Mutations in the Spike Protein of Middle East Respiratory Syndrome Coronavirus Transmitted in Korea Increase Resistance to Antibody-Mediated Neutralization

Polymorphisms D510G and I529T in MERS-S of Korean patients reduce DPP4 binding but allow efficient entry into cells expressing large amounts of DPP4.

We first investigated whether polymorphisms identified during the Korean MERS outbreak, D510G and I529T (13, 14) (Fig. 1), are compatible with robust S protein expression and S protein-driven host cell entry. For comparison, we analyzed polymorphisms found in MERS-S from viruses from the Arabian Peninsula, L411F and F473S (15–19) (Fig. 1); these changes had previously not been investigated for their impact on MERS-S-driven entry. For our analyses of MERS-S-mediated entry, we employed a previously described vesicular stomatitis virus (VSV)-based pseudotyping system (20), which faithfully mimics central aspects of MERS-CoV entry into _target cells (21).

Analysis of 293T cells transfected to express the S proteins under study followed by immunoblot and signal quantification revealed that wild-type (WT) MERS-S and all S protein variants were comparably expressed (Fig. 2A). Similarly, all S proteins were proteolytically processed (Fig. 2A, top), in agreement with the finding that furin processes the S protein in MERS-S-transfected and MERS-CoV-infected cells. Finally, all S protein mutants were efficiently incorporated into VSV particles, although incorporation of mutant F473S was slightly reduced compared to that of MERS-S WT (Fig. 2B). Thus, the polymorphisms studied did not impact S protein expression, proteolytic processing, or, with the exception of mutant F473S, incorporation into VSV particles.

Next we analyzed whether the polymorphisms impacted entry into the colon-derived cell line Caco-2, the African green monkey kidney cell line Vero E6, and the human adrenal gland-derived cell line 293T (either control transfected or transfected with DPP4 expression plasmid [293T + DPP4]). These cell lines were chosen for analysis because they are permissive to MERS-CoV infection and were previously used to study host cell interactions of MERS-CoV (22–25). The selected cell lines were highly permissive to transduction by rhabdoviral particles harboring VSV glycoprotein (VSV-G; positive control), while transduction by particles bearing no glycoprotein (negative control) was within background levels, as expected (Fig. 3A). Changes L411F and F473S observed in MERS-CoV from humans and camels in the Middle East had no effect on transduction efficiency. In contrast, changes I529T and, to a lesser degree, D510G observed in Korean MERS patients reduced entry into Vero E6 and 293T cells (Fig. 3A) and diminished binding to DPP4 (Fig. 3B), in agreement with published data (14). However, these mutations had only a modest effect on transduction of Caco-2 and 293T cells expressing saturating amounts of DPP4 (293T + DPP4), respectively (Fig. 3A and data not shown). The finding that directed expression of DPP4 could largely rescue the inhibitory effect of D510G and I529T on transduction of 293T cells prompted us to investigate whether the impact of these changes on S protein-driven entry was dependent on the level of DPP4 expression. Indeed, quantitative reverse transcription-PCR (Fig. 3C) and flow cytometry (Fig. 3D) revealed that Caco-2 and 293T + DPP4 cells, which can be readily transduced by MERS-S D510G and I529T variants, expressed substantially higher levels of DPP4 than 293T and Vero E6 cells, which are not readily susceptible to transduction mediated by S protein mutants D510G and I529T (Fig. 3C and D). Collectively, our results confirm that D510G and I529T reduce S protein binding to DPP4 but also suggest that this reduction translates into diminished S protein-driven cell entry only when DPP4 expression is low.

D510G and I529T do not alter sialic acid dependency of viral entry.

A recent study showed that MERS-S can bind to sialic acids, glycans presented on plasma membrane proteins and lipids, and that sialic acid engagement promotes S protein-driven entry (9). We enzymatically removed sialic acids by neuraminidase (NA) treatment to investigate whether the Korean polymorphisms or the polymorphisms found in the Middle East impact sialic acid dependence. Neuraminidase treatment markedly reduced influenza A virus hemagglutinin (HA)-driven entry but had no effect on entry driven by VSV-G (Fig. 4), as expected. Moreover, entry driven by MERS-S WT and S protein variants was comparably inhibited by neuraminidase treatment (Fig. 4), indicating that none of the amino acid changes in MERS-S altered sialic acid dependence of viral entry.

D510G and I529T do not alter the choice of MERS-S activating host cell protease.

CatL and TMPRSS2 activate MERS-S in cell culture (10, 24), and the activity of TMPRSS2 or related enzymes is required for SARS-CoV spread in the host (26). Therefore, we investigated whether the polymorphisms under study modulated the capacity of MERS-S to employ CatL and TMPRSS2 for activation. For this, we first employed 293T cells, which express endogenous CatL but not TMPRSS2 and allow MERS-S-driven entry in a CatL-dependent fashion (10, 24). The cells were transfected with DPP4 plasmid and either TMPRSS2 encoding or empty plasmid (control), incubated with the CatL inhibitor MDL28170, and then analyzed for S protein-driven transduction. Incubation of control cells with MDL28170 reduced entry driven by MERS-S WT and S protein variants, and this effect was fully rescued by directed expression of TMPRSS2 (Fig. 5A). Next, we investigated S protein activation in Caco-2 cells, which express endogenous TMPRSS2 (27) and afforded the opportunity to visualize a potentially more subtle impact of the S protein polymorphisms on activation by TMPRSS2. However, incubation of Caco-2 cells with rising concentrations of camostat mesylate, a TMPRSS2 inhibitor, comparably reduced entry driven by MERS-S WT and S protein variants and had no effect on VSV-G-mediated entry (Fig. 5B), as expected. These findings suggest that the polymorphisms under study did not compromise S protein activation by CatL and TMPRSS2 and did not endow the S protein with the ability to use low levels of TMPRSS2 activity with increased efficiency for S protein activation.

D510G and I529T do not modulate IFITM sensitivity.

Expression of IFITM proteins is IFN inducible and inhibits MERS-S-driven entry into _target cells (28), most likely at the stage of membrane fusion. We investigated whether the S protein polymorphisms under study modulated susceptibility of MERS-S-driven entry to blockade by IFITM proteins. For this, we employed 293T cells stably expressing IFITM1, IFITM2, and IFITM3. Expression of IFITM proteins had little effect on entry driven by the Machupo virus glycoprotein (MACV-GPC), while both IFITM2 and IFITM3 markedly reduced entry by the influenza A virus hemagglutinin (Fig. 6), in accordance with published data (28). Moreover, expression of IFITM2 but not IFITM1 and IFITM3 diminished MERS-S WT-mediated host cell entry, as expected, and similar effects were observed for the S protein variants analyzed (Fig. 6). Finally, comparable results were obtained with 293T cells transiently transfected with escalating amounts of IFTIM2 plasmid, although variant F473S showed a slightly reduced IFITM2 sensitivity under these conditions (data not shown). In sum, the RBD polymorphisms observed in Korean patients and MERS-CoV from the Middle East did not markedly alter IFITM sensitivity of viral entry.

D510G and I529T increase resistance to antibody-mediated neutralization.

The antibody response significantly contributes to control of MERS-CoV infection in the host. Therefore, we asked whether the polymorphisms under study impact antibody-mediated neutralization. We focused our analysis on D510G and I529T since these changes modulated the efficiency of host cell entry. First, we tested whether D510G and I529T alter inhibition of S protein-driven entry by the murine antibody F11, which binds to an epitope encompassing amino acid 509, and macaque antibody JC57-14, which recognizes an epitope including amino acid 535 (29). Indeed, exchange D510G rendered MERS-S-driven entry insensitive to inhibition by F11, while I529T reduced blockade of S protein-driven entry by JC57-14 (Fig. 7A). These findings encouraged us to investigate whether D510G and I529T alter susceptibility of MERS-S-driven host cell entry to inhibition by human sera obtained from MERS patients. For this, we studied sera of three patients who had acquired MERS-CoV infection in the Middle East and were admitted to German hospitals in Essen (serum CSS16), Osnabrück (CSS53), and Munich (CSS23) (Table 1). One serum sample was of sufficient quantity for detailed analysis (CSS53), while the others could be tested only at a single dilution (CSS16 and CSS23). Remarkably, MERS-S harboring polymorphism D510G showed a significantly increased resistance against neutralization by one serum sample (CSS16), and polymorphism I529T diminished neutralization by two sera (CSS16 and CSS53 [Fig. 7B and C]). Finally, neither mutation had any impact on neutralization by serum sample CSS23. However, the overall neutralizing activity of this particular serum sample was reduced compared to those of the other sera tested and might have been too low to allow for differential neutralization of MERS-S WT and mutants D510G and I529T. In summary, our results suggest that increased resistance against humoral immune responses might have driven the emergence of the D510G and I529T variants.

Plasmids.

We employed expression plasmids encoding the VSV glycoprotein (VSV-G) (41), Machupo virus glycoprotein (MACV-GPC) (42) (kindly provided by M. Farzan), influenza A virus strain A/WSN/33 (H1N1) hemagglutinin and neuraminidase (WSN-HA/NA) (43), and WT MERS-S (10), which have been described elsewhere. In addition, we employed overlap extension PCR to generate MERS-S mutants harboring single (L411F, F473S, D510G, and I529T) mutations in the RBD (primer sequences and detailed information on the cloning procedure are available upon request). For all S proteins, we also generated versions containing a C-terminal V5 epitope for detection by immunoblotting. Expression plasmids for TMPRSS2 N-terminally fused to a cMYC epitope and DPP4 N-terminally fused to DsRed (EU827527.1 and DsRed-DPP4) were constructed by amplifying the genetic information from existing expression plasmids and cloning the respective open reading frame (ORF) into the pQCXIP plasmid (kindly provided by A. L. Brass). For the TMPRSS2 expression vector, we further exchanged the selection marker for puromycin resistance by that for blasticidin resistance, which was taken from plasmid pcDNA6/TR vector (8, 44). All PCR-amplified sequences were subjected to automated sequence analysis (Microsynth SeqLab) to verify their integrity.

Cell culture.

293T (human) and Vero E6 (African green monkey) cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; PAN-Biotech). In addition, the human colorectal adenocarcinoma cell line Caco-2 was grown in minimum essential medium (MEM; Thermo Fisher Scientific). 293T cells stably expressing IFITMs or chloramphenicol acetyltransferase (CAT), 293T-IFITM1, 293T-IFITM2, 293T-IFITM3, and 293T-CAT (45), were cultivated as for the parental 293T cell line with the exception that they additionally received 0.5 μg/ml of puromycin (Biomol). All media were supplemented with 10% fetal bovine serum (FBS; PAN-Biotech) as well as 1× penicillin and streptomycin from a 100× stock solution (PAN-Biotech). All cell lines were cultivated in a humidified atmosphere at 37°C and 5% CO₂. Transfection of 293T cells was performed by calcium phosphate precipitation.

Protease inhibitors.

We employed inhibitors _targeting cathepsin L and related proteases (MDL28170; Sigma-Aldrich) or TMPRSS2 and related proteases (camostat mesylate; Sigma-Aldrich). _target cells for transduction experiments were treated with the respective inhibitor (specific concentrations are indicated in the legends of the respective figures) for 2 h prior to inoculation with rhabdoviral transduction vectors.

Neuraminidase treatment of _target cells.

To remove terminal sialic acids from macromolecules on the cell surface, _target cells were preincubated with 100 mU of recombinant neuraminidase (Sigma-Aldrich) for 2 h before being inoculated with rhabdoviral transduction vectors.

Production of rhabdoviral transduction vectors and transduction of _target cells.

We employed rhabdoviral transduction vectors (pseudoparticles/pseudotypes) based on a replication-deficient vesicular stomatitis virus that lacks the genetic information for VSV-G but contains ORFs for enhanced green fluorescent protein (eGFP) and firefly luciferase (fLuc), VSV*ΔG-fLuc (46). For production of VSV-based pseudotypes (VSVpp), 293T cells were transfected with expression plasmids for MERS-S WT, mutant MERS-S harboring RBD polymorphisms, MACV-GPC, H1N1(WSN)-HA/NA, VSV-G (positive control) or empty expression vector (negative control). At 24 h posttransfection, cells were inoculated with VSV-G-transcomplemented VSV*ΔG-fLuc (Indiana strain, kindly provided by G. Zimmer) at a multiplicity of infection of 3 and incubated for 1 h at 37°C and 5% CO₂. Next the inoculum was removed, cells were washed with phosphate-buffered saline (PBS), and standard culture medium which contained anti-VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC) was added to neutralize residual input virus (cells transfected with VSV-G expression plasmid received culture medium without anti-VSV-G antibody). The cells were further incubated for 24 h before the supernatant was harvested, freed from cellular debris by centrifugation (3,000 × g for 10 min), and either stored at −80°C or used for transduction experiments. For the latter, _target cells were grown in 96-well plates. If necessary, _target cells were previously transfected with expression plasmids for DsRed-DPP4 and/or TMPRSS2 (24 h in advance) or were pretreated with protease inhibitors or neuraminidase (2 h in advance). For transduction, the culture medium was aspirated and the rhabdoviral transduction vectors were added to the cells. Transduction efficiency was quantified by measuring the virus-encoded fLuc activity in cell lysates using commercial kits (Beetle Juice; PJK) and a plate luminometer (Hidex, Turku, Finland) as described elsewhere (47).

Analysis of MERS-S expression and incorporation into VSVpp.

293T cells were transfected with expression plasmids for C-terminally V5-tagged versions of the MERS-S WT or MERS-S mutants or were control transfected with empty plasmid (negative control). At 48 h posttransfection, the culture supernatant was removed, cells were washed with PBS, and whole cells lysates (WCL) were prepared as follows. First, 2×SDS sample buffer (0.03 M Tris-HCl, 10% glycerol, 2% SDS, 5% beta-mercaptoethanol, 0.2% bromophenol blue, 1 mM EDTA) was added to the cells, which were then incubated for 10 min before the sample was transferred to 1.5-ml reaction tubes and heated to 95°C for additional 10 min. For analysis of S protein incorporation into VSV particles, the respective culture supernatants were pelleted by high-speed centrifugation (25,000 × g, 120 min, and 4°C) through a 20% (wt/vol) sucrose cushion, mixed with 2× SDS sample buffer, and heated to 95°C for additional 10 min. Following SDS-PAGE, proteins were transferred onto nitrocellulose membranes (Hartenstein GmbH) by immunoblotting. The membranes were then blocked by incubation in 5% skim milk in PBS–0.5% Tween 20 (PBS-T) for 30 min at room temperature (RT) and then incubated with primary antibody solution (anti-V5, anti-ACTB, or anti-VSV-M) overnight at 4°C. Next membranes were washed with PBS-T and further incubated with secondary antibody solution (anti-mouse-horseradish peroxidase [HRP]) for 1 h at RT. After additional washing steps, the membranes were finally developed using an in house-made chemiluminescence reagent in combination with the ChemoCam imaging system and ChemoStar Professional software (Intas Science Imaging Instruments GmbH). For quantification of the signal intensity, the program ImageJ (FIJI distribution) (48) was used. S protein signals detected in the WCL and supernatants were normalized against the respective signals of the loading control, either ACTB or VSV-M.

Antibodies for protein detection in immunoblot analysis.

The following antibodies were used as primary antibodies: anti-V5 (mouse, 1:1,000; Thermo Fisher Scientific), anti-β-actin (ACTB, mouse, 1:1,000; Sigma-Aldrich), and anti-VSV-M (mouse, 1:1,000; Kerafast). As a secondary antibody, an HRP-coupled anti-mouse antibody was used (goat, 1:5,000; Dianova). All antibodies were diluted in PBS-T (Carl Roth) and 5% skim milk (Carl Roth).

Analysis of MERS-S/DPP4 interaction and DPP4 surface expression by flow cytometry.

For the detection of MERS-S/DPP4 interaction, 293T cells were transfected with expression plasmids for MERS-S WT, MERS-S mutants, or VSV-G or empty plasmid (both negative controls). At 48 h posttransfection, the cells were washed with PBS and then resuspended in 1% bovine serum albumin (BSA)-PBS. Next, the cells were pelleted by centrifugation (5 min at 600 × g at 4°C) and resuspended in 1% BSA-PBS containing soluble DPP4 equipped with a C-terminal human Fc tag (solDPP4-Fc, 1:200; ACROBiosystems). After incubation for 1 h at 4°C, the cells were washed with 1% BSA-PBS and then resuspended in 1% BSA-PBS containing an Alexa Fluor 488-conjugated anti-human antibody (1:500; Thermo Fisher Scientific). After another incubation period (as described above), cells were washed with 1% BSA-PBS, fixed with 4% paraformaldehyde solution, and analyzed by flow cytometry as described below. For the analysis of DPP4 surface expression by flow cytometry, 293T (untransfected or transfected with expression plasmid for DPP4), Caco-2, and Vero E6 cells were detached by resuspension in 1% BSA-PBS (293T cells) or by incubation in citric saline (PBS containing 0.135 M potassium chloride and 0.015 M sodium citrate; Caco-2 and Vero E6 cells). Cells were then pelleted and washed as described above, successively incubated with anti-DPP4 (mouse, 1:200; Abcam) and Alexa Fluor 488-conjugated anti-mouse (goat, 1:500; Thermo Fisher Scientific) antibodies, and fixed. Finally, all samples were analyzed using an LSR II flow cytometer and the FACS Diva software (both from BD Biosciences). For further data analysis, FCS Express 4 Flow research software (De Novo software) was employed.

Quantification of DPP4-specific transcript levels by qPCR.

Total cellular RNA was extracted from 293T, Caco-2, and Vero E6 cells using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions. Then 1 μg of RNA was treated with DNase (New England BioLabs) and reverse transcribed into cDNA using the SuperScript III first-strand synthesis system (Thermo Fisher Scientific), both following the manufacturers’ specifications. Subsequently, 1 μl of each sample was subjected to quantitative PCR (qPCR) using the QuantiTect SYBR green PCR kit (Qiagen) and a Rotor-Gene Q platform (Qiagen). All samples were analyzed for β-actin (housekeeping gene control) and DPP4 (gene of interest) transcripts in triplicates. Finally, data were analyzed based on the threshold cycle (2^−ΔΔCT) method (49) using 293T cells as a reference.

Neutralization experiments.

Rhabdoviral transduction vectors harboring MERS-S WT, MERS-S mutants, or VSV-G were normalized for comparable transduction of Caco-2 cells (∼10⁵ luminescent counts/s), incubated for 30 min at RT with increasing concentrations of monoclonal antibodies _targeting distinct epitopes within the MERS-S RBD, JC57-14 (_targeting an epitope around amino acid residue 535 in MERS-S, isolated from vaccinated nonhuman primates [PDB code 6C6Y]) and F11 (_targeting an epitope around amino acid residue 509 in MERS-S, isolated from vaccinated mice) (29) or with different dilutions of serum from a MERS patient who traveled from the Arabian Peninsula to Germany. Furthermore, sera from two additional MERS patients, CSS16 and CSS23, were tested at a dilution of 1:200 (since the amount of those sera was limited, they were tested at a fixed dilution). Please see Table 1 for further information on the sera and the patient histories. Afterwards, the mixtures of VSVpp and antibodies/serum were inoculated onto Caco-2 cells that were further incubated at 37°C and 5% CO₂. At 18 h posttransduction, transduction efficiency was quantified (as described above). For normalization, transduction efficiency of rhabdoviral transduction vectors that were incubated in the absence of antibodies/serum was set as 100%.

Statistical analysis.

If not stated otherwise, unpaired or paired, two-tailed Student t tests were performed to test statistical significance of data originating from single representative or multiple combined experiments, respectively. In figures, statistical significance is represented as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; and ns, not significant.