Bat SARS-Like WIV1 coronavirus uses the ACE2 of multiple animal species as receptor and evades IFITM3 restriction via TMPRSS2 activation of membrane fusion

Abstract

Diverse SARS-like coronaviruses (SL-CoVs) have been identified from bats and other animal species. Like SARS-CoV, some bat SL-CoVs, such as WIV1, also use angiotensin converting enzyme 2 (ACE2) from human and bat as entry receptor. However, whether these viruses can also use the ACE2 of other animal species as their receptor remains to be determined. We report herein that WIV1 has a broader tropism to ACE2 orthologs than SARS-CoV isolate Tor2. Among the 9 ACE2 orthologs examined, human ACE2 exhibited the highest efficiency to mediate the infection of WIV1 pseudotyped virus. Our findings thus imply that WIV1 has the potential to infect a wide range of wild animals and may directly jump to humans. We also showed that cell entry of WIV1 could be restricted by interferon-induced transmembrane proteins (IFITMs). However, WIV1 could exploit the airway protease TMPRSS2 to partially evade the IFITM3 restriction. Interestingly, we also found that amphotericin B could enhance the infectious entry of SARS-CoVs and SL-CoVs by evading IFITM3-mediated restriction. Collectively, our findings further underscore the risk of exposure to animal SL-CoVs and highlight the vulnerability of patients who take amphotericin B to infection by SL-CoVs, including the most recently emerging (SARS-CoV-2).

Keywords: ACE2 receptor; IFITM; SARS-like coronavirus WIV1; TMPRSS2; viral entry.

Figure 1.

Multiple ACE2 orthologues supported spike protein-mediated entry and membrane fusion of SARS-CoV and bat SL-CoV WIV1. (A) Transient expression of ACE2 orthologs in 293 T cells. The cell lysates were detected by western blot assay, using an anti-C9 monoclonal antibody. (B) Detection of WIV1 and Tor2 S expression (left panel) and pseudotype incorporation (right panel) by western blot using an antibody targeting the N-terminal myc tag added to the viral S proteins. β-Actin (cell lysates) and p24 (pseudoviral particles) served as loading controls. (C) HIV-1-Luc-based pseudotyped virus entry. 293 T cells were transfected with empty vector pcDNA3.1 or ACE2s orthologs. At 48 h. post-transfection, the cells were infected by pseudotyped virus particles of SARS-CoV Tor2 (Tor2pp) or WIV1 (WIV1pp). At 48 h. post-infection, luciferase signal of each set of Tor2pp or WIV1pp infection was measured and normalized to human ACE2, respectively. Error bars reveal the standard deviation of the means from four repeats.

Figure 2.

Multiple ACE2 orthologues supported WIV1 spike protein-mediated membrane fusion. (A) and (B) Syncytia formation assay. 293 T cells transfected with plasmid DNA of S gene of Tor2 (A) or WIV1 (B) were mixed at a 1:1 ratio with those cells transfected with plasmids encoding different animals ACE2s. Twenty-four hours later, syncytia formation was observed. (C) 293 T cells cotransfected with plasmids encoding either ACE2 orthologs and T7 polymerase or spike protein and T7/luciferase. For syncytium formation, the cells were mixed at a 1:1 ratio and cultured for 24 h. Luciferase activities in the cell lysates were determined, normalized to the human ACE2 mixed with S, and then expressed as means ± standard deviations (n = 4).

Figure 3.

Human ACE2 is not an efficient receptor for WIV1. (A) Phylogenetic tree of SL-CoVs and SARS-CoVs based on the amino acid sequence of S protein. (B) Pseudotyped virus entry. 293 T cells transfected with plasmid DNA of human ACE2 were infected by similar amount of pseudotyped virus particles of SARS-CoVs (a severe isolate Tor2 and a mild isolate GD03), WIV1, and civet cat SL-CoVs (SZ03, B039G and A022G). At 48 h. post-infection, the luciferase activities were measured and normalized to Tor2pp. Error bars reveal the standard deviation of the means from four repeats. (C) The interactions of human ACE2 and receptor-binding domain (RBD) of S proteins of SARS-CoVs and SL-CoVs were detected by IP assay by using anti-C9 antibody and anti-myc antibody. (D). Sequence alignment of 14 amino acid residues in the RBD that are critical to human ACE2 binding.

Figure 4.

IFITM proteins restrict SL-CoV spike-mediated viral entry. (A) FLIP-IN T-Rex 293 cells which expressed CAT or indicated IFITM proteins were cultured in DMEM with/without 1μg/ml tetracycline. Twenty-four hours later, expression of IFITM proteins was tested by western blot assay by using anti-FLAG antibody. (B) ACE2-transfected FLIP-IN T-Rex 293 cells, which expressed CAT or IFITMs proteins, were cultured in the existence or of absence of tetracycline (1μg/ml) in 96-well plate, and then were infected with similar amount of pseudotyped virus particles of SARS-CoVs and SL-CoVs. Influenza virus A virus and Lassa virus (LASV) were used as positive and negative control, respectively. The luciferase activity was measured at 48 h post-infection. Relative infection activity refers to the ratio of luciferase efficiency in cells grown in 1μg/ml Tet over which in cells grown without Tet. Error bars reveal the standard deviation (n = 4).

Figure 5.

TMPRSS2 promotes WIV1 entry by activating WIV1 S-mediated membrane fusion and partially bypassed IFTIM3-mediated restriction. (A), (B) and (C) Enhancement of TMPRSS2 on cell entry mediated by glycoproteins from Tor2, WIV1 and VSV, respectively. Tor2pp, WIV1pp and VSV-Gpp were used to infect T-Rex 293-ACE2 cells or T-Rex 293-ACE2-TMPRSS2 cells. At 48 h post-infection, luciferase activities were measured. Error bars reveal the standard deviation of the means from four repeats. (D) Syncytia formation between cells expressing S protein of Tor2 or WIV1 or VSV-G and cells expressing human ACE2 in the presence or absence of TMPRSS2. 293 T cells transfected with plasmids containing S gene were mixed with 293 T cells transfected with plasmids expressing human ACE2 and pCAGGS vector or TMPRSS2 plasmid at a 1:1 ratio. Twenty-four hours later, syncytia formation was observed. (E) ACE2-transfected FLIP-IN T-Rex 293-IFITM3 cells were cultured in the presence or absence of tetracycline (1μg/ml) for 24 h, then transduced with Tor2pp or WIV1pp. The transduced cells were treated with 5 μg/ml TPCK-trypsin or DMSO at 37°C for 13 min. Luciferase activities were measured at 48hpi. Relative infection refers to the ratio of the luciferase activity in T Rex293 cells cultured with Tet over that in cells cultured without Tet. Error bars reveal the standard deviation (n = 4). (F) T- Rex 293-ACE2 cells and T-Rex 293-ACE2-TMPRSS2 cells were cultured in the presence or absence of tetracycline (1 μg/ml) for 24 h, then transduce with Tor2pp and WIV1pp. Luciferase activities were measured at 48hpi. Relative infection refers to the ratio of the luciferase activity in T Rex293 cells cultured with Tet over that in cells cultured without Tet. Error bars reveal the standard deviation (n = 4).

Figure 6.

Amphotericin B treatment enhances the entry of SL-CoVs and SARS-CoVs and abrogates IFITM3-mediated restriction. (A) T Rex 293 cells transfected with ACE2 were infected with Tor2pp, GD03pp, SZ3pp, WIV1pp, IAVpp or LASVpp in the presence or absence of 1μM AmphoB. Luciferase efficiency was measured at 48 hr post-infection, and was normalized to DMSO-treated cells. The error bars refer to standard deviations (n = 4). (B) and (C) T-Rex 293 cells stably expressing IFITM1 or IFITM3 proteins were cultured in the presence or absence of Tet, and then were treated with 1μM AmphoB or DMSO. Twenty-four hours later, the cells above-mentioned were infected with Tor2pp, GD03pp, SZ3pp, WIV1pp, IAVpp or LASVpp. Luciferase efficiency was measured at 48 hr post-infection and normalized to cells cultured in the absence of Tet. The error bars refer to standard deviations (n = 4).