EMBO reports - Peer Review Process File - EMBO-2016-43764
Manuscript EMBO-2016-43764
Vesicular Stomatitis Virus N Protein-Specific Single-Domain Antibody Fragments Inhibit Replication Leo Hanke, Florian I. Schmidt, Kevin E. Knockenhauer, Benjamin Morin, Sean P.J. Whelan, Thomas U. Schwartz, and Hidde L. Ploegh Corresponding author: Hidde Ploegh, Boston Children's Hospital
Review timeline:
Submission date: Editorial Decision: Revision received: Editorial Decision: Revision received: Accepted:
06 December 2016 02 January 2017 10 February 2017 26 February 2017 08 March 2017 13 March 2017
Editor: Achim Breiling Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.)
1st Editorial Decision
02 January 2017
Thank you for the submission of your research manuscript to EMBO reports. We have now received reports from the three referees that were asked to evaluate your study, which can be found at the end of this email. As you will see, all referees acknowledge the potential interest of the findings. However, all three referees have raised a number of concerns and suggestions to improve the manuscript, or to strengthen the data and the conclusions drawn, which all need to be addressed during a revision. As the reports are below, I will not detail them here. Moreover, as referee #1 has some doubts regarding the conceptual advance of your study, we wonder if you can add data that would increase the general understanding of VSV transcription/replication mechanisms, e.g. that would shed some light on the role of P during transcription and/or replication. Or, do you have or could get data that would help to explain the antiviral mechanisms of the other two VHHs? Given the constructive referee comments, we would like to invite you to revise your manuscript with the understanding that all referee/editor concerns must be fully addressed in the revised manuscript and in a complete point-by-point response. Acceptance of your manuscript will depend on a positive outcome of a second round of review. It is EMBO reports policy to allow a single round of revision only and acceptance or rejection of the manuscript will therefore depend on the completeness of your responses included in the next, final version of the manuscript. REFEREE REPORTS
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Referee #1: In their manuscript, Hanke et al. have analyzed the inhibition of VSV replication by three specific single-domain antibody fragments which are expressed in the cytosol of the target cells. These antibody fragments were derived from the variable region of the heavy chain of camelid heavy chain-only antibodies (VHHs). These VHHS have been previously identified and described by the same group (Nature Microbiology, 2016). The study presented in this manuscript is therefore a follow-up of the previous work. Here, the authors have determined the crystalline structure of two complexes of a decameric N ring bound to VHHs (VHH 1004 and VHH 1307) and thus accurately defined the epitope which is recognized. For the three VHHs, they also selected virus variants that escape VHH inhibition. As mentioned by the authors, these VHHs might constitute new tools to further investigate VSV replication mechanisms. However, the referee is left with the feeling that this study does not contribute much to our understanding of VSV transcription/replication mechanisms. Clearly, the authors demonstrate that VHH 1307 competes with P binding but this gives no clues about the way P acts during transcription and/or replication. Concerning the two others VHHs, the identification of their binding sites do not even explain their antiviral mechanism. For this reason, it is not completely clear to me that the manuscript provides the expected level of advance for EMBO reports publication. Other concerns: 1) In figure 1A, the MOI used for the infection in absence of doxycycline is not indicated. 2) In the text, line 107-108, the authors wrote "levels for VHH 1004 were barely detectable". I would rather say that VHH 1004 expression is not detectable at all (Figure 1B) which is really problematic in view of the results presented in figure 6A. Is the HA tag really accessible in the HAVHH1004 construction? 3) The resolution of the VSV N:VHH 1004 complex structure is quite low (5.45 ≈). It would be good for the authors to provide some views of the electron density map in the interaction area but also omit maps which will convince the reader that the model is unambiguous. 4) In figure 3B, a quantification of the ratio of N and P detected in the gel after Coomassie staining is required and the experiment has to be done in triplicate. Indeed, there is much less N bound to VHH 1307 than to VHH 1001 which may explain why less P is detected in the gel. Furthermore, in presence of VHH 1004, it seems that more P is associated with N upon N binding to VHH1004 than upon N binding to VHH 1001. This really needs to be quantified. 5) The figure 5 is important but poorly illustrative as such. In 5A and 5B, it would be better to show the full protomer and a close-up view of the interaction area. In 5B and 5C, it is not easy to distinguish the two N protomers. Grey and silver are probably not the optimal colors choice. 6) In the abstract, the authors wrote "The determined structures pave the way for targeted antiviral intervention". This is probably excessive: even knowing the crystal structure of a protein-protein complex, it is very difficult to extrapolate the structure of a small molecule which might mimic the interaction area of one partner. Referee #2: This study is part of an attempt to develop antiviral treatment against negative-sense RNA viruses by targeting components of the replication machinery with nanobodies. In a previous work, four single-domain camel VHHs antibody fragments were identified that protect human cells from infection by vesicular stomatitis virus. These VHHs interacted with the nucleoprotein and blocked viral mRNA transcription. In this new study, the authors aimed to determine the mechanism of action of these different VHHs and for that, they tried to localize their binding sites on the nucleoprotein by X-ray crystallography or by generating escape mutants. The paper present crystal structures for two complexes as well as additional evidences from pulldown experiments and escape mutants that map the binding site on N. For the third one, the binding site was localized by the mapping mutations encountered in escape mutants to the surface of the nucleoprotein. For one VHH that binds to the C-terminal domain of N, the mechanism of action is evident. For the other two VHHs, thinks are less clear.
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Therefore, they found that with VHH1004 and VHH1307 the retrieve of some N mutants is stronger than that of WT, and they found that the rate of dissociation of the complexes with mutated N is faster than with WT N. If it seems plausible, as proposed by the authors, that polymeric N-RNA complexes bind to multiple immobilized VHHs, leading to an "avidity effect", it is not clear why this should be different between WT and mutants. Moreover, intuitively it seems that an increase of the dissociation rate (not entirely compensated by an increase of the association rate) should result in a decrease of the affinity. The paper is clearly written and the results are important for understanding the mechanism of the replication complex of these viruses, in particular for understanding the change of conformation occurring in N upon passage of the polymerase and the dynamical interactions between N and L. However, the authors should improve their explanation (hypothesis) of how a change in the dynamic of the interaction between N and the VHH (by mutating N) could affect the avidity between these two partners. Referee #3: Hanke and colleagues present a manuscript on their recent work with nanobodies targeting the nucleocapsid protein (N) of VSV. This body of work is a further characterization of the nanobodies identified in their previous manuscript (Schmidt et al. Nature Microbiology 2016) and provides detail on location of epitope sites through structural studies of nucleocapsid-like particle-nanobody complexes and isolation/studies of escape variants. With this information, the authors have identified a rational explanation for the inhibitory mechanism of one nanobody (VHH 1307), while others remain unclear. The characterizations are a necessary progression toward the development of VHHs as tools to further investigate this nonsegmented RNA virus at key points along the continuum of viral replication. The interpretation at times seems focused toward one model (physical prevention of an enzymatic reaction) and seems narrow. There is room to interpret the results in other ways (including defects on capsid/progeny template assembly) with careful consideration of previous structural data on VSV N protein complexes. For this reason, the manuscript could be strengthened with consideration of the points below, prior to publication: Page 6, line 131 (See also Page 11, line 261): "Since in vitro replication was not affected, it is likely that VHH 1004 only affects genome replication, but not mRNA transcription. This would substantially reduce the amount of genomes that serve as templates for mRNA transcription in infected cells." Do the authors mean "Since in vitro transcription"? It is true that the VHH 1004 has the potential to occlude L protein association with the template but would this really differentiate transcription and genomic replication? The authors overlook additional steps in viral replication that could be affected by the binding of this nanobody to this site. 1) The associations between N subunits on the viral template are different than those observed in the small rings of nucleocapsid in the study (see Ge et al. Science 2010). Accordingly, the edge of the N binding-site for VHH 1004, mainly residues 111-113(KAL), is in much closer proximity to neighboring subunits in larger nucleocapsid complexes than it is in the decameric rings presented here. If associated with an antibody, N monomers could be precluded from assembling properly (or at all) on the RNA template or potentially forming a proper template for transcription. If assembly is not possible then the little to no transcription levels observed in infected cells could be due to this, ie. no progeny templates no quantifiable transcription. 2) This region overlaps with the site of M protein binding in virus assembly. Thus antibody binding here could prevent viral assembly and mature virion formation. In the end, this could suggest that VHH1004 is responsible for disrupting multiple stages in virus replication: transcription, replication and assembly. This could be the reason for such high potency at low expression levels. The authors should look at this site in the context of the virion structure from Ge et al. Science 2010 and adjust their analysis as warranted. Page12, line 273: From these studies, the mechanism of action of VHH 1001 is unknown. As suggested, if VHH 1001 displaces the N-terminal arm, upon binding to the nucleocapsid-like particles or prevents proper assembly during the infection of VHH 1001 expressing cells, either case could result in a loss or lack of genomic RNA and, thus, loss of transcription activity. The function of the N-terminal arm is less about stabilizing the interactions between protomers, as suggested by the interpretation of bipartite binding site, and is more about formation of the RNA encapsidation cavity. Absence of the N-terminal arm, as with other assembly defective mutants, results in a loss of
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RNA binding (or maintenance). Stabilizing the interaction between the arm and the C-lobe, would reduce accessibility to genomic RNA, as the authors note. Still the method of action is more difficult to interpret, as the escape mutants do not give a direct reference on which residues are critical for the nanobodies mechanism of action. An alternate point could be related to P protein binding and assembly. E243 resides within 3.5 angstrom of the No-binding region of P as shown by the work of Leyrat et al., Plos Pathogens 2011. Antibody binding could potentially block this interaction subsequently preventing nucleocapsid assembly and, in turn, preventing downstream replication as noted above. Secondary to this, VHH 1001 binds to a surface of the nucleocapsid that is nestled between successive turns of the nucleocapsid in the condensed form. Assuming that assembly of nucleocapsids with RNA occurs, VHH 1001 could lead to imperfection in the formation of the condensed nucleocapsid "bullet" and probably lead to defects in the route to build mature virions. In much the same way as VHH 1004, there could be several modes of action for this nanobody. The authors should interpret as realistic with reference to this manuscript. In several cases, the methods are intertwined with the results, and no official method sections are provided for the reader (see escape mutants, immunoprecipitations, etc). This makes it seem like a note rather than article. The lack of information leads to confusion as to which reagents were used for what experiments. The authors should correct this. Minor points: Page 6, Line 123: "The VHH contacts residues that are dispersed over three polypeptide segments of the same N protomer, which suggests that VHH 1004 should also be capable of binding to monomeric N0." The three segments are folded in proximity on the on one surface of the N protein. The authors should be more specific on how the pattern of binding leads to the assumption of unassembled N-binding capability. The authors should provide more detail for the refinement of the crystal structures. Credit should be given to the appropriate software and authors. PDB codes need to be added to Table 2. Figure 5. 1) It would be helpful to readers with less knowledge of the N structure to have perspective on the views (ex. Sup. Fig 1b) in figure 5, especially for 5C. 2) The shades in figure 5 degenerate when rendered with depth due to grey shadowing. This is especially evident in the blown up window to the right in figure 5C and makes it hard to distinguish the individual monomers. Could the authors explore alternate coloring. 3) In 5C, could the model be tilted backward a few degrees to expose the other escape mutant 257. The perspectives in 5C are not identical currently anyway, and this could give better perspective on how the escape mutants cluster. Is the buried surface area of VHH 1307 comparable to that for 1004? It is reported for one but not the other. Add for the other. Page 6, line 121 and 141, could the authors note which atoms are used for RMSD analysis (ie. CA, mainchain, etc.). Page 7, line 160: "In contrast, VHH 1307 only retrieved N from the complexes, indicating that the VHH displaced P from the complex." P protein is evident in the N/P+ lane for VHH 1307. The wording here should be less black and white.
1st Revision - authors' response
10 February 2017
We thank the reviewers for reading the manuscript and for their constructive criticism. We have prepared an updated version of the manuscript based on the reviewers’ comments: The new version includes updated figures, a thorough quantification of the immunoprecipitation experiments, and a more extensive discussion on the inhibitory mechanism of VHH 1001 and 1307. While the overall conclusions remain the same, we think that the revision has improved the manuscript. We thank the reviewers for their input.
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Referee #1: In their manuscript, Hanke et al. have analyzed the inhibition of VSV replication by three specific single-domain antibody fragments which are expressed in the cytosol of the target cells. These antibody fragments were derived from the variable region of the heavy chain of camelid heavy chain-only antibodies (VHHs). These VHHS have been previously identified and described by the same group (Nature Microbiology, 2016). The study presented in this manuscript is therefore a follow-up of the previous work. Here, the authors have determined the crystalline structure of two complexes of a decameric N ring bound to VHHs (VHH 1004 and VHH 1307) and thus accurately defined the epitope which is recognized. For the three VHHs, they also selected virus variants that escape VHH inhibition. As mentioned by the authors, these VHHs might constitute new tools to further investigate VSV replication mechanisms. However, the referee is left with the feeling that this study does not contribute much to our understanding of VSV transcription/replication mechanisms. Clearly, the authors demonstrate that VHH 1307 competes with P binding but this gives no clues about the way P acts during transcription and/or replication. Concerning the two others VHHs, the identification of their binding sites do not even explain their antiviral mechanism. For this reason, it is not completely clear to me that the manuscript provides the expected level of advance for EMBO report publication. Other concerns: 1) In figure 1A, the MOI used for the infection in absence of doxycycline is not indicated. We have indicated the MOI used. 2) In the text, line 107-108, the authors wrote, "levels for VHH 1004 were barely detectable". I would rather say that VHH 1004 expression is not detectable at all (Figure 1B) which is really problematic in view of the results presented in figure 6A. Is the HA tag really accessible in the HAVHH1004 construction? Although the expression levels are so low that they cannot be detected by flow cytometry, the antiviral effect of VHH 1004 is strong, even at high MOIs, but only upon addition of doxycycline. Based on the structure of VHH 1004 and the well-conserved VHH framework regions, we expect the C-terminal HA tag of VHHs to be accessible. We have now included an immunoblot to show that VHH 1004 is expressed in A549 cells after doxycycline addition. 3) The resolution of the VSV N:VHH 1004 complex structure is quite low (5.45 Å). It would be good for the authors to provide some views of the electron density map in the interaction area but also omit maps that will convince the reader that the model is unambiguous. We have now included electron density map and omit maps (see EV1). 4) In figure 3B, a quantification of the ratio of N and P detected in the gel after Coomassie staining is required and the experiment has to be done in triplicate. Indeed, there is much less N bound to VHH 1307 than to VHH 1001 which may explain why less P is detected in the gel. Furthermore, in presence of VHH 1004, it seems that more P is associated with N upon N binding to VHH1004 than upon N binding to VHH 1001. This really needs to be quantified. We thank the reviewer for this suggestion. We have now quantified the band intensities in the Coomassie-stained gels from three independent experiments. As pointed out by the reviewer, the band intensity of P normalized by N is not only reduced when immunoprecipitated with VHH 1307, but also when immunoprecipitated with VHH 1001. This leads to the conclusion that VHH 1001 might overlap with the reported binding site of the P N-terminus (now included in the revised manuscript). The observed phenotypes thus suggest that the second N-P interaction site is also important during polymerase activity and not only as a chaperone for newly synthesized N. This may have further implications for the dynamic interactions of P with N-RNA during processive polymerase activity. 5) Figure 5 is important but poorly illustrative as such. In 5A and 5B, it would be better to show the
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full protomer and a close-up view of the interaction area. In 5B and 5C, it is not easy to distinguish the two N protomers. Grey and silver are probably not the optimal colors choice. We thank the reviewer for this suggestion. We have edited the Figure 5 accordingly. 6) In the abstract, the authors wrote "The determined structures pave the way for targeted antiviral intervention". This is probably excessive: even knowing the crystal structure of a protein-protein complex, it is very difficult to extrapolate the structure of a small molecule that might mimic the interaction area of one partner. We have modified/toned down this statement. Referee #2: This study is part of an attempt to develop antiviral treatment against negative-sense RNA viruses by targeting components of the replication machinery with nanobodies. In a previous work, four single-domain camel VHHs antibody fragments were identified that protect human cells from infection by vesicular stomatitis virus. These VHHs interacted with the nucleoprotein and blocked viral mRNA transcription. In this new study, the authors aimed to determine the mechanism of action of these different VHHs and for that, they tried to localize their binding sites on the nucleoprotein by X-ray crystallography or by generating escape mutants. The paper present crystal structures for two complexes as well as additional evidences from pulldown experiments and escape mutants that map the binding site on N. For the third one, the binding site was localized by the mapping mutations encountered in escape mutants to the surface of the nucleoprotein. For one VHH that binds to the C-terminal domain of N, the mechanism of action is evident. For the other two VHHs, thinks are less clear. Therefore, they found that with VHH1004 and VHH1307 the retrieve of some N mutants is stronger than that of WT, and they found that the rate of dissociation of the complexes with mutated N is faster than with WT N. If it seems plausible, as proposed by the authors, that polymeric N-RNA complexes bind to multiple immobilized VHHs, leading to an "avidity effect", it is not clear why this should be different between WT and mutants. Moreover, intuitively it seems that an increase of the dissociation rate (not entirely compensated by an increase of the association rate) should result in a decrease of the affinity. We thank the reviewer for careful reading of the manuscript. Indeed, the increased dissociation rate for 1307 results in a much-reduced affinity of VHH 1307 to the D374N mutant of N (Table 3). We now emphasize this more clearly in the text. The paper is clearly written and the results are important for understanding the mechanism of the replication complex of these viruses, in particular for understanding the change of conformation occurring in N upon passage of the polymerase and the dynamical interactions between N and L. However, the authors should improve their explanation (hypothesis) of how a change in the dynamic of the interaction between N and the VHH (by mutating N) could affect the avidity between these two partners. The 10-mer N-RNA ring is presumably bound to multiple copies of the immobilized VHHs. Disruption of a single N-RNA-VHH interaction is unlikely to allow dissociation/removal of the whole oligomer. The dissociation rate is thus negligible in this scenario, and the amount of retrieved N is merely a measure of the association rate. This is why we see increased binding for the mutants (high association rate). We now describe this phenomenon more clearly in the text. Referee #3: Hanke and colleagues present a manuscript on their recent work with nanobodies targeting the nucleocapsid protein (N) of VSV. This body of work is a further characterization of the nanobodies identified in their previous manuscript (Schmidt et al. Nature Microbiology 2016) and provides detail on location of epitope sites through structural studies of nucleocapsid-like particle-nanobody complexes and isolation/studies of escape variants. With this information, the authors have
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identified a rational explanation for the inhibitory mechanism of one nanobody (VHH 1307), while others remain unclear. The characterizations are a necessary progression toward the development of VHHs as tools to further investigate this nonsegmented RNA virus at key points along the continuum of viral replication. The interpretation at times seems focused toward one model (physical prevention of an enzymatic reaction) and seems narrow. There is room to interpret the results in other ways (including defects on capsid/progeny template assembly) with careful consideration of previous structural data on VSV N protein complexes. For this reason, the manuscript could be strengthened with consideration of the points below, prior to publication. We thank the reviewer for his/her careful reading and his/her suggestions to improve the manuscript. Page 6, line 131 (See also Page 11, line 261): "Since in vitro replication was not affected, it is likely that VHH 1004 only affects genome replication, but not mRNA transcription. This would substantially reduce the amount of genomes that serve as templates for mRNA transcription in infected cells." Do the authors mean "Since in vitro transcription"? We apologize for the error. We have corrected it. It is true that the VHH 1004 has the potential to occlude L protein association with the template but would this really differentiate transcription and genomic replication? This would be the case if L engages the N-RNA template differently for transcription than for replication, which is one conceivable option. However, we agree with the reviewer that interference with the formation of a usable template is a plausible alternative for how 1004 inhibits the virus, as elaborated below. We have updated the text of the manuscript accordingly. The authors overlook additional steps in viral replication that could be affected by the binding of this nanobody to this site. We thank the reviewer for highlighting additional steps that our VHHs might inhibit. Considering the versatile role of the N protein in the virus life cycle, our VHHs may interfere with virus replication in multiple ways and so we agree: we think that all of the reviewers’ suggestions are plausible. However, the majority of suggested additional means of interference occur at late steps of the virus life cycle. Our manuscript primarily focuses on the VHH binding sites and how the virus is inhibited when it enters a cell that expresses a VHH, that is to say, we have focused on the early steps of the life cycle. We agree with the reviewer’s suggestions: the VHHs we describe in our paper should be excellent tools to analyze later steps of VSV infection. We have not addressed these yet. We now elaborate on possible additional inhibitory effects in the discussion part that could be subject of future experiments, in agreement with the reviewer’s suggestions. Of note, using VHHs we have successfully investigated later stages of influenza replication, by carefully timing their expression [1]. 1) The associations between N subunits on the viral template are different than those observed in the small rings of nucleocapsid in the study (see Ge et al. Science 2010). Accordingly, the edge of the N binding-site for VHH 1004, mainly residues 111-113(KAL), is in much closer proximity to neighboring subunits in larger nucleocapsid complexes than it is in the decameric rings presented here. If associated with an antibody, N monomers could be precluded from assembling properly (or at all) on the RNA template or potentially forming a proper template for transcription. If assembly is not possible then the little to no transcription levels observed in infected cells could be due to this, ie. no progeny templates no quantifiable transcription. We superposed N in complex with VHH 1004 on the nucleocapsid assembly of Ge et al. and included a picture of the superposition for the reviewers reference below. Based on this superposition, VHH 1004 (green) should still be capable of binding to larger nucleocapsid structures (cyan). However, it is still possible that N, when bound by VHH 1004, fails to correctly interact with the RNA and thus hampers the generation of progeny templates. This would also explain why mRNA transcription is not affected by this VHH (no encapsidation necessary). We thank the reviewer for this plausible suggestion that we have now included in the manuscript.
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2) This region overlaps with the site of M protein binding in virus assembly. Thus antibody binding here could prevent viral assembly and mature virion formation. In the end, this could suggest that VHH1004 is responsible for disrupting multiple stages in virus replication: transcription, replication and assembly. This could be the reason for such high potency at low expression levels. The authors should look at this site in the context of the virion structure from Ge et al. Science 2010 and adjust their analysis as warranted. We now describe this possibility in the discussion. However, we do not think that it would explain the high potency of VHH1004 despite low expression levels, because cells are infected for only 4 hours, which largely excludes the possibility of superinfections. Page12, line 273: From these studies, the mechanism of action of VHH 1001 is unknown. As suggested, if VHH 1001 displaces the N-terminal arm, upon binding to the nucleocapsid-like particles or prevents proper assembly during the infection of VHH 1001 expressing cells, either case could result in a loss or lack of genomic RNA and, thus, loss of transcription activity. The function of the N-terminal arm is less about stabilizing the interactions between protomers, as suggested by the interpretation of bipartite binding site, and is more about formation of the RNA encapsidation cavity. Absence of the N-terminal arm, as with other assembly defective mutants, results in a loss of RNA binding (or maintenance). Stabilizing the interaction between the arm and the C-lobe, would reduce accessibility to genomic RNA, as the authors note. Still the method of action is more difficult to interpret, as the escape mutants do not give a direct reference on which residues are critical for the nanobodies mechanism of action. An alternate point could be related to P protein binding and assembly. E243 resides within 3.5 angstrom of the No-binding region of P as shown by the work of Leyrat et al., Plos Pathogens 2011. Antibody binding could potentially block this interaction subsequently preventing nucleocapsid assembly and, in turn, preventing downstream replication as noted above. Secondary to this, VHH 1001 binds to a surface of the nucleocapsid that is nestled between successive turns of the nucleocapsid in the condensed form. Assuming that assembly of nucleocapsids with RNA occurs, VHH 1001 could lead to imperfection in the formation of the condensed nucleocapsid "bullet" and probably lead to defects in the route to build mature virions. In much the same way as VHH 1004, there could be several modes of action for this nanobody. The authors should interpret as realistic with reference to this manuscript. We thank the reviewer for these excellent suggestions. Indeed, the binding region of the P Nterminus on N0 is in proximity of the putative binding site of VHH 1001 and might compete with this part of P for binding. This is in line with the new quantification of the N and P polypeptides in Figure 3. The possible imperfection of the nucleocapsid “bullet” as a consequence of VHH 1001 binding is what we consider antiviral effects of the VHHs in late steps of the infection (these are not the focus of this manuscript; addressing them would be beyond the scope of this paper). Because these VHHs might indeed be good tools to further dissect these processes, we added a paragraph to
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the discussion that highlights possible additional inhibitory effects during the later stages of infection as suggested by the reviewer, and how they could be analyzed. In several cases, the methods are intertwined with the results, and no official method sections are provided for the reader (see escape mutants, immunoprecipitations, etc). This makes it seem like a note rather than article. The lack of information leads to confusion as to which reagents were used for what experiments. The authors should correct this. We have now extended the methods section. Minor points: Page 6, Line 123: "The VHH contacts residues that are dispersed over three polypeptide segments of the same N protomer, which suggests that VHH 1004 should also be capable of binding to monomeric N0." The three segments are folded in proximity on the on one surface of the N protein. The authors should be more specific on how the pattern of binding leads to the assumption of unassembled N-binding capability. In contrast to VHH 1307 and VHH 1001, the binding site of VHH 1004 is composed of a single N protomer. The N0-P complex crystallizes almost identically to the N-RNA ring. With that in mind, we assume that 1004 is able to bind N0. We have now expressed this more clearly in the text. The authors should provide more detail for the refinement of the crystal structures. Credit should be given to the appropriate software and authors. We added additional refinement information and included all used software and authors. PDB codes need to be added to Table 2. PDB IDs are now added to Table 1 Figure 5: 1) It would be helpful to readers with less knowledge of the N structure to have perspective on the views (ex. Sup. Fig 1b) in figure 5, especially for 5C. 2) The shades in figure 5 degenerate when rendered with depth due to grey shadowing. This is especially evident in the blown up window to the right in figure 5C and makes it hard to distinguish the individual monomers. Could the authors explore alternate coloring. 3) In 5C, could the model be tilted backward a few degrees to expose the other escape mutant 257. The perspectives in 5C are not identical currently anyway, and this could give better perspective on how the escape mutants cluster. We thank the reviewer for this suggestion and have edited the Figure 5 accordingly. Is the buried surface area of VHH 1307 comparable to that for 1004? It is reported for one but not the other. Add for the other. We now include the buried surface area also for VHH 1307. Page 6, line 121 and 141: could the authors note which atoms are used for RMSD analysis (ie. CA, mainchain, etc.)? We now specify that Cα atoms were used for RMSD analysis. Page 7, line 160: "In contrast, VHH 1307 only retrieved N from the complexes, indicating that the VHH displaced P from the complex." P protein is evident in the N/P+ lane for VHH 1307. The wording here should be less black and white. We have now adjusted the text accordingly.
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References: 1. Hanke L, Knockenhauer KE, Brewer RC, Diest E Van, Schmidt FI, Schwartz TU, Ploegh HL (2016) The Antiviral Mechanism of an Influenza A Virus Nucleoprotein- Specific Single-Domain Antibody Fragment. MBio 7: 1–11.
2nd Editorial Decision
26 February 2017
Thank you for the submission of your revised manuscript to our editorial offices. We have now received the reports from the referees that were asked to re-evaluate your study (you will find enclosed below). As you will see, all three referees now support the publication of your manuscript in EMBO reports. However, referee #3 has a couple of further suggestions to improve the manuscript, which we ask you to address in a final revised version. Before we can proceed with formal acceptance, I also have a few editorial requests: The abstract is currently too long. Please shorten it to not more than 175 words. Further, please add a running title and up to five key words to the main manuscript text. It seems the structures in Figs. 3 and EV1 are cut. Is there missing something or did you do that on purpose? The schematic summary figure you provided has not the right format and is rather crowded. Most details would not be discernible in the final online version. Could you please provide a more simplified figure (in jpeg or tiff format with the exact width of 550 pixels and a height of about 400 pixels) that can be used as part of a visual synopsis of the paper on our website? I look forward to seeing the final revised version of your manuscript when it is ready. Please let me know if you have questions or comments regarding the revision. -----------------------------REFEREE REPORTS Referee #1: In their revised version of the manuscript, the authors have addressed my previous concerns. Particularly, they have quantified the ratio of N to P in figure 3B and improved figure 5 which is now much more informative. It is still not clear to me whether this study contributes to our understanding of VSV transcription/replication mechanisms. As mentioned by referee 3, there is room to interpret the data in several ways. However, as the two other referees were rather in favor of publication and as the characterized VHHs constitute new tools to further investigate VSV replication mechanisms, my feeling is that the manuscript is now suitable for publication. Referee #2: The authors properly answered to the requests and the paper is now suitable for publication in EMBO reports. Referee #3: Hanke and colleagues present a revised manuscript on their studies with single- domain antibody fragments targeting of the nucleocapsid protein of vesicular stomatitis virus. These studies expand on the groups previous publications to provide the location of epitope sites and inferences on mechanisms of viral inhibition. The revision has addressed many of the points from the previous reviewers. There are a few remaining points that should be corrected before publication. Page 6, Line 123: "The VHH contacts residues that are dispersed over three polypeptide segments of the same N protomer, which suggests that VHH 1004 should also be capable of binding to
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monomeric N0." The three segments are folded in proximity on the on one surface of the N protein. The authors should be more specific on how the pattern of binding leads to the assumption of unassembled N-binding capability. Authors: "In contrast to VHH 1307 and VHH 1001, the binding site of VHH 1004 is composed of a single N protomer. The N0-P complex crystallizes almost identically to the N-RNA ring. With that in mind, we assume that 1004 is able to bind N0. We have now expressed this more clearly in the text." VHH 1004 does not potentially bind to N0 because it achieves binding to three regions of N protein primary sequence. Stating that the No-P complex from previous studies crystallizes in a way to fit the narrative is incorrect, since that assembly among other reasons is made possible by removal of the N-terminal arm in those studies. The argument should be simple and less overreaching to state that VHH 1004 binds to a single N protein in the crystallized assembly and thus could potentially bind to No. Page 11, line 263 Reference needs to be updated. Page 12, line 290 "Based on its binding site, VHH 1307 is likely to prevent the correct assembly of the 'bullet' structure in the virion by disturbing the interaction between the C-lobe of N and the Nlobe of an N protomer of the adjacent turn." This statement is unclear and potentially wrong. VHH 1307 binds to the C-lobe/loop. From the initial spiral of the bullet to the trunk region, there are no stacking associations between C- and N-lobe of N proteins between. There could potentially be steric occlusion of building the bullet of the nanobody but the description of interactions should be re-examined and updated. The resolution of the VSV N:VHH 1004 complex structure is quite low (5.45 ≈). It would be good for the authors to provide some views of the electron density map in the interaction area but also omit maps which will convince the reader that the model is unambiguous. Authors: We have now included electron density map and omit maps (see EV1). Fig. EV1a: What type of omit map is this? What was omitted? What programs were used to generate this and the composite omit map? For EV1a, Assuming this is an Fo-Fc map, the sigma level is too low at 2.2, and probably inflates the volume to an unrealistic level. What does the map look like at more conservative value, 2.7? As long as there is a characteristic volume there, this should be fine. The current image should be adjusted.
2nd Revision - authors' response
08 March 2017
Thank you for the opportunity to submit a revised version of our manuscript EMBOR-201643764V2. We found (most of) the reviewersí comments constructive and they have helped to improve the manuscript. Our overall conclusions remain the same and we have further strengthened the conclusions of our work in the revised manuscript. We hope that you will find the revised version of this manuscript acceptable for publication in EMBO reports. Referee #1: In their revised version of the manuscript, the authors have addressed my previous concerns. Particularly, they have quantified the ratio of N to P in figure 3B and improved figure 5 which is now much more informative. It is still not clear to me whether this study contributes to our understanding of VSV transcription/replication mechanisms. As mentioned by referee 3, there is room to interpret the data in several ways. However, as the two other referees were rather in favor of publication and as the characterized VHHs constitute new tools to further investigate VSV replication mechanisms, my feeling is that the manuscript is now suitable for publication. Referee #2: The authors properly answered to the requests and the paper is now suitable for publication in EMBO reports.
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EMBO reports - Peer Review Process File - EMBO-2016-43764
Referee #3: Hanke and colleagues present a revised manuscript on their studies with single- domain antibody fragments targeting of the nucleocapsid protein of vesicular stomatitis virus. These studies expand on the groups previous publications to provide the location of epitope sites and inferences on mechanisms of viral inhibition. The revision has addressed many of the points from the previous reviewers. There are a few remaining points that should be corrected before publication. Page 6, Line 123: "The VHH contacts residues that are dispersed over three polypeptide segments of the same N protomer, which suggests that VHH 1004 should also be capable of binding to monomeric N0." The three segments are folded in proximity on the on one surface of the N protein. The authors should be more specific on how the pattern of binding leads to the assumption of unassembled N-binding capability. Authors: "In contrast to VHH 1307 and VHH 1001, the binding site of VHH 1004 is composed of a single N protomer. The N0-P complex crystallizes almost identically to the N-RNA ring. With that in mind, we assume that 1004 is able to bind N0. We have now expressed this more clearly in the text." VHH 1004 does not potentially bind to N0 because it achieves binding to three regions of N protein primary sequence. Stating that the No-P complex from previous studies crystallizes in a way to fit the narrative is incorrect, since that assembly among other reasons is made possible by removal of the N-terminal arm in those studies. The argument should be simple and less overreaching to state that VHH 1004 binds to a single N protein in the crystallized assembly and thus could potentially bind to No. We have improved on the precision of our previous phrasing. We now state more clearly that VHH 1004 may bind to N0 because it binds to a single N molecule. Page 11, line 263 Reference needs to be updated. We have updated the reference. Page 12, line 290 "Based on its binding site, VHH 1307 is likely to prevent the correct assembly of the 'bullet' structure in the virion by disturbing the interaction between the C-lobe of N and the Nlobe of an N protomer of the adjacent turn." This statement is unclear and potentially wrong. VHH 1307 binds to the C-lobe/loop. From the initial spiral of the bullet to the trunk region, there are no stacking associations between C- and N-lobe of N proteins between. There could potentially be steric occlusion of building the bullet of the nanobody but the description of interactions should be re-examined and updated. This is correct. We have modified our statement. The resolution of the VSV N:VHH 1004 complex structure is quite low (5.45 ≈). It would be good for the authors to provide some views of the electron density map in the interaction area but also omit maps which will convince the reader that the model is unambiguous. Authors: We have now included electron density map and omit maps (see EV1). Fig. EV1a: What type of omit map is this? What was omitted? What programs were used to generate this and the composite omit map? For EV1a, Assuming this is an Fo-Fc map, the sigma level is too low at 2.2, and probably inflates the volume to an unrealistic level. What does the map look like at more conservative value, 2.7? As long as there is a characteristic volume there, this should be fine. The current image should be adjusted. Both omit map and composite omit map were generated with tools from the Phenix suite. We now specify that we show electron density from a Sigma-A weighted difference (mFo-DFc) omit map for the centered VHH in EV1 A, which is now contoured at 2.7.
3rd Editorial Decision
13 March 2017
I am very pleased to accept your manuscript for publication in the next available issue of EMBO reports. Thank you for your contribution to our journal.
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