April 21, 2018

EFFECTIVE FILOVIRUS ENTRY MAY DEPEND UPON CONSERVED TANDEM ASPARTIC ACID RESIDUES WITHIN TARGET NPC1: A CROSS-SPECIES COMPARATIVE STUDY

Introduction

Filovirus (Ebolavirus, Marburgvirus, Cuevavirus) binds to receptors on the cell surface through the viral spike glycoprotein. Prior studies have demonstrated that the virus initially enters the host cells by using different uptake mechanisms including lipid raft, receptor mediated clathrin-dependent endocytosis and/or macropinocytosis [1,2].

After internalization of the virus following macropinocytosis into the endosomes of the host cell, proteolysis of the viral trimeric glycoprotein (GP1/GP2) by two cysteine proteases, CTSB/cathepsin B and CTSL/cathepsin L, further induce a conformational change of GP2. This allows its binding of the virus to the host entry endosome receptor NPC1 at its loop 2 domain C site. This unmasks the filovirus GP2 fusion peptide to initiate fusion with the endosome membrane [3] which results from proteolytic cleavage of GP1 by the endosomal proteases cathepsin B and cathepsin L [4].

The binding of the viral glycoprotein (GP) to the host endosomal NPC1 appears to be position specific. Ng et al [5] identified a critical aspartic acid residue (Asp502) in domain C of loop 2 (371-615) of NPC1 which, when substituted by Phe502, significantly diminished infectivity within the African straw-colored fruit bats (Eidolon helvum). Hoffman et al [6] also identified variable infectivity with the straw-colored fruit bat and concluded that filoviruses rely on the same host cell factors for entry into human and fruit bat cells, although the efficiency of the usage of these factors might differ between filovirus species via the interactome with bat NPC1 loop 2.

Data:

The NPC1 DDFF Motif is Conserved Across Multiple Species

Our BLASTp alignment identified 41 of 159 (25%) unique species with the conserved DDFF motif in NPC1 loop 2. 54 species were identified (34%) with the conserved tandem DD residues within the DDFF motif. The alignment also appeared to demonstrate significant covariance with a substitution of Asp502->Glu502 (n=62) that may or may not have significant effect on infectivity. Further studies remain to be studied as to the effect of that covariant nonsynonymous substitution on filovirus infectivity.

Ebola Glycoprotein/NPC1 Interactive Model

PDB file 5JNX (Ebola Zaire) was obtained from the protein databank and visualized with UCSF Chimera. Chain E (EBOV GP2) was isolated along with Chain A|NPC1 loop 1 (1-316) and loop 2 (317-615)-[Image 1]. We identified 15 residues in EBOV GP2 that interacted with NPC1 [Image 3]. 14 interactions were with loop 2 and a single surface interaction was identified between GP2 (Tyr137) and loop 1 of NPC1 (Gly178).
The Lys84 in GP2 formed extensive electrostatic interactions with the tandem aspartic acid residues (Asp501-Asp502) in NPC1 in addition to Gln421.

We also show the Asp502 residue is located within a highly conserved DDFF motif of the host NPC1 loop 2 Domain C. Asp502 in host NPC1 appears to form significant Van der Waals surface interactions with the Ebolavirus Phe88, Gly89, and Trp86 facing residues. Possibly more significantly, our computational modeling demonstrates extensive electrostatic interactions with the Ebola GP Lys84 residue within loop 2. This interaction is also shared with the adjacent Asp501 residue which very likely may further enhance the binding capacity. NPC1 Asp501 also appears to form a single hydrogen bond with the Ebola GP Thr83 residue. The host target NPC1 Phe503 residue appears to form primarily Van der Waals surface interactions with Ebola Gly87 and Trp88. NPC1 Phe504 appears to have only a minor surface interaction with Ebola GP Ser142. It would therefore appear that the tandem Asp-Asp residues (Asp501->Asp502) within the DDFF motif of NPC1 loop 2, domain C, form the greatest contribution to the binding between filovirus GP and host NPC1 and are critical to infectivity. Our study also suggests that a substitution of Lys84 within the Ebola GP interactome mat also have a significant effect on binding capacity due to its extensive electrostatic interaction with the NPC1 tandem Aspartic acid residues at positions 501-502.

NPC1 alignment

Filovirus SsGP Alignment

Representative small-secreted GP sequences for EBOV, SUDV, BDBV, TAFV, RESTV, MARV, RAVN, and LLOV were obtained by BLASTp using EBOV as the query at NCBI and aligned using Clustal Omega in Jalview. Interacting residues identified in the model were marked for identification and conservation across species. Lys84 was 100% conserved across all species of filovirus.

Summary

From prior published reports, this modeling exercise would support the conclusion by Ng et al that the host DDFF motif in the filovirus receptor NPC1 containing Asp502 is a factor with infectivity. The extensive electrostatic interactions between the GP2 Lys84 and NPC1 (Asp501-Asp502, Gln421) would support Hoffman et al that an Ebolavirus species specific substitution of Lys84 would very likely effect infectivity even if the tandem Asp residues (Asp501-Asp502) were conserved in target NPC1. It must be mentioned that the Lys84 residue is 100% conserved across all species of Filoviridae further suggesting either host NPC1 molecular interacting residues or other interacting residues besides the tandem aspartic acid residues play a more significant role in infectivity.

Effective Filovirus Entry May Depend upon Conserved Tandem Aspartic Acid Residues within Target NPC1: CV