Lysosomal Proteases Are a Determinant of Coronavirus Tropism

FIG 5

Cleavage of cell surface-expressed MERS-CoV spike using purified lysosomal extracts. (A) Cleavage of cell surface-expressed MERS-CoV spike using lysosomal extracts from a number of cell lines. MERS-CoV spike was exogenously expressed on the surfaces of h-HEK293T cells and then treated with 50 µg/ml lysosomal extracts (from different types of cells) at pH 5.6 for 30 min. The cleavage state of MERS-CoV spike was detected through Western blotting using an antibody recognizing its C-terminal C9 tag. (B) Cleavage of cell surface-expressed MERS-CoV spike using 100 µg/ml lysosomal extracts (from two types of cells) at pH 5.6 in a time-dependent manner (10, 30, and 60 min). The experiments were repeated five times, and representative results are shown.

To further compare the coronavirus spike-processing activities of human and bat lysosomal proteases, we examined whether lysosomal extracts from human and bat cells process the spike from a MERS-like bat coronavirus, HKU4, differently. Previously, we showed that HKU4 spike contains a glycosylated lysosomal protease site at the S1-S2 boundary and that it mediates virus entry into bat cells, but not human cells (44). Here, we investigated direct biochemical evidence for the different HKU4 spike-processing activities of human and bat lysosomal proteases. To this end, we purified lysosomal extracts from h-HEK293T cells and b-Tb1-Lu cells and incubated them individually with HKU4 spike expressed on the surfaces of h-HEK293T cells. The results showed that lysosomal extracts from b-Tb1-Lu cells, but not their counterparts from h-HEK293T cells, cleaved HKU4 spike containing a glycosylated lysosomal protease motif to produce S2 (Fig. 6A). Next, we introduced an N762A mutation into HKU4 spike; the mutation had been shown to remove the glycosylation from the lysosomal protease motif in HKU4 spike (44). The result showed that lysosomal extracts from both h-HEK293T cells and b-Tb1-Lu cells cleaved the mutant HKU4 spike to produce S2 (Fig. 6B). These results provided direct biochemical evidence demonstrating that lysosomal extracts from b-Tb1-Lu cells, but not their counterparts from h-HEK293T cells, can process the glycosylated lysosomal protease motif in HKU4 spike, whereas lysosomal extracts from both h-HEK293T cells and b-Tb1-Lu cells can process the unglycosylated lysosomal protease motif in HKU4 spike.

FIG 6

Cleavage of cell surface-expressed HKU4 spike using purified lysosomal extracts. The experiments were performed in the same way as for Fig. 5A, except that HKU4 spike (either wild type [WT] or containing an N762 mutation that removed a glycosylation site from the lysosomal protease motif) replaced MERS-CoV spike. The experiments were repeated five times, and representative results are shown.

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DISCUSSION

The tropism of coronaviruses includes species and tissue tropisms (1). Lysosomal proteases play a critical role in coronavirus entry (8, 10, 11), but their roles in coronavirus tropism have not been established. In contrast, extracellular proteases and other cellular proteases have been shown to be important determinants of coronavirus tropism (22, 23, 26, 29,–31). We and others previously showed that a MERS-like coronavirus from bats, HKU4, uses the same host receptor, DPP4, as MERS-CoV (24, 41), and we also showed that cellular proteases from bat and human cells support HKU4 entry differently (24, 44). However, two factors can complicate the roles of lysosomal proteases in coronavirus tropism: human and bat DPP4 molecules have different activities as coronavirus receptors, and other proteases may also play significant roles in the cell entry process of coronaviruses. In the current study, we quantified and controlled the contributions from host receptor and other proteases to coronavirus entry so that the roles of lysosomal proteases in coronavirus entry into cells from different origins could be clearly defined. To this end, we screened a number of cell lines originating from different tissues and host species and found three types of cells that were suitable for studying the roles of lysosomal proteases in coronavirus tropism: human HEK293T cells, human HeLa cells, and bat Tb1-Lu cells. These three types of cells share the following common features: they are naked for endogenously expressing very low levels of coronavirus receptor or cell surface proteases, and they can be easily transfected to exogenously express the coronavirus receptor from a single host species. As a result, lysosomal proteases likely function as the only or the main contributor to coronavirus spike-mediated entry. The above-described approach and findings may be extended to study the roles of lysosomal proteases in the entry of other viruses.

The current study investigated the roles of lysosomal proteases from the above-mentioned human and bat cells in coronavirus entry using a combination of pseudovirus entry, cell-cell fusion, and biochemical assays. To this end, we exogenously expressed human DPP4 in different types of cells and performed MERS-CoV spike-mediated pseudovirus entry and cell-cell fusion. In the presence of DPP4 from the same species and in the absence of extracellular proteases and other cellular proteases, lysosomal proteases and lysosomal extracts from bat cells supported MERS-CoV spike-mediated membrane fusion more efficiently than their counterparts from human cells. These observations were then extended to SARS-CoV spike-mediated pseudovirus entry and cell-cell fusion. Moreover, we prepared lysosomal extracts from human and bat cells and showed that lysosomal extracts from bat cells cleaved MERS-CoV spike more efficiently than their counterparts from human cells. We also showed that lysosomal extracts from bat cells cleaved HKU4 spike, which contains a glycosylated lysosomal protease motif, more efficiently than their counterparts from human cells. These results demonstrated that the spikes from MERS-CoV, SARS-CoV, and HKU4 all mediated viral entry into bat cells at higher efficiency than into human cells, due to or mainly due to the higher coronavirus spike-processing activities of bat lysosomal proteases.

The correlation between lysosomal protease activities and coronavirus tropism is a novel finding in virology. Previous studies had already shown that the expression levels of lysosomal proteases vary among different tissues within the same host species due to the different physiological functions of the tissue cells (7, 45). Our study demonstrates that lysosomal protease activities may also vary among different mammalian species, indicating that adaptation of coronaviruses to new species may occur through adaptation to different lysosomal protease activities. The physiological reason for different lysosomal protease activities among mammalian species is not clear, but it could be due to the different lifestyles of these species. For instance, bats are the only flying mammals, and hence, the enhanced lysosomal protease activities of bat cells may provide fast turnover of metabolic products and also produce high levels of nutrients, although this is speculative. In this sense, supporting coronavirus entry efficiently could be a by-product of the enhanced lysosomal protease activities of bat cells. It is worth noting that, due to the difficulty in culturing bat tissue cells, this study was performed using bat cell lines. Although cell lines usually maintain many features of original tissue cells, these findings need to be confirmed using bat tissue cells. Our study suggests that no matter whether cells are from different host species or from different tissues of the same host species, cells with higher lysosomal protease activities in general support coronavirus entry more efficiently than cells with lower lysosomal protease activities. It remains to be further investigated whether the higher lysosomal protease activities in some cells are due to enhanced enzymatic activity, elevated expression levels, or other changes to their lysosomal proteases. Nevertheless, our study has established that different lysosomal proteases from different types of cells have direct impacts on coronavirus entry, which has implications for the tissue and species tropism of coronaviruses.

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MATERIALS AND METHODS

Cell lines and plasmids.

HEK293T cells (human embryonic kidney cells), HeLa cells (human cervical epithelial cells), A549 cells (human alveolar epithelial cells), Vero cells (monkey kidney cells), MRC5 cells (human lung cells), and Tb1-Lu cells (Triatoma brasiliensis bat lung cells) were obtained from the ATCC (American Type Culture Collection). RSKT cells (Rhinolophus sinicus bat kidney cells), PESU-B5L cells (Perimyotis subflavus bat lung cells), and BKD9 cells (Myotis davidii bat kidney cells) were purchased from Sigma-Aldrich. Huh-7 cells (human hepatoma cells) were kindly provided by Charles M. Rice (Rockefeller University). All the cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies). The full-length genes of MERS-CoV spike (GenBank accession number AFS88936.1), SARS-CoV spike (GenBank accession number AFR58742), human DDP4 (GenBank accession number NM_001935.3) and human ACE2 (GenBank accession number NM_021804) were synthesized (GenScript Biotech) and subcloned into the pcDNA3.1(+) vector (Life Technologies) with a C-terminal C9 tag (i.e., a tag corresponding to the 9 amino acids at the C terminus of human complement component C9 protein). Plasmids (pFR-Luc and pBD-NF-κB) for cell-cell fusion were kindly provided by Zhaohui Qian (Chinese Academy of Medical Sciences and Peking Union Medical College).

Coronavirus spike-mediated pseudovirus entry into human and bat cells.

Retroviruses pseudotyped with MERS-CoV or SARS-CoV spike (i.e., MERS-CoV pseudoviruses or SARS-CoV pseudoviruses, respectively) were generated as described previously (24). Briefly, HEK293T cells were cotransfected with a plasmid carrying an Env-defective, luciferase-expressing HIV-1 genome (pNL4–3.luc.R-E-) and a pcDNA3.1(+) plasmid encoding MERS-CoV or SARS-CoV spike. Pseudoviruses were harvested 72 h after transfection and used to enter human and bat cells. For screening of cell lines expressing no or low levels of coronavirus receptor, different types of cells were seeded in 96-well plates and infected immediately with pseudoviruses. To study the roles of lysosomal proteases in coronavirus entry, cells were transfected with the pcDNA3.1(+) plasmid encoding human DPP4 or human ACE2; 24 h after the transfection, the cells expressing the receptor were seeded in 96-well plates and then infected with pseudoviruses. After incubation at 37°C for 6 h, the medium was replaced with fresh DMEM. After another 60 h, the cells were washed with phosphate-buffered saline (PBS) and lysed. Aliquots of the cell lysates were transferred to an Optiplate-96 plate (PerkinElmer Life Sciences), and then luciferase substrate (Promega) was added. Relative luciferase units were measured using an EnSpire plate reader (PerkinElmer Life Sciences) and normalized for exogenous expression levels of the corresponding receptor in the cell membranes (see below).

Inhibition of pseudovirus entry using various protease inhibitors was carried out as described previously (50). Briefly, target cells were preincubated with medium containing a final concentration of 50 μM camostat mesylate (Sigma-Aldrich), 50 μM E-64d (Sigma-Aldrich), 50 μM chloromethyl ketone (Enzo), or DMSO (dimethyl sulfoxide) (negative control) at 37°C for 1 h. The cells were subsequently infected with pseudoviruses. The cells were incubated at 37°C for 6 to 8 h, and then the medium was replaced with fresh DMEM. After another 48 h, the cells were lysed and measured for luciferase activity.

Exogenous expression of coronavirus receptor in cells and on cell surfaces.

To examine the exogenous expression level of coronavirus receptor in whole-cell lysates, cells were transfected with pcDNA3.1(+) plasmid encoding human DPP4 or human ACE2 containing a C-terminal C9 tag; 48 h after transfection, the cells were lysed using ultrasonication, and aliquots of cell lysates were subjected to Western blotting. The C9-tagged coronavirus receptors were detected using an anti-C9 tag monoclonal antibody (Santa Cruz Biotechnology). The current assay measures the total expression level of coronavirus receptor in a certain number of cells without specifying how many of the cells were transfected or how much protein was expressed in each transfected cell.

To examine the exogenous expression level of coronavirus receptor in cell membranes, the cells expressing the receptor were harvested as described above, and all membrane-associated proteins were extracted using a membrane protein extraction kit (Thermo Fisher Scientific). Briefly, cells were centrifuged at 300 × g for 5 min and washed with cell wash solution twice. The cell pellets were resuspended in 0.75 ml permeabilization buffer and incubated at 4°C for 10 min. The supernatant containing cytosolic proteins was removed after centrifugation at 16,000 × g for 15 min. The pellets containing membrane-associated proteins were resuspended in 0.5 ml solubilization buffer and incubated at 4°C for 30 min. After centrifugation at 16,000 × g for 15 min, the membrane-associated proteins from the supernatant were transferred to a new tube. The expression level of membrane-associated C9-tagged coronavirus receptor among the membrane-associated proteins was then measured using Western blot analysis as described above and used for normalizing the results from pseudovirus entry assays. Although the current assay could not differentiate between plasma membrane-associated proteins and internal-membrane-associated proteins, ACE2 and DPP4 are known to be strongly associated with plasma membranes due to their respective plasma membrane-targeting signal peptides (51, 52).

MTT assay.

Cells were seeded in 96-well plates and treated with DMSO or protease inhibitors dissolved in DMSO at 37°C. After incubation for 6 h, the medium was replaced with fresh DMEM. After incubation for 70 h at 37°C, 10 μl MTT solution (Biotium) was added to each well and mixed with the medium. After incubation at 37°C for 2 h, 200 μl DMSO or protein inhibitor dissolved in DMSO was added to each well and mixed with the medium. The MTT signal was measured as absorbance at 570 nm using a Synergy 2 multimode microplate reader (BioTek Instruments).

Preparation of lysosomal extracts.

Lysosomal extracts from human or bat cells were prepared according to the lysosome isolation kit procedure (Sigma-Aldrich). Briefly, cells were harvested and washed with PBS buffer and then resuspended in 2.7 packed cell volumes (PCV) of extraction buffer. The cells were broken in a 7-ml Dounce homogenizer using a loose pestle (i.e., pestle B) until 80% to 85% of the cells were broken (protease inhibitors from the kit were omitted in our procedure). The samples were centrifuged at 1,000 × g for 10 min, and the supernatants were transferred to a new centrifuge tube and centrifuged at 20,000 × g for another 20 min. The supernatants were removed, and the pellets were resuspended in extraction buffer as the crude lysosomal fraction (CLF). The CLF was diluted in buffer containing 19% Optiprep density gradient medium solution and further purified using density gradient centrifugation at 150,000 × g for 4 h to yield lysosomal extracts. The concentrations of the lysosomal extracts were measured using a NanoDrop 8000 (Thermo Fisher Scientific) and calculated according to their absorbance at 280 nm. The purities of the lysosomal extracts were examined using the assays described below.

CPR is an ER marker. For evaluation of the potential contamination of the purified lysosomal extracts by ER proteins, the cytochrome P450 reductase activities of the purified lysosomal extracts were measured using a cytochrome P450 reductase assay kit (Biovision). Briefly, a glucose-6-phosphate (G6P) standard curve was first calculated by mixing a series of volumes of 1 mM G6P standard solution with 5 μl NADPH substrate and 5 μl G6P standard developer to make the final volume 100 μl/well. The well contents were then mixed and incubated at room temperature for 30 min (protected from light). Absorbance at 460 nm was measured. Then, 5 μl lysosomal extracts from different cell lines was mixed with 55 μl CPR assay buffer. After adding 30 μl of the assay reaction mixture to each well and incubating the solutions at room temperature for 5 min, 10 μl of the 20 mM G6P solution was added to each well. Absorbance at 460 nm was measured immediately in kinetic mode at 25°C for 25 min using a Synergy 2 multimode microplate reader (BioTek Instruments). Calculation of the cytochrome P450 reductase activity was performed according to the manufacturer’s manual.

ALP is a plasma enzyme marker. For evaluation of the potential contamination of the purified lysosomal extracts by plasma proteins, the alkaline phosphatase activity of the purified lysosomal extracts was measured using an alkaline phosphatase assay kit (Abnova). Briefly, a standard curve was first calculated by mixing a series of concentrations of 4-methylumbelliferyl phosphate disodium salt (MUP) standard with 10 μl ALP enzyme solution. The reaction mixtures were incubated at 25°C for 30 min (protected from light). The ALP enzyme can convert MUP substrate to an equal molar amount of fluorescent 4-methylumbelliferone (4-MU). Hence, 20 μl 0.5 mM MUP substrate solution was added to each well containing 5 μl lysosomal extracts from different cell lines. After mixing and incubating at 25°C for 30 min (protected from light), all the reactions were stopped by adding 20 μl stop solution to each reaction mixture. Then, the fluorescence intensities at excitation and emission wavelengths of 360 and 440 nm, respectively, were measured using a Synergy 2 multimode microplate reader (BioTek Instruments). Calculation of the alkaline phosphatase activity was performed according to the manufacturer’s manual.

Coronavirus spike-mediated cell-cell fusion.

Cell-cell fusion was performed as described previously (53). Briefly, to produce cells expressing one of the coronavirus spikes, HEK293T cells were cotransfected with the plasmid pFR-Luc, which contains a synthetic promoter with five tandem repeats of the Saccharomyces cerevisiae GAL4 binding sites that controls expression of the luciferase gene, and the pcDNA3.1(+) plasmid encoding one of the coronavirus spikes. To produce cells expressing one of the corresponding coronavirus receptor proteins, HEK293T cells were cotransfected with pBD-NF-κB, which encodes a fusion protein with the DNA binding domain of GAL4 and the transcription activation domain of NF-κB, and a pcDNA3.1(+) plasmid encoding one of the corresponding coronavirus receptor proteins. After culturing for 24 h, the spike-expressing HEK293T cells were lifted, centrifuged, and then resuspended in low-pH medium containing 10 mM sodium citrate, pH 5.6. Subsequently, the spike-expressing HEK293T cells were treated with purified lysosomal extracts (100 µg/ml) in the low-pH medium. After incubation at 37°C for 30 min, the spike-expressing cells were centrifuged, resuspended in fresh neutral pH medium, and then overlaid onto receptor-expressing HEK293T cells at a ratio of 1:2. When cell-cell fusion occurred, the expression of the luciferase gene was activated through binding of the GAL4–NF-κB fusion protein to GAL4 binding sites at the promoter of the luciferase gene. After incubation for 24 h, the cells were lysed, aliquots of the cell lysates were transferred to an Optiplate-96 plate (PerkinElmer Life Sciences), and then luciferase substrate (Promega) was added. Relative luciferase units were measured using an EnSpire plate reader (PerkinElmer Life Sciences).

Cleavage of coronavirus spikes using purified lysosomal extracts.

HEK293T cells were transfected with pcDNA3.1(+) plasmid encoding MERS-CoV spike or HKU4 spike; 48 h after transfection, the cells were harvested and washed with PBS buffer. The cells were then treated with 50 µg/ml purified lysosomal extracts at pH 5.6 for 30 min or 100 µg/ml purified lysosomal extracts at pH 5.6 for different periods of time (i.e., 10, 30, or 60 min). After treatment, the cells were lysed and boiled for Western blotting. The C9-tagged spikes were detected using an anti-C9 tag monoclonal antibody (Santa Cruz Biotechnology).

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ACKNOWLEDGMENTS

This work was supported by NIH grants R01AI089728 (to F.L.) and R01AI110700 (to F.L. and R.B.).

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