This argument would be strengthened by data from an A36 mutant that binds even less Nck, which might be expected to be less efficient/induce slower comet tails.Of course lack of Nck binding gives no comet tails, but another data point would be helpful here.
6. Do the authors know why the stoichiometry of Nck binding to A36 in CEV is roughly 0.5?Some potential explanations include substoichiometric A36 phosphorylation, or the local concentration of Nck being near the Kd for binding to A36.

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Response to reviewer comments:
Please note that the line numbers men oned here correspond to the final text (without track changes marked-up).
Reviewer #1 (Comments for the Author): Orthopoxviruses such as vaccinia virus u lize ac n polymerisa on for the forma on of ac n tails to propel newly released virions away from infected cells.Ac n tail forma on has served as a useful paradigm for ac n polymeriza on.In the manuscript by Basant and Way, they u lize this system to make careful calcula ons of the stoichiometry of the various proteins involved in ac n tail forma on.The manuscript is well-wri en and the conclusions jus fied.I feel that there are only a few small issues that need to be addressed.
1) It should be noted in the text that the addi on of TagGFP2 to A36, B5 and F13 significantly reduces that amount of these proteins that are expressed (Suppl1).
The following has been added in line 138 of the revised text "The overall expression levels of the tagged proteins in infected cells are reduced when compared to untagged controls (Fig S1 )." 2) Wording is a bit tricky when to referring to where the various proteins are localized.Technically, none of these are "on" CEV but lie underneath them.
We agree that it is challenging to appropriately describe the localisa on of protein with respect to virus par cles in each case.We have rephrased sentences where possible for more clarity, for eg. in line 158 of the revised text "Nck and N-WASP are essen al for this process, but how many molecules of each are recruited beneath CEV on the plasma membrane?"and line 224 of the revised text "To test whether these differences are caused by varying levels of A36 associated with CEV, we generated recombinants expressing A36(132F)-TagGFP2 and A36△NPF-TagGFP2 at the endogenous locus." 3) The authors should explain how "inclusion of an ac n marker ensures that only CEV are analyzed" (line 117).
We have now clarified this point in line 118 of the revised text which reads as follows "Colocalisa on of A36-TagGFP2 and RFP-A3 signals occurs on IEV and CEV, but the inclusion of an ac n marker ensures that only CEV are analysed, as ac n tails are not generated by IEV in the cytoplasm of infected cells (Reitdorf et al., 2001& Hollinshead et al., 2001)."4) Viruses A36-Y132 and A36∆NPF are not described in the text nor the materials and methods.Exactly how they were created should be described (or cited).
We apologise for this omission in the original submission.These viruses were previously generated in the Way lab.The relevant papers are now cited in line 526 of the revised text "Recombinant viruses expressing RFP-A3 with untagged variants A36(132F) and A36△NPF were generated previously in the Way lab (Weisswange et al., 2009& Snetkov et al., 2016)." 5) The sentence on line 228 in incomplete.
We apologise for this omission in the original submission.This has now been corrected to read "Previous studies have taken advantage of the known structure of viruses to generate fluorescence calibra on standards by tagging defined numbers of viral capsid components with fluorescent proteins" (line 246 in the revised text).
6) Looking at the diagram in Fig. 1, the assump on (line 246) that the area under the CEV (plasma membrane) is equal to the size of the outer IEV membrane is inaccurate.Once the IEV fuses with the outer membrane, much of the IEV membrane (lipids and proteins) would likely diffuse away and only the part that is directly under the CEV virion would contain proteins interac ng with the CEV.This assump on is also made on line 293.
We think that there is insufficient data available to ascertain the extent to which lipids and proteins diffuse away when IEV fuse with the plasma membrane.In line 308 of the revised text, we make clear the assump ons being made "Assuming that no membrane is lost, and proteins do not diffuse significantly when IEVs fuse with the plasma membrane, our data indicate that A36 is present at a density of 0.2-0.5 molecules per 100 nm 2 based on the outer surface of 405,037 nm 2 for IEV." Reviewer #2 (Comments for the Author): This manuscript by Basant and Way con nues this group's longstanding dissec on of ac n-based mo lity in the Vaccinia virus system.Here, they rigorously quan fy the number of A36, Nck, and N-WASP molecules recruited to cell-associated enveloped virus par cles (CEV) associated with ac n comets in infected cells.They do this both in human HeLa cells and mouse embryonic fibroblasts (MEF), which must be used to quan fy Nck and N-WASP levels.Somewhat surprisingly, they find that A36 levels associated with CEV are higher in MEF cells than HeLa, perhaps due to the slower kine cs of infec on in murine cells (allowing more me for A36 to accumulate).Quan fica on in MEF cells demonstrated that A36:Nck:N-WASP ra os were approximately 4:2:1 using wt virus.Muta on of the Grb2 SH2 binding site in A36 modestly decreased Nck binding, while muta on of the intersec n binding site more strongly decreased Nck binding; both muta ons strongly decreased N-WASP binding to CEV.Finally, and perhaps most surprisingly, using these mutants the speed of ac n mo lity on CEV was shown to be inversely propor onal to the level of Nck binding.
Overall, the experiments are technically sound and carefully interpreted, and thus provide valuable new informa on for understanding the design principles that underlie localized ac n polymeriza on.Solid data on numbers of molecules will likely be valuable in quan ta ve modeling studies as well.
Specific points: 1.One poten al technical issue with the quan fica on of Nck and N-WASP is that it is impossible to measure more than one of the three molecules (A36, Nck, N-WASP) in the same cells--not only is only one tag used for quan fica on, but Nck and N-WASP must be quan fied in knockout cells recons tuted with tagged Nck and N-WASP.This to some extent weakens the ability to correlate amounts of the three different molecules and their ra os.In principle, recons tu on with tagged Nck and N-WASP could affect other components associa ng with CEV.I am not sure this can be experimentally addressed, but the issue should at least be discussed briefly in the text.
This is now discussed in line 198 of the revised text "This ra o is our best es mate, as unfortunately due to the nature of the fluorescent tag and lack of double Nck/N-WASP null cells, we cannot simultaneously measure the numbers of any pair of the three molecules in a single cell." 2. Similarly, it would be useful to quan fy the number of mutant A36 molecules (Y132F and deltaNPF) associated with CEV, to ensure that differen al expression of the mutants is not what is driving differences observed in Nck and N-WASP recruitment.
To address this issue, we generated two new recombinant viruses where the A36 Y132F and A36△NPF mutants were tagged with TagGFP2.Data shown in Fig 6C and line 226 of the revised manuscript demonstrate that the number of A36 molecules present at CEV inducing ac n tails does not vary significantly across mutants.
3. A36-Nck-Grb2-NWASP complexes (perhaps also including WIP, intersec n, etc.) involve the type of rela vely weak mul valent interac ons that have been shown to lead to forma on of biomolecular condensates.Indeed, the first in vitro studies of such condensates used constructs that mimic mul valent Nck/N-WASP interac ons.It might be interes ng to discuss poten al effects of condensate forma on on dwell-me of components, and how this might affect the extent of ac n polymeriza on and its speed.I know this group has previously looked at apparent off-rates for various components in the Vaccinia system, which may be relevant.
Our recent eLife paper (Basant & Way, 2022) suggests that spa al constraints and network organiza on are important parameters in determining signalling output.The role of condensates in Vaccinia ac n tail forma on remains to be inves gated.Moreover, previous work on Nck/N-WASP interac ons in condensate forma on have used in vitro systems or situa ons that lack spa al constraints, which are clearly important for Vaccinia.Given this, we felt it was not appropriate to discuss the impact of condensates in the context of our results especially in the absence of any analysis of protein dynamics.We have, however, added "It will be interes ng to inves gate how these parameters influence the turnover of Nck and N-WASP beneath CEV (Weisswange et al., 2009)" (line 282) in the discussion of the revised text to bring up this issue to the reader.
4. The authors are careful to provide mul ple valida ons for extrapola ng outside the exis ng standard curve to quan fy A36 levels (Fig. 2).However, the quan fica on of B5 and F13 are much further outside the standard curve.The authors should at minimum comment on the reasons they feel this extrapola on is jus fied.
As the images for B5 and F13 intensi es were obtained in the linear response range of the camera i.e. without any satura ng pixels, we think our es mates are reliable.A comment on this has been added in line 147 of the revised text. 5. P. 7, line 199: The statement of changes in ra os of A36 to Nck to N-WASP should perhaps be phrased differently; currently it says differences in NPF mutants are "significantly reduced" while differences in Y132F mutants are "reduced."If the intent is to discuss sta s cal significance, my guess is only Nck binding to the NPF mutant would be significant.
To address this, lines 209-211 of the revised text now read as follows "Our measurements show that muta on of Tyr132 to phenylalanine (loss of Grb2) reduced the level of Nck and N-WASP by ~24% and 44% respec vely N-WASP (Fig 6A , B).However, in the absence of the A36 NPF mo fs, Nck numbers were ~61% lower while N-WASP was reduced by ~36% (Fig 6A , B)." 5. Discussion, p. 9: The authors speculate that for the deltaNPF mutant, the level of Nck binding is in a "sweet spot" to maximize ac va on of Arp2/3 by N-WASP.This argument would be strengthened by data from an A36 mutant that binds even less Nck, which might be expected to be less efficient/induce slower comet tails.Of course lack of Nck binding gives no comet tails, but another data point would be helpful here.
We agree that a fourth datapoint would indeed strengthen our argument.We tested other A36 mutants as candidates, but we were unable to find a condi on where Nck levels at CEV par cles were lower than those observed in the A36△NPF virus.
6. Do the authors know why the stoichiometry of Nck binding to A36 in CEV is roughly 0.5?Some poten al explana ons include substoichiometric A36 phosphoryla on, or the local concentra on of Nck being near the Kd for binding to A36.
We currently do not have evidence on the stoichiometry of A36 phosphoryla on, or the local concentra on of Nck at virus par cles (Kd for Nck1 binding to the A36 phosphopep de in vitro has been calculated to be ~50uM (Frese at al., JBC 2006) Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.You will be notified when your proofs are ready to be viewed.
One reviewer suggested that it is more accurate to state that " the amount of Nck correlates with velocity."so please modify accordingly.
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Wen Chang Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred ). 1st Revision -Editorial Decision