The Ebola virus was first discovered in 1976 ¹ , and has been since known as a rare, but deadly disease ² . However, the current outbreak in West African countries (Guinea, Liberia, Nigeria, Sierra Leone and Senegal) has rapidly deteriorated into a full blown epidemic ³ , and poses grave humanitarian dangers to these countries ⁴ . Ebola, along with the Marburg virus, belongs to the Filoviridae family ⁵ , and causes haemorrhagic fever ² by quickly suppressing innate antiviral immune responses to facilitate uncontrolled viral replication ⁶ .

Interestingly, the genome of the Ebola virus encodes seven proteins ⁷ , although their extreme ‘plasticity allows multiple functions’ ^{8, 9} . Protein structures are formed by well ordered local segments, of which the most prevalent are alpha helices (AH) and β sheets. AHs are right-handed spiral conformations which have a hydrogen bond between the carbonyl oxygen (C=O) of each residue and the alpha-amino nitrogen (N-H) of the fourth residue away from the N-terminal. AH domains are often the target of peptides designed to inhibit viral infections ^{10– 12} . Recently, we have provided open access to software that has reproduced previously described computational methods ¹³ to compute the hydrophobic moment of AHs (PAGAL ¹⁴ ).

In the current work, we characterize the helices in the Ebola proteome using PAGAL, and demonstrate that the helices with characteristically unique feature values are involved in critical interactions with the host proteins. The PDB database is queried for the keyword ‘Ebola’, and the structures obtained are analyzed using DSSP ¹⁵ for identifying AHs. We process all PDB structures, and do not filter out redundant structures based on sequence. These helices are analyzed using PAGAL, and the results are sorted based on three criteria - hydrophobic moment and high proportion of positive or negative residues. The helices that are ranked highest in these sorting criteria are involved in critical interactions with either antibodies or host proteins. For example, the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain has an AH with the largest hydrophobic moment in all helices analyzed ¹⁶ . This helix has part of the epitope recognized by the neutralizing antibody (KZ52) derived from a human survivor of the 1995 Kikwit outbreak, emphasizing the critical nature of this helix in the virulence of Ebola ¹⁷ . Another example, obtained by choosing the helix with the highest proportion of negatively charged residues, is the interaction between the human karyopherin alpha nuclear transporters C terminus and the Ebola virus VP24 protein (eVP24) ¹⁸ , which suppresses tyrosine-phosphorylated STAT1 nuclear import ¹⁹ . These helices probably are, or can be, the target of molecules designed to inhibit AH mediated protein-protein interactions ²⁰ . Our method provides a comprehensive list of such targets. Further, each protein can be individually queried using PAGAL, and thus identify helices that might have a poor global rank, but still be critical in the particular proteins context.

Although, Ebola and Marburg viruses are members of the Filoviridae family ²¹ , they have different antigenicity of the virion glycoprotein ²² . By comparing the AHs in proteins of Marburg and Ebola viruses, we are able to elicit subtle changes in the proteins that might render them ineffective against previously successful drugs. These differences are not apparent from a simple sequence or structural alignment. Thus, in the current work, we elucidate a simple methodology that can aid rational design of drugs and vaccine, an important aspect of the global effort to counter the deadly Ebola epidemic.

We searched for the keyword ‘Ebola’ in the PDB database ( Table 1). Subsequently, each protein was split based on the chain id, resulting in 146 single chained proteins (See ALPHA.zip in Dataset 1). We have not reduced the set based on sequence similarity since the proteins might have different conformations based on their ligands. Note, this list might include non-Ebola proteins which might have been co-crystallized with the Ebola protein. However, they have been put through the same analysis since they might provide insights into the Ebola proteins themselves.

These proteins were then analyzed using DSSP ¹⁵ , and resulted in 758 helices in all (See ALPHA.zip in Dataset 1). These helices were then analyzed using PAGAL. The PAGAL algorithm has been detailed previously ¹⁴ . 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 helix makes one full circle. We compute the hydrophobic moment by connecting the center to the coordinate of the residue and give it a magnitude obtained from the hydrophobic scale (in our case, this scale is obtained from ¹³ ). These vectors are then added to obtain the final hydrophobic moment.

The color coding is as follows: all hydrophobic residues are colored red, while hydrophilic residues are colored in blue: dark blue for positively charged residues, medium blue for negatively charged residues and light blue for amides.

The raw file generated by analyzing all 146 proteins through PAGAL is provided as PAGALRAWDATA.txt ( Dataset 1), and contains the hydrophobic moment, percent of positive charges and the total number of charged residues for every helix. These are then sorted based on the charge (negative or positive) or the hydrophobic moment. We ignore the helices that have none or a single charged residue, and those that are smaller than 10 residues in length. The proportion of charged residues is computed based on the total number of charged residues, and not the length of the helix.

All protein structures were rendered by PyMol ( http://www.pymol.org/). The sequence alignment was done using ClustalW ²³ . The alignment images were generated using Seaview ²⁴ . Protein structures have been superimposed using MUSTANG ²⁵ .

The ability of a genome as small as the Ebola virus to inflict a dishearteningly high percentage of mortality in human subjects is a humbling experience in the context of the tremendous technological advancements achieved in the last few decades ^{3, 4} . The Ebola virus potently suppresses the human immune response ^{2, 6, 43} by binding with key human proteins involved in the immune pathway ¹⁸ . These protein-protein interactions are often mediated through well structured secondary regions within the protein structures (alpha helices), and the design of molecules that inhibit these ‘hotspots’ ^{20, 44} has been a well known strategy to develop drugs to counter bacterial and viral infections ^{10– 12} . For example, synthetic peptides derived from the oligomerization domain of polymerase subunits has been shown to inhibit viral proteins ^{45, 46} . In addition, there might exist other protein domains that might be exploited by non-native viral peptides to obstruct viral functionality. In the current work, we characterize alpha helices in the Ebola virus proteome using a recently implemented open access software (PAGAL) ¹⁴ , thus identifying potential targets for inhibition of the helix mediated interactions. Through several examples, we demonstrate that helices with unique features are involved in interactions with host proteins (either antibodies from survivors, or proteins regulating the immune response). Further, we also provide an alternate way of analyzing differences in related proteins (from the Marburg virus) by focusing on the properties of corresponding helices. As future work, we intend to develop methodologies to design peptides that would target these ‘hotspots’ ⁴⁴ . It has to be kept in mind that it has been a challenge to design small ligands that disrupt protein-protein interactions, and designers resort to several innovative techniques to overcome thermodynamic instability or proteolytic susceptibility ^{47– 50} . These helices can essentially be epitopes ^{51, 52} for developing antibodies against the virus ^{53, 54} . Interestingly, ZMapp, a cocktail of three antibodies has shown reversion of advanced Ebola symptoms in non-human primates ⁵⁵ , and uses only glycoprotein-specific epitope generated antibodies ^{52, 56} . It is interesting to hypothesize that additions to this cocktail with antibodies derived from other epitopes (for example, 4U2XA.HELIX5 from VP24 that is involved in immune response suppression) could prove more effective. Thus, we provide a comprehensive list of potential targets within the small proteome of the Ebola virus that can directed rational design to quickly innovate therapies.