Correlating the ability of VP 24 protein from Ebola and Marburg viruses to bind human karyopherin to their immune suppression mechanism and pathogenicity using computational methods

Immune response suppression is crucial for viral invasion. The protein VP24 is pivotal in achieving this in Ebola, although interestingly the mechanism of immune suppression is different in the closely related Marburg virus. Here, we illustrate that a possible molecular basis for this difference emanates from two alpha helical structures ( 5 and 6) in VP24 involved in binding human α α karyopherin (KPNA) (PDBid:4U2X), wherein the Ebola and Marburg viruses have distinctly different charged properties in 5. 6 is absent in Marburg, and α α has a different hydrophobic moment in the Reston Ebola (REBOV) species, which is surprisingly non-pathogenic in humans. Based on the hypothesis that REBOV is not immunosuppressive, which is in turn is due to its inability to bind KPNA, we show by docking KPNA to the REBOV VP24 that the single amino acid substitution R140S is responsible for this difference between REBOV and Zaire Ebola strains. Such a scenario of getting a virulent REBOV through a single mutation is particularly worrisome, since the REBOV, once found only in monkeys, has been recently detected in pigs. We also reiterate the potential of using these helices as potential epitopes for generating protective antibodies against Ebola. This article is included in the Disease Outbreaks


Introduction
Viruses from the family Filoviridae are negative-stranded RNA viruses having a filamentous shape 1 . The first member of this family (Marburg) was discovered in 1967 2 , while the Ebola virus was first discovered in 1976 3 . Public attention has been drawn to this rare, but deadly disease 4 ever since the current outbreak in West African countries threatened to rapidly deteriorate into a full-blown epidemic 5,6 . Both viruses cause haemorrhagic fever by quickly suppressing innate antiviral immune responses 7 . However, quite surprisingly, the Reston Ebola (REBOV) strain, first identified in monkeys that were imported into Reston in the United States from the Philippines 8 , is non-pathogenic in humans 9,10 .
Previously, we have characterized α-helical (AH) structures in Ebola proteins using PAGAL 11 , and demonstrated that the AHs with characteristically unique feature values are involved in critical interactions with host proteins 12 . We showed that the AH from Ebola virus membrane fusion subunit GP2 13 , which is disrupted by a neutralizing antibody derived from a human survivor of the 1995 Kikwit outbreak 14 , has a very large hydrophobic moment compared to other AHs in Ebola proteins 12 . Similarly, another AH with the highest proportion of negatively charged residues is the binding site of the human karyopherin (KPNA) to the Zaire Ebola (ZEBOV) virus VP24 (ezVP24) protein 15 .
In spite of sharing a common ancestry 16 , Marburg and Ebola have different antigenicity of the virion glycoprotein 17 . Furthermore, the mechanism of immunosuppression is different in these viruses 18 . These differences are probably the reason for the reduced mortality observed in Marburg outbreaks. In Ebola, the crucial role of host immune system evasion is accomplished by two proteins: VP35 and VP24 19 . Ebola VP24 inhibits interferon (IFN) signaling by hindering the nuclear accumulation of tyrosine-phosphorylated STAT1 by binding KPNA 20,21 . In contrast, the Marburg virus abrogates the host immune response by inhibiting IFN-induced tyrosine phosphorylation of STAT1 and STAT2 18 via a moonlighting function matrix protein, VP40 22 . Specifically, ezVP24 binds KPNA via two AHs (α5 and α6) 15 . In Marburg VP24 (mVP24), α5 has distinctively different properties (not easily identified by a sequence or structural alignment), while α6 is just a small turn 23 . This explains why mVP24 is not immunosuppressive.
We investigated these AHs in VP24 from the REBOV strain (erVP24). While α5 in erVP24 was similar to that in ezVP24, α6 in erVP24 had different properties caused by the presence of a serine in the place of arginine (S140R). We modeled the apo erVP24 (PDBid:4D9OA) using the ezVP24 in complex with KPNA as a template (PDBid:4U2X) by SWISS-MODEL 24 , and then docked KPNA to this structure using DOCLASP 25 . The docked structure helped visualize the ability of Arg140 in ezVP24 to make the correct electrostatic interaction with two glutamic acids, one residing on α5 in VP24, and the other in KPNA. The effect of single mutations in modulating virulence has been well established 26-28 . However, our methodology provides a more rational way of finding such critical residues. The possibility of a REBOV mutant gaining immunosuppressive capabilities is particularly disconcerting since the isolation of the REBOV strains from pigs [29][30][31] . We also highlight the possibility of using α5 and α6 from VP24 as epitopes for generating antibodies 32 or designing compounds and peptides to inhibit protein-protein interaction 33 .

Materials and methods
AHs in proteins were identified using DSSP 34 . These AHs were then analyzed using PAGAL 11 . Briefly, the Edmundson wheel is computed by considering a wheel with centre (0,0), radius 5, first residue coordinate (0,5) and advancing each subsequent residue by 100 degrees on the circle, as 3.6 turns of the AH makes one full circle. We compute the hydrophobic moment by connecting the center to the coordinate of the residue and giving it a magnitude obtained from the hydrophobic scale obtained from 35 . These vectors were then added to calculate the final hydrophobic moment. The color coding for the Edmundson wheel was as follows: all hydrophobic residues were colored red, while hydrophilic residues were colored in blue: dark blue for positively charged residues, medium blue for negatively charged residues and light blue for amides.
The protein structures used in the current work were all identified using the PDBid, and are available at www.rcsb.org. We used the SWISS-MODEL program to model the erVP24 (PDBid:4D9OA) structure using the ezVP24 (PDBid:4U2XA) in complex with KPNA as template. See 4D9OA4U2XA.pdb in Dataset 1 Note the residue numbering is not conserved by SWISS-MODEL. For example, Glu113 in PDBid:4D9OA corresponds to Glu97 in PDBid:4D9OA4U2XA. We used DOCLASP 25 to dock KPNA to the modelled structure of erVP24 (See Pymol script 'docking-KPNAtoRestonVP24.p1m' for human KPNA and 'RESTONVP-24mouse.p1m' for mouse KNPA in Dataset 1). '4U2XA.4U2XD. maxdist.out.sort' in Dataset 1 lists the closest atoms of the residues of VP24 (PDBid:4U2XA) that make contact with human karyopherin (PDBid:4U2XD), sorted based on distances.
All protein structures were rendered by PyMol (http://www.pymol. org/). The sequence alignment was done using ClustalW 36 . The alignment images were generated using SeaView 37 . Protein structures were superimposed using MUSTANG 38 .

Amendments from Version 1
In this version, based on the suggestion of a reviewer (Dr McIntosh), we have docked the modelled structure of VP24 from Reston Ebola (erVP24) virus to the structure of mouse KPNA (PDBid:1Y2AC) (there are no known models of KPNA from other non-human primates that are susceptible to Reston Ebola virus). However, inspite of sequence difference (50.3\% identity and 77.8\% similar) between human and mouse KPNA, the residues that interact with erVP24 is the same. However, Reston Ebola is known to be non-virulent in mice, although viral replication does occur (de Wit E, et al.). Virulence in mice is caused only by a STAT1 knockout, corroborating the inability of erVP24 to suppress immune response, as hypothesized by us as a major cause for its non-pathogenicity.
The title of the article has been changed, Dataset 1 has been updated and a new Figure (Figure 5) has been added. Differences in α5 in Ebola and Marburg viruses: explaining why Marburg VP24 is not immunosuppressive ezVP24 has a 39.6% identity (73.8% similar) with mVP24 (Figure 1a), and there is significant structural homology among VP24 proteins from different strains of Ebola and Marburg ( Figure 1b). Yet, the mechanism of immune response suppression is different in these viruses from the Filoviridae family 18 . 'Reasons why Marburg virus VP24 is not immunosuppressive remain elusive' 23 . Therefore, we sought to investigate the differences in residues involved in binding KPNA in the ezVP24 and mVP24. ezVP24 binds KPNA via two AHs (α5 and α6), residues on loops and a Lys on a β-sheet (Table 1). In mVP24, α5 has different properties ( Figure 2a,b and Table 2), while α6 is just a small turn (Figure 1c). These differences in the properties of AHs involved in binding      KPNA in eVP24 to those in mVP24 strongly indicates that mVP24 is not immunosuppressive, as is widely accepted 18 (or at least it does not use the same mechanism).

S140R substitution in α6 may explain why Ebola Reston strain is non-pathogenic in humans
The REBOV strain 'does not represent an immediate public health menace on the scale of the African Ebola virus' 9 , possibly due to the generation of antibodies against this strain 39 . Also, gene expression study of infected cells showed that the ZEBOV and Marburg viruses has fewer activated IFN-inducible genes relative to REBOV 40 . Thus, most likely, the REBOV strain does not have the same immunosuppressive capabilities as the ZEBOV or Sudan strain. While α5 of erVP24 has properties similar to ezVP24 (Figure 2c), α6 in REBOV VP24 (erVP24) is clearly different in hydrophobic moment and residue composition ( Figure 3). For example, Arg140 in ezVP24 is replaced with Ser140 in erVP24.
To better visualize and quantify this difference, we docked KPNA to erVP24. First, we modelled the apo erVP24 (PDBid:4D9OA) using the ezVP24 complexed with KPNA (PDBid:4U2X) using SWISS-MODEL 24 . Subsequently, KPNA was docked to this protein using DOCLASP 25 . Figure 4 shows ezVP24 and erVP24 docked to KPNA. In ezVP2, KPNA binding is primarily facilitated by electrostatic attraction between the negatively charged Asp124 in α5 and Lys481 in KPNA (at 3.9 Å) 12 , and a hydrogen bond between Arg140 (α6) and Glu475 of KPNA (among other hydrogen bonds, Table 3). Also, the ezVP24 itself is stabilized by an electrostatic bond between the negatively charged Glu113/OE1 (α5) and the positively charged Arg140/NH1 (α6) at 3.4 Å. Note, that this pair is at distance of 12.8 Å in the apo ezVP24 (PDBid:4M0QA). This 8 Å conformational change in these AHs emphasizes the role of plasiticity in binding KPNA. In contrast, in the erVP24, the distance between Glu113/OE1 and Ser140/OG changes from 14 Å in the apo enzyme to 6.2 Å in the docked model. Also, the Ser140/OG atom is not positively charged unlike Arg140/NH1. Further, the possibility of Ser140/OG making a hydrogen bond with Glu475 of KPNA is remote, since they are 6.7 Å apart. Thus, we conclude that the mutation R140S is likely to be one of the critical factors for the non-pathogenic nature of REBOV, since this mutation renders erVP24 incapable of binding KPNA. Other factors might include the different susceptibilities of the glycoproteins of ZEBOV and REBOV for furin cleavage 41 .

Role of intrinsically disordered stretches in VP24
It is interesting to note that the apo α5 (PDBid:4M0QA) is extended by two residues towards the N-terminal (Figure 2c, Glu113 and Pro114) in the ezVP24 complex with KPNA (PDBid:4U2XA). Notably, Pro and Glu are the two most disorder-promoting residues 45 . The peptide stretch preceding Glu113 in the Sudan Ebola VP24 (PDBid:3VNEA) is also disordered, and residues in that stretch are  unassigned in the crystal structure ( Figure 1a). Quite interestingly, the α6 (Figure 3a) is also extended by two residues (towards the C-terminal) in the ezVP24 complex ( Figure 3d). As mentioned earlier, this stretch is not a helix in mVP24. In the apo Sudan Ebola VP24, α6 (Figure 3c) is similar to the ezVP24 complex (Figure 3b), and is already extended. This is probably due to the fact that Glu is replaced by Asp, which is not disordered-generating. Also, the hydrophobic moment of all three AHs have (almost) the same direction and magnitude (Figure 3a-c). These observations emphasizes the role of intrinsically disordered regions in viral functionality 46,47 .

Conclusions
The ability of a single mutation to significantly alter the immunosuppressive properties of the Ebola proteins is well established 26,27,48 .  Sequence-based methods (whole genome profiling) are typically used to identify these critical mutations 26 . Structural studies provide an alternative, and possibly more rational, method to identify such mutations. For example, while double (and not single) mutations are required in VP35 to inhibit protein kinase R activation, it is difficult to rationalize this based on sequence data only 28 . In the current work, we build on previous work that characterized AH structures in Ebola proteins to rationalize the lack of immunosuppressive properties in the mVP24. ezVP24 binds to KPNA via two AHs (α5 and α5), loops and a residue on a β-sheet. We attribute the lack of immunosuppressive properties of mVP24 to its inability to bind KPNA, which emanates from different characteristics of mVP24 α5 compared to ezVP24 α5. Subsequently, we demonstrate that a single mutation in α6 in the erVP24 might endow it with immunosuppressive properties. We corroborate this conclusion by modelling the apo structure of the erVP24 based on the structure of ezVP24 in complex with KPNA using SWISS-MODEL 24 , and by docking KPNA to the modelled structure using DOCLASP 25 . The REBOV strain, first identified in monkeys and imported into the United States from the Philippines 8 , has never caused disease in humans 9,10 . However, the isolation of the REBOV strains from pigs in the Philippines 29,30 , and recently in China 31 , highlights the significance of finding preventive therapies in the probable scenario that a mutant REBOV for VP24 with immunosuppressive capabilities gets transferred to human handlers. Such a difference does not exist in the VP35 protein, where REBOV VP35 has been used as a model to show how they could silence and sequester double-stranded RNA, which is a key event in immunosuppression 49 . We also reiterate the potential of using these AHs from VP24 as epitopes 50,51 for generating antibodies 32,52,53 , or innovating drugs to inhibit proteinprotein interaction 33,54-58 . The presence of two intrinsically disordered residues proximal to these AHs in the apo structure that gain a AH structure upon binding should encourage antibody search to use both apo and complexed AHs. It is certainly worth investigating whether supplementing ZMapp, a cocktail of three antibodies that has shown reversion of advanced Ebola symptoms in non-human primates 59 , with more antibodies would prove more effective. Author contributions SC wrote the computer programs. All authors analyzed the data, and contributed equally to the writing and subsequent refinement of the manuscript.

Competing interests
No competing interests were disclosed.

Qiaoying Zeng
College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, China Zoonotic transmission of Ebola virus (EBOV) to humans causes a severe haemorrhagic fever in affected humans. Neither vaccines nor therapeutics are available at present. To devise antiviral strategies, it is important to understand the pathogenicity and molecular basis of EBOV infection. Among all the 7 proteins including NP, VP35, VP40, GP, VP30, VP24 and L of EBOV, structural proteins VP24 and VP35 have already been found playing a key role in interference with proper functioning of host interferon system. Present computational analysis offered insights into potentially underlying mechanisms of VP24.

Suggestions for revision:
The title seems too long. Single-point mutation in VP24 ---one of the key molecular mechanisms underlying the pathogenicity of filovirus.
The writing needs to be substantially improved. There are grammar errors, illogical expression, inaccurate, undefined and misleading descriptions, and some biased or questionable conclusions. Just take the abstract as an example where questionable words by the authors were marked in and my opinion labeled in . bold italics "Immune response suppression is crucial for viral invasion.

in the Reston Ebola (REBOV) species, something else?] has a different hydrophobic moment which is surprisingly [what makes "non-pathogenic in humans" so surprising? The authors know why, and I know why, because we know background information related to Ebola, but the point is, "you know and I know" doesn't necessarily mean all the readers know why. The background information should be clearly presented with the least words. In all the 5 Ebola species, outbreaks of ZEBOV, SEBOV, CIEBOV and BEBOV have been recorded. However, REBOV has just been detected in swine] non-pathogenic in humans [the only one non-pathogenic in humans out of 5 Ebola species including ZEBOV, SEBOV, CIEBOV and BEBOV. This information actually should
. Based on the hypothesis that REBOV is not be given in the beginning of this abstract] immunosuppressive, which due to its is in turn is [ . Such a scenario of getting a virulent REBOV through a single mutation is particularly STAT1] worrisome, since the REBOV, once found only in monkeys, has been recently detected in pigs. We also reiterate the potential of using these helices as potential epitopes for generating protective antibodies against Ebola." The abstract should be logically organized, starting from background information to methods, direct analyzed results, conclusion and finally the significance of present research.
In the introduction, some key information is missing which is indispensable for clear, accurate and logical understanding of the following analysis, related discussion and correlation between analysis and observed facts. For example: There are Five EBOV species that have been defined, Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Coˆte d'Ivoire ebolavirus (CIEBOV), Bundibugyo ebolavirus (BEBOV) and Reston ebolavirus (REBOV). They have shown different pathogenicity up to date. Outbreaks of ZEBOV, SEBOV, CIEBOV and BEBOV have been recorded. However, REBOV has just been detected in swine.
"In Ebola, the crucial role of host immune system evasion is accomplished by two proteins: VP35 and VP24." ---What about Marburg? It's also dependent on VP35 and VP24 or just on VP24? because we are going to compare between Ebola and Marburg.
The main part of the article -computer modeling and analysis of VP24 and its interactions to other molecules -is reliable and sufficient. However, what makes the present analysis valuable is whether these analysis explain observed facts including pathogenicity between Marburg and Ebola virus, and among different Ebola species, and what about experimental findings by others? In other words, are there any experimental observations supporting present analysis?
In the Conclusion part of this article, the authors did not actually conclude their main analyzed results and corresponding significance. This "Conclusion" is actually a discussion.
About the discussion:

About the discussion:
As both VP35 and VP24 contribute to "immune evasion" as described in "Introduction", how could you get an accurate and reliable conclusion just based on the analysis of VP24? Change your angle of view.
All previous experimental observations and conclusions by other scientists about VP24 should be included in discussion, giving a comprehensive and impartial comparative analysis. However, some key studies are obviously missing in this part. For example: The IFN system can protect immune-competent mice from lethal EBOV infection. Adaptation of ZEBOV to lethal infection of mice was associated with mutations in VP24 and NP ( However, the different, even These findings are opposites of the present statement. opposite opinions on the same topic by different scientists are normal phenomenon in scientific community. The most important thing is how to analyze, to explain these differences and finally get a scientific conclusion and evaluation of you own work, without ignoring those opposite findings or opinions.
No competing interests were disclosed. 1.

2.
That all EBOV species employ VP24 to subvert the host innate immune response by binding KPNA.
That a single point mutation R140S can explain a lack of pathogenesis by REBOV in humans through a lost capacity to bind KPNA. Neither of these assumptions have been experimentally verified by the authors or elsewhere. At a minimum, it seems these assumptions would need to be addressed in silico through analysis of the KPNA for a susceptible host to REBOV.

Minor concerns:
Abstract "which is surprisingly non-pathogenic" ...to .."which is notably non-pathogenic in humans." "which is in turn is due to its inability to bind" ..to.."which is in turn due to its inability to bind ." Results and Discussion: Dataset 1 title and legend appear to be mislabeled as "search methodology to identify plant alpha helica-antimicrobial peptides in the PDB dataset"...should this not be labeled as."to identify filovirus VP24 alpha helices"? Major Concerns: Regarding Figure 1 and The added experiment of docking mouse KPNA to erVP24 is appreciated but does not address the important question of whether or not non-human primate KPNA has compensatory substitutions to restore the potential for binding Reston VP24. Following this line of thought, such compensatory substitutions would conversely not be expected to reduce binding with VP24 from other African species of EBOV. While the ability of single point mutations to abrogate protein-protein interactions is indeed well established, the ability of compensatory substitutions to restore intermolecular interactions is also well established. Would it not be more prudent to sequence KPNA from a non-human primate host susceptible to hemorrhagic disease caused by REBOV and test the hypothesis in silico? Understandably, access to non-human primate sequence is limiting making it difficult to address this concern. In light of the inability to validate these findings either experimentally or in silico with a susceptible host species for fatal disease with REBOV, I suggest that the observation of the R140S subsititution in REBOV and its forecast impact on pathogenicity, while intriguing, remains highly speculative.
No competing interests were disclosed.

Competing Interests:
I confirm that I have read this submission and believe that I have an appropriate level of I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. In this article, in addition to gross charge and structural differences in two alpha helices (a5 and a6) of VP24 between EBOV and Marburg viruses, possibly explaining the different mechanisms of INF response suppression, the authors hypothesis that a single substitution R140S in VP24 between the pathogenic Zaire ebolavirus (ZEBOV) and non-pathogenic Reston ebolavirus (REBOV) alters charged properties of the a5 alpha helix leading to a lack of binding to human KPNA by REBOV VP24. This substitution in REBOV VP24 is hypothesized to be responsible for the lack of REBOV pathogenesis in humans. The authors further express concern regarding the potential for a single amino acid substitution in REBOV, previously observed in domestic swine, to perhaps lead to a more pathogenic virus in the future.

Article Content:
The study employs computational modeling of the primary VP24 amino acid sequences of different EBOV species and Marburg virus onto the previously resolved crystal structure of ZEBOV VP24 bound to KPNA5 (Xu , 2014). The direct comparisons between potential binding sites of KPNA and VP24 et al. from different species of EBOV are intriguing but the study unfortunately lacks experimental verification either through binding or functional studies In addition there are concerns regarding the accuracy in vitro of theoretical modeling of primary VP24 sequences from various EBOV species to the known crystal structure of ZEBOV VP24 and KPNA5 peptides. Without experimental verification it is not possible to draw the conclusion that the R140S substitution present in REBOV affects binding to KPNA or that it is 1.

2.
structure of ZEBOV VP24 and KPNA5 peptides. Without experimental verification it is not possible to draw the conclusion that the R140S substitution present in REBOV affects binding to KPNA or that it is responsible for the absence of pathogenicity in humans. One approach not tried is modeling of a KPNA5 homolog from non-human primates as REBOV is known to still be pathogenic in non-human primates. In concept, it seems unlikely that a single mutation could be wholly responsible for the observed differences in pathogenicity between REBOV and other EBOV species. Various mechanisms not involving VP24 including EBOV glycoprotein and VP35-mediated mechanisms of immune suppression as well as a potential host genetic differences are likely to have critical influences on EBOV pathogenesis beyond the specific mechanism of VP24-mediated suppression of activated STAT1 nuclear localization and expression of INF triggered host antiviral mechanisms.
Of minor importance, invasion should be replaced with pathogenesis in the first sentence of the abstract and minor typographical errors should be corrected.
No competing interests were disclosed. Competing Interests: I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.  'We would like to thank you for taking the time to review this paper, and for your insightful comments. While our method is computational, and there is no easy way to get around that fact for us with respect to Ebola, we do believe that dissemination of such information can provide direction in the effort to understand, and finally abrogate, the mechanism of pathogenesis of the Ebola virus. Recently, we have used the PAGAL software to design anti-microbial peptides that work against plant pathogens .
The logical thread of our hypothesis in this manuscript follows the inability of the VP24 from Marburg to bind KPNA owing to the difference in two helices (analzyed using PAGAL) that bind KPNA in the Zaire Ebola virus. We believe this point is irrefutable. A small difference in one of the helices (alpha6) in the VP24 from Reston Ebola virus results in two computationally arrived differences.
Different hydrophobic moment in the Edmundson wheel (Fig3) (on a known structure, so confirmed). This difference is also visible in a multiple sequence alignment of the protein from different species.
Different charged interactions of the residues in KPNA and VP24, after docking (on a modelled structure, possible inaccuracies). These differences might not have drawn attention, if Reston Ebola was not known to be non-pathogenic to humans. We have taken care at each point to clearly indicate that this is a possibility, and not a foregone conclusion. In fact, studying the 'Reston-pathogenicity puzzle' using deuterium exchange mass spectrometry (DEMS) methods, Zhang . (2012) have identified et. al putative sites which includes a 'cluster of Reston-specific residues in VP24 is L136, R139 and S140' . It is possible that these differences would not lead to loss of binding when such experiments are finally done, and we would have to revise our hypothesis (which the 1 2 3 experiments are finally done, and we would have to revise our hypothesis (which the F1000Research model allows us to do). We emphasize on the role of computational methods to make intelligent and informed decisions, enabling biologist to design experiments, and minimizing human effort and cost -something that has been sorely missing in the Ebola effort.
In this context, and also in response to your comment on the unlikelihood of a single mutation resulting in pathogenicity, we would like to cite recent work that identifies two mutations (one in VP24 and the other in the nucleoprotein) resulting in the acquisition of high virulence in mice . The VP24 mutation is Thr50, and lies on a beta-sheet, and its importance in the structure has not been completely understood to date, although this residue is another putative site in the DEMS study . Our group, that has focused on the importance of alpha-helices, but not beta-sheets , is trying to rationalize the overwhelming significance of this mutation.
We also appreciate your idea of using KPNA from a non-human primate. However, only mice and rats have solved KPNAs. We have now included data on docking of a mouse KPNA to the Reston VP24 after conducting a similar analysis, and found no difference in their interactions (Fig. 5). Interestingly, we have also come across a study which concludes that only a STAT1 knockout mouse is susceptible to Reston Ebola virus . This strongly points towards the lack of immunosuppressive properties of the Reston Ebola virus in mice.
We have also made the suggested minor corrections, and had the manuscript corrected for typographical errors (Mary Mendum has been acknowledged). We hope that we have addressed your concerns by the changes that we have made.