Measles Virus Hemagglutinin Protein Establishes a Specific Interaction With the Extreme N-Terminal Region of Human Signaling Lymphocytic Activation Molecule to Enhance Infection

Abstract

Measles virus (MV) is a human pathogen that is classified in the genus Morbillivirus in the family Paramyxoviridae together with several non-human animal morbilliviruses. They cause severe systemic infections by using signaling lymphocytic activation molecule (SLAM) and poliovirus receptor-like 4 expressed on immune and epithelial cells, respectively, as receptors. The viral hemagglutinin (H) protein is responsible for the receptor-binding. Previously determined structures of MV-H and SLAM complexes revealed a major binding interface between the SLAM V domain and MV-H with four binding components (sites 1-4) in the interface. We studied the MV-H and human SLAM (hSLAM) complex structure in further detail by in silico analyses and determined missing regions or residues in the previously determined complex structures. These analyses showed that, in addition to sites 1-4, MV-H establishes a unique interaction with the extreme N-terminal region (ExNTR) of hSLAM. The first principles calculation-based fragment molecular orbital computation method revealed that methionine at position 29 (hSLAM-Met29) is the key residue for the interaction. hSLAM-Met29 was predicted to establish a CH-π interaction with phenylalanine at position 549 of MV-H (MVH-Phe549). A cell-cell fusion assay showed that the hSLAM-Met29 and MVH-Phe549 interaction is important for hSLAM-dependent MV membrane fusion. Furthermore, Jurkat cell lines expressing hSLAM with or without Met29 and recombinant MV possessing the H protein with or without Phe549 showed that the hSLAM-Met29 and MVH-Phe549 interaction enhanced hSLAM-dependent MV infection by ~10-fold. We speculate that in the evolutionary history of morbilliviruses, this interaction may have contributed to MV adaptation to humans because this interaction is unique for MV and only MV uses hSLAM efficiently among morbilliviruses.

Keywords: extreme N-terminal; hemagglutinin; measles virus; receptor; signaling lymphocytic activation molecule.

Figure 1

Determination of the N-terminal amino acid residues of human SLAM (hSLAM). (A) The left, middle, and right panels show amino acid profiles of the first, second, and third cycles of Edman degradation, respectively. (B) The extreme N-terminal amino acid sequence of hSLAM. The three patterns of the N-terminal sequence are shown. The first, second, and third residues are shown in red, blue, and green, respectively.

Figure 2

Modeled MV H protein (MV-H) and hSLAM complex structure. (A–C) The MV-H (amino acids 152–606) and hSLAM (amino acids 21–137) complex structure was modeled using the Molecular Operating Environment (MOE) program. A reported structure (PDB ID: 3ALW; Hashiguchi et al., 2011) was used as the initial template. Missing hydrogen atoms were added using the Protonate 3D utility of MOE utilizing the Amber10:extended Hückel theory (EHT) force-field with solvation energy accounted for via the Born model. The resultant complex structure was optimized with Amber10:EHT force-field. The structure was visualized using PyMOL. (A) The overall complex structure of MV-H (gray) and hSLAM (cyan). The extreme N-terminal region (ExNTR) of hSLAM is shown in red. (B) The previously identified four binding components (sites 1–4; Hashiguchi et al., 2011) are shown in magenta, light green, yellow, and orange. The newly identified interaction using ExNTR of hSLAM is shown in red. (C) The modeled structure showed that hSLAM-Met29 and hSLAM-Met30 in the loop domain of hSLAM ExNTR interact with MVH-Lys185, MVH-Ser548, and MVH-Phe549. In particular, the interaction of hSLAM-Met29 was established with MVH-Phe549 through a CH-π interaction. (D) Fragment molecular orbital (FMO) interaction energies calculated between hSLAM-ExNTR residues and MV-H. Interfragment interaction energies (IFIEs) of a SLAM residue denote the sum of its interactions with all the MV-H residues.

Figure 3

Importance of hSLAM-Met29 and MVH-Phe549 shown by cell-cell fusion assays. (A–E) A quantitative cell fusion assay using the dual split protein (DSP) system. (A) Cells were transfected with MV-H and MV-F expression plasmids together with a non-mutated hSLAM‐ or mutated (M29S or M29F) hSLAM-expressing plasmid. (B) Cells were transfected with canine distemper virus (CDV)-H/P541S and CDV-F expression plasmids together with a non-mutated hSLAM-, mutated (M29S) hSLAM-, or dog SLAM-expressing plasmid. (C–E) Cells were transfected with non-mutated or mutated (F549S, F549N, F549H, F549Q, F549V, F549I, or F549M) MV-H and MV-F expression plasmids together with (C) an hSLAM-expressing plasmid, (D) a dog SLAM-expressing plasmid, or (E) a mutated (M29S) hSLAM-expressing plasmid. (A,B) For control cells [SLAM (−)], the SLAM-expression plasmid was omitted from the transfection mixture. (C–E) For control cells [H (−)], the MV-H-expression plasmid was omitted from the transfection mixture. At 1 day post transfection, the Renilla luciferase activity was measured. Error bars indicate the standard deviations of triplicate wells. (A–E) ^*** p < 0.001; ns, not significant.

Figure 4

Importance of Met29^hSLAM and Phe549^MVH shown by virus infection assays. (A) SLAM expression profile of the parental Jurkat, Jurkat/hSLAM, Jurkat/hSLAM-M29F, and Jurkat/hSLAM-M29S cells. Three independent clones of Jurkat cells expressing each hSLAM were generated. The parental Jurkat cells (filled gray profile) and three individual clones (red, blue, and green empty profiles) were stained with the anti-SLAM monoclonal antibody (IPO-3) followed by an Alexa Fluor 546-conjugated anti-mouse IgG. (B,C) The parental Jurkat, Jurkat/hSLAM, Jurkat/hSLAM-M29F, Jurkat/hSLAM-M29S, and Vero.DogSLAMtag cells were infected with IC323-enhanced green fluorescent protein (EGFP) (B) or IC323-EGFP-F549S (C) and cultured with the fusion block peptide. At 1 day post-infection (PI), the cell infectious unit (CIU) was determined by counting the number of EGFP-expressing cells. (B,C) ^* p < 0.03; ns, not significant.