Fig 1.
hDPP4-binding property and pseudovirus entry efficiency.
(A) Phylogenetic trees derived from the amino acid sequences of merbecoviruses for Spike and RBD. Viruses characterized in this study are shown with a blue triangle in front of the taxon names and with sequence similarities backward. (B) Binding curves of immobilized hDPP4 with MERS-CoV, 2014-422, and GX2012 RBDs. Data are shown as red crosses, and the best fit of the data to a 1:1 binding model is shown as black lines. (C) The cell entry efficiency of pseudoviruses as measured by luciferase activity. Equivalent amounts of MERS-CoV, 2014-422, and GX2012 pseudoviruses were used to infect Huh-7 cells. 293T cells were used as mock. Data are presented as mean values ± SD of five replicates. (D) MERS-CoV, 2014-422, and GX2012 pseudoviruses were treated with indicated concentrations of trypsin (5, 15, and 45 µg/mL) prior to infection of Huh-7 cells. Data are presented as mean values ± SD of five replicates. ****P < 0.0001 (two-tailed unpaired t-test). (E) Pseudovirus entry assays of MERS-CoV, 2014-422, and GX2012 were performed in WT Huh-7 cells or Huh-7 cells overexpressing TMPRSS2. Data are presented as mean values ± SD of five replicates.
Fig 2.
Overall structures of the 2014-422 and GX2012 S trimers.
(A) (Top) Overall structures of 2014-422 and GX2012 S trimers are shown in side views, with cryo-EM maps presented as semitransparent surfaces. (Bottom) Top views of 2014-422 and GX2012 S trimers are shown in surface. Three protomers are colored coral, green, and slate blue, respectively. The locations of CTDs are indicated. (B) Segmentation of 2014-422 and GX2012 S protomer. Structural segments are shown as boxes with the width related to the length of the amino acid sequence. The position of the S1/S2 and S2′ cleavage sites are indicated by arrows. (C) Overall structure of the 2014-422 S protomer. As 2014-422 and GX2012 spikes have similar overall structures, only the 2014-422 protomer was thus used to show detailed structures.
Fig 3.
Structural comparisons between 2014-422, GX2012, and MERS-CoV S trimers.
(A) The RBD and S2 trimers of closed state of 2014-422, GX2012, and MERS-CoV are shown in the top view. The buried interface calculated by Protein Interfaces, Surfaces and Assemblies (PISA) is listed in the table. (B) GX2012 and MERS-CoV S protomers are aligned by the S2 subunit. The GX2012 S protomer is in blue, and the MERS-CoV S protomer is in magenta. The measurement of structural movements in NTD, RBD, SD1 and SD2 subdomain are shown in magnify details. PDB code: MERS-CoV S, 8IFN.
Fig 4.
Structures of the 2014-422 and GX2012 RBD-hDPP4 complex.
(A) The core subdomains of 2014-422 RBD and GX2012 RBD are individually labeled and highlighted in green and blue. The external subdomains of the RBDs are in red and the β-strands are specified by ESPript. The β-propeller and hydrolase domains of DPP4 are in orange and dark grey. The blade IV and blade V of β-propeller that engage RBD are shown in semitransparent surfaces. (B) The binding angles of 2014-422 RBD, GX2012 RBD and MERS RBD with hDPP4. Three RBDs are colored green, blue and magenta, respectively. (C) Structural comparison of RBD displacement in the RBD-hDPP4 complex. β5, β7 and β8 strands are labeled. (D) The binding interface of 2014-422 RBD-hDPP4, GX2012 RBD-hDPP4, and MERS-CoV RBD-hDPP4 (PDB:4KR0). Three RBDs and hDPP4 are shown as surfaces. The AA sequences of 2014-422 and MERS-CoV RBD are renumbered based on the GX2012 RBD for convenient structural comparison.
Fig 5.
Structural details of the 2014-422 RBD-hDPP4, GX2012 RBD-hDPP4, and MERS-CoV RBD-hDPP4 complexes.
(A) The three complexes are structurally aligned, and their interaction networks are divided into Patch1 and Patch2 for comparison analyses. The AA sequences of the 2014-422 and MERS-CoV RBDs were renumbered for convenient structural comparison based on the GX2012 RBD. hDPP4 is colored in dark grey, and the 2014-422, GX2012, and MERS-CoV RBDs are colored in green, blue, and magenta, respectively. The black dashes present H-bonds and the black solid lines present salt bridge. (B) The RBDs interactions with N321 glycan of hDPP4. (C) The RBDs Interactions with N229 glycan of hDPP4.