Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) currently spreads in humans and causes ∼ 36% fatality in infected patients. Believed to have originated from bats, MERS-CoV is genetically related to bat coronaviruses HKU4 and HKU5. To understand how bat coronaviruses transmit to humans, we investigated the receptor usage and cell entry activity of the virus-surface spike proteins of HKU4 and HKU5. We found that dipeptidyl peptidase 4 (DPP4), the receptor for MERS-CoV, is also the receptor for HKU4, but not HKU5. Despite sharing a common receptor, MERS-CoV and HKU4 spikes demonstrated functional differences. First, whereas MERS-CoV prefers human DPP4 over bat DPP4 as its receptor, HKU4 shows the opposite trend. Second, in the absence of exogenous proteases, both MERS-CoV and HKU4 spikes mediate pseudovirus entry into bat cells, whereas only MERS-CoV spike, but not HKU4 spike, mediates pseudovirus entry into human cells. Thus, MERS-CoV, but not HKU4, has adapted to use human DPP4 and human cellular proteases for efficient human cell entry, contributing to the enhanced pathogenesis of MERS-CoV in humans. These results establish DPP4 as a functional receptor for HKU4 and host cellular proteases as a host range determinant for HKU4. They also suggest that DPP4-recognizing bat coronaviruses threaten human health because of their spikes' capability to adapt to human cells for cross-species transmissions.

Fig. 1.

Binding interactions between HKU4 spike and DPP4. (A) AlphaScreen assay showing the direct binding interactions between the coronavirus spike S1 domains and hDPP4 or bDPP4. Binding affinity was characterized as AlphaScreen counts. Error bars indicate SEM (two-tailed t test, *P < 0.05, ***P < 0.001; n = 3). (B) Pull-down assay showing the direct binding interactions between the coronavirus S1 domains and hDPP4 or bDPP4. His₆-tagged hDPP4 or bDPP4 was incubated with each of the Fc-tagged coronavirus S1 domains. The S1 domain/DPP4 complex was then precipitated with nickel-nitrilotriacetic acid beads. Binding affinity was characterized as Western blotting on the precipitated Fc-tagged coronavirus S1 domains. (C) Inhibition of MERS-CoV-spike-mediated pseudovirus entry by the HKU4 S1 domain. HEK293T cells expressing hDPP4 or bDPP4 were preincubated with gradient concentrations of purified coronavirus S1 domains and then infected by MERS-CoV-spike-packaged pseudoviruses expressing luciferase. The pseudovirus entry efficiency was characterized as luciferase activity accompanying the entry and normalized relative to the entry in the absence of any coronavirus S1 domain. Error bars indicate SEM (n = 3).

Fig. 2.

HKU4- and MERS-CoV-spike-mediated pseudovirus entry into human cells. Retroviruses pseudotyped with HKU4 spike (A), MERS-CoV spike (B), or no spike (mock) (C) were incubated with gradient concentrations of trypsin and used to enter HEK293T cells (exogenously expressing hDPP4, bDPP4, or no DPP4, respectively). The pseudovirus entry efficiency was characterized as luciferase activity accompanying the entry. Error bars indicate SEM (n = 4).

Fig. 3.

Effects of human TMPRSS2 on HKU4- or MERS-CoV-spike-mediated pseudovirus entry into human cells. Retroviruses pseudotyped with HKU4 spike or MERS-CoV spike were pretreated with no trypsin, 20 µg/mL trypsin, or 100 µg/mL trypsin and used to enter Huh-7 cells or Huh-7 cells exogenously expressing human TMPRSS2. The pseudovirus entry efficiency was characterized as luciferase activity accompanying the entry. Error bars indicate SEM (two-tailed t test, N.S., P > 0.05; n = 4).

Fig. 4.

Role of human endosomal proteases in HKU4- and MERS-CoV-spike-mediated entry into human cells. Huh-7 cells were first preincubated with endosomal acidification inhibitor NH₄Cl or endosomal protease inhibitor E-64d at the indicated concentrations. Then the cells were infected by HKU4- or MERS-CoV-spike-pseudotyped retroviruses that had been pretreated or not pretreated with 100 μg/mL trypsin. The pseudovirus entry efficiency was characterized as luciferase activity accompanying the entry. Error bars indicate SEM (n = 3).

Fig. 5.

HKU4- and MERS-CoV-spike-mediated pseudovirus entry into bat cells. Retroviruses pseudotyped with HKU4 spike (A), MERS-CoV spike (B), or no spike (mock) (C) were incubated with gradient concentrations of trypsin and used to enter Tb1-Lu bat cells (exogenously expressing hDPP4, bDPP4, or no DPP4, respectively). The pseudovirus entry efficiency was characterized as luciferase activity accompanying the entry. Error bars indicate SEM (n = 4).