Identification of residues on human receptor DPP4 critical for MERS-CoV binding and entry

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) infects host cells through binding the receptor binding domain (RBD) on its spike glycoprotein to human receptor dipeptidyl peptidase 4 (hDPP4). Here, we report identification of critical residues on hDPP4 for RBD binding and virus entry through analysis of a panel of hDPP4 mutants. Based on the RBD-hDPP4 crystal structure we reported, the mutated residues were located at the interface between RBD and hDPP4, which potentially changed the polarity, hydrophobic or hydrophilic properties of hDPP4, thereby interfering or disrupting their interaction with RBD. Using surface plasmon resonance (SPR) binding analysis and pseudovirus infection assay, we showed that several residues in hDPP4-RBD binding interface were important on hDPP4-RBD binding and viral entry. These results provide atomic insights into the features of interactions between hDPP4 and MERS-CoV RBD, and also provide potential explanation for cellular and species tropism of MERS-CoV infection.

Keywords: Amino-acid residue substitution; MERS-CoV; RBD; hDPP4.

Fig. 1

The amino-acid residue interaction details at the binding interface. (A) Two patches of the binding interface. Patch 1 interface is characterized by interactions between the C-terminal end of the long linker connecting the RBD β6/β7 strands (light magenta) and the hDPP4 blade 4 (cyan). In patch 2, a gently concaved outer surface in RBD (light magenta) accommodates a linker containing a short α helix between hDPP4 blades 4 and 5 (cyan). (B) and (C) Hydrophilic residues of RBD and hDPP4 interact through polar contacts in patch 1. RBD D539 has salt-bridge interaction with hDPP4 residue K267 (B). DPP4 residue R336 forms hydrogen bond with RBD residue Y499 (C). The polar contacts (salt-bridge and hydrogen bond) are drawn as black dashed sticks. (D) Hot spot residues in the hydrophobic core and hydrophilic periphery of patch 2.

Fig. 2

SPR assay for affinity binding between RBD and wide-type or mutant hDPP4. hDPP4 and its mutants (R336A, R317A, Q344A, K267A, K267E, L294A+I295A, L294D+I295D) were injected at a series of concentrations (shown on the right of the respective profile) in a buffer containing 10 mM HEPES, pH 7.2, 150 mM NaCl, and 0.005% (v/v) Tween-20. The kinetic fits are shown in blue.

Fig. 3

Infection efficiency of MERS-CoV pseudoviruses into COS7 cells expressing wide-type or mutant hDPP4. (A) hDPP4 expression on COS7 cells. COS7 cells transiently transfected with different hDPP4 constructs were used as target cells for pseudovirus infection. The mean fluorescence intensity (MFI) of target cells and control cells (without hDPP4 transfection) incubated with fluorescent antibody was determined by flow cytometer. The result shown is representative of three independent experiments conducted in triplicate. The actual residue mutants in hDPP4 are indicated below the horizontal axis. (B) The entry efficiency (%) of pseudovirus was calculated on the basis of luciferase activity. And the percentages of pseudovirus entry efficiency shown for mutant hDPP4 were luciferase activity values versus that of the wide-type hDPP4, as the entry efficiency for wide-type hDPP4 was defined as 100%. Data shown were corrected for the expression of different hDPP4 constructs by the parameter of MFI. Error bars represent standard errors of the means of three independent experiments. Student’s t-test; ^⁎P<0.05; ^⁎⁎P<0.01.

Fig. 4

Amino acid sequence alignment of DPP4 blades IV and V from different species. The mutated positions are highlighted in red boxes.