Two Mutations Were Critical for Bat-to-Human Transmission of Middle East Respiratory Syndrome Coronavirus

Abstract

To understand how Middle East respiratory syndrome coronavirus (MERS-CoV) transmitted from bats to humans, we compared the virus surface spikes of MERS-CoV and a related bat coronavirus, HKU4. Although HKU4 spike cannot mediate viral entry into human cells, two mutations enabled it to do so by allowing it to be activated by human proteases. These mutations are present in MERS-CoV spike, explaining why MERS-CoV infects human cells. These mutations therefore played critical roles in the bat-to-human transmission of MERS-CoV, either directly or through intermediate hosts.

FIG 1

Domain structure of MERS-CoV and HKU4 spike proteins. The spikes contain a receptor-binding S1 subunit, a membrane-fusion S2 subunit, a transmembrane anchor (TM), and an intracellular tail (IC). S1 contains the receptor-binding domain (RBD) that binds DPP4 receptor. S2 contains the fusion peptide (FP), heptad repeat 1 (HR1), and heptad repeat 2 (HR2), all of which are essential structural elements for the membrane fusion process. The S1/S2 boundary in MERS-CoV spike (defined as the region between the RBD and the fusion peptide) contains one established human protease motif that is recognized by proprotein convertases (hPPC) (15, 16); it also contains one established human protease motif that is recognized by endosomal cysteine proteases (hECP) (17, 18). Sequence alignments of these regions in MERS-CoV and HKU4 spikes (GenBank accession no. AFS88936.1 for MERS-CoV spike; ABN10839.1 for HKU4 spike) are shown, with the critical residue differences labeled in red. Arrows indicate the predicted sites of cleavage by human proteases in MERS-CoV spike.

FIG 2

Characterization of two protease motifs in MERS-CoV and HKU4 spike proteins. (A) Western blot analysis of HKU4 and MERS-CoV spikes in pseudovirus particles. Retroviruses pseudotyped with HKU4 spike (i.e., HKU4 pseudoviruses) or MERS-CoV spike (i.e., MERS-CoV pseudoviruses) were prepared in HEK293T (human embryonic kidney) cells as previously described (14). The incorporations of wild-type (WT) and mutant HKU4 and MERS-CoV spikes into pseudovirus particles were measured by Western blotting using antibody against their C-terminal C9 tags. Plots below the Western blot images correspond to quantifications of the band intensities from the cleaved and uncleaved spikes combined. The numbers below the plots indicate the relative amounts of spikes incorporated into pseudovirus particles compared to wild-type HKU4 and MERS-CoV spikes, respectively. All quantifications were done using ImageJ software (National Institutes of Health). (B) Glycosylation state of HKU4 spike at the hECP motif. HEK293T cells exogenously expressing wild-type (WT) and mutant HKU4 spikes were lysed and subjected to Western blot analysis. To improve the separation of high-molecular-mass spikes, 3 to 8% NuPAGE Tris-acetate gels (Life Technologies) were used for gel electrophoresis. To improve the accuracy of the result, each of the mutant and wild-type spikes was run in two lanes that alternated between samples. The experiment was also repeated in a separate gel. Compared with wild-type HKU4 spike, the downward shift in the band of HKU4 spike bearing the mutated hECP motif (i.e., mutation N762A) is consistent with the removal of glycosylation. (C) Glycosylation state of MERS-CoV spike at the hECP motif. Compared with MERS-CoV spike bearing the reengineered hPPC motif (i.e., mutation R748S), the upward shift in the band of MERS-CoV spike bearing both the reengineered hPPC and hECP motifs (i.e., mutations R748S/A763N/F764Y/N765T) indicates the introduction of glycosylation.

FIG 3

HKU4- and MERS-CoV-spike-mediated pseudovirus entry in human cells. (A) HKU4 pseudoviruses bearing no mutation, the reengineered hPPC motif (S746R), the reengineered hECP motif (N762A), or both of the reengineered motifs (S746R/N762A) were prepared in HEK293T (human embryonic kidney) cells and were then used to infect HEK293T cells exogenously expressing human DPP4 (GenBank accession no. NP_001926.2). The infections were carried out in the presence or absence of exogenous trypsin. The pseudovirus entry efficiencies were characterized by analysis of the levels of luciferase activity accompanying the entry and were normalized against the relative amounts of spikes incorporated into pseudovirus particles (Fig. 2A). The pseudovirus entry mediated by HKU4 spike bearing both of the reengineered motifs in the absence of exogenous trypsin was taken as 100%. (B) Pseudovirus-producing HEK293T cells were treated with proprotein convertase (PPC) inhibitor (dec-RVKR-CMK) 5 h after transfection of plasmids that encode HKU4 spike containing the reengineered hPPC motif (S746R). Pseudovirus-targeting HEK293T cells were treated with endosomal cysteine protease (ECP) inhibitor (E-64d) before being infected by HKU4 pseudoviruses bearing the reengineered hECP motif (N762A). Both of the inhibitors were used for HKU4 pseudoviruses bearing both of the mutations. (C) MERS-CoV pseudoviruses bearing no mutation, the mutated hPPC motif (R748S), the mutated hECP motif (A763N/F764Y/N765T), or both of the mutated motifs (R748S/A763N/F764Y/N765T) were used to infect HEK293T cells exogenously expressing human DPP4. The pseudovirus entry mediated by the wild-type MERS-CoV spike in the absence of exogenous trypsin was taken as 100%. (D) Pseudovirus-producing HEK293T cells were treated with dec-RVKR-CMK 5 h after transfection of plasmids that encode MERS-CoV spike containing the mutated hPPC motif (R748S). Pseudovirus-targeting HEK293T cells were treated with E-64d before being infected by MERS-CoV pseudoviruses bearing the mutated hECP motif (A763N/F764Y/N765T). Both of the inhibitors were used for MERS-CoV pseudoviruses bearing both of the mutated motifs. Error bars indicate standard errors of the means (SEM) (n = 4).

FIG 4

HKU4- and MERS-CoV-spike-mediated pseudovirus entry into bat cells. (A) HKU4 pseudoviruses bearing no mutation or the reengineered hECP motif (N762A) were prepared in HEK293T cells and were then used to infect RSKT cells endogenously expressing bat DPP4 (Rhinolophus sinicus bat kidney cells [24]). The cells were pretreated with indicated concentrations of ECP inhibitor (E-64d) before being infected by pseudoviruses. (B) The same HKU4 pseudoviruses were used to infect Tb1-Lu cells (Tadarida brasiliensis bat lung cells) exogenously expressing bat DPP4 (GenBank accession no. KC249974). (C) MERS-CoV pseudoviruses bearing no mutation or the mutant hECP motif (A763N/F764Y/N765T) were prepared in HEK293T cells and were then used to infect RSKT cells. (D) The same MERS-CoV pseudoviruses were used to infect Tb1-Lu cells. The pseudovirus entry efficiencies were characterized by analysis of the levels of luciferase activity accompanying the entry and were normalized against the relative amounts of spike proteins incorporated into pseudovirus particles. The pseudovirus entry mediated by the wild-type HKU4 spike (for panels A and B) or wild-type MERS-CoV spike (for panels C and D) in the absence of the inhibitor was taken as 100%. Error bars indicate SEM (n = 4).