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– 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 .

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 ‘dockingKPNAtoRestonVP24.p1m’ for human KPNA and ‘RESTONVP24mouse.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 .

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 protein-protein 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.

The data referenced by this article are under copyright with the following copyright statement: Copyright: � 2014 Chakraborty S et al.

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

F1000Research: Dataset 1. Version 2. Data used for SCALPEL search methodology to identify plant alpha helical - antimicrobial peptides in the PDB database.

10.5256/f1000research.5666.d40354 ⁶⁰