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

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) poses a threat to public health. The virus is endemic in the Middle East but can be transmitted to other countries by travel activity. The introduction of MERS-CoV into the Republic of Korea by an infected traveler resulted in a hospital outbreak of MERS that entailed 186 cases and 38 deaths. The MERS-CoV spike (S) protein binds to the cellular protein DPP4 via its receptor binding domain (RBD) and mediates viral entry into target cells. During the MERS outbreak in Korea, emergence and spread of viral variants that harbored mutations in the RBD, D510G and I529T, was observed. Counterintuitively, these mutations were found to reduce DPP4 binding and viral entry into target cells. In this study, we investigated whether they also exerted proviral effects. We confirm that changes D510G and I529T reduce S protein binding to DPP4 but show that this reduction only translates into diminished viral entry when expression of DPP4 on target cells is low. Neither mutation modulated S protein binding to sialic acids, S protein activation by host cell proteases, or inhibition of S protein-driven entry by interferon-induced transmembrane proteins. In contrast, changes D510G and I529T increased resistance of S protein-driven entry to neutralization by monoclonal antibodies and sera from MERS patients. These findings indicate that MERS-CoV variants with reduced neutralization sensitivity were transmitted during the Korean outbreak and that the responsible mutations were compatible with robust infection of cells expressing high levels of DPP4.IMPORTANCE MERS-CoV has pandemic potential, and it is important to identify mutations in viral proteins that might augment viral spread. In the course of a large hospital outbreak of MERS in the Republic of Korea in 2015, the spread of a viral variant that contained mutations in the viral spike protein was observed. These mutations were found to reduce receptor binding and viral infectivity. However, it remained unclear whether they also exerted proviral effects. We demonstrate that these mutations reduce sensitivity to antibody-mediated neutralization and are compatible with robust infection of target cells expressing large amounts of the viral receptor DPP4.

Keywords: MERS; antibody; neutralization; spike; virus entry.

FIG 1

Schematic illustration of the MERS-CoV spike glycoprotein and location of the receptor binding domain (RBD) polymorphisms. The MERS-CoV spike glycoprotein (MERS-S) consists of two subunits (S1 and S2). The S1 subunit contains an N-terminal signal peptide (SP) and an RBD, which binds to the receptor DPP4. The S2 subunit harbors the functional elements required for membrane fusion, a fusion peptide (FP), and two heptad repeats (HR1 and HR2), as well as the transmembrane domain (TD) and a cytoplasmic tail (CT). Below the scheme, the locations of the four amino acid polymorphisms investigated in this study (L411F, F473S, D510G, and I529T) are highlighted (bold letters).

FIG 2

All MERS-S variants analyzed were robustly expressed and incorporated into rhabdoviral particles. (A, top) Expression plasmids for the indicated S proteins or empty plasmid (control) were transfected into 293T cells. Whole-cell lysates (WCL) were prepared from transfected cells, and S protein expression was analyzed by SDS-PAGE and immunoblotting using anti-V5 antibody reactive against the V5 epitope at the C terminus of the S proteins. Detection of β-actin (ACTB) served as a negative control. Similar results were obtained in three separate experiments. (B, top) Expression plasmids for the indicated S proteins or empty plasmid (control) were transfected into 293T cells and the cells were then used to produce rhabdoviral particles. The particles were subsequently pelleted by centrifugation through a sucrose cushion and analyzed for S protein expression by SDS-PAGE and immunoblotting. Detection of vesicular stomatitis virus matrix protein (VSV-M) served as a loading control. (A and B, bottom) Immunoblots conducted for panels A (n = 3) and B (n = 6) were subjected to quantitative analysis using ImageJ software. S protein signals were normalized against the corresponding signals for ACTB or VSV-M, and expression and particle incorporation, respectively, of MERS-S WT were set as 100%. For S proteins that yielded two bands (S0 and S2), the two signals were combined before normalization. Mean values are shown; error bars indicate SEMs. Statistical significance of differences in particle expression or incorporation efficiency between MERS-S WT and variants was analyzed by paired two-tailed Student t tests.

FIG 3

Polymorphisms found in the S proteins of Korean MERS patients allow robust entry into cells expressing large amounts of DPP4. (A) Equal volumes of rhabdoviral particles harboring MERS-S WT, the indicated S protein mutants, VSV-G (positive control), or no glycoprotein (negative control) were inoculated onto Caco-2, Vero E6, 293T, and 293T cells overexpressing DPP4. Transduction efficiency was quantified at 18 h posttransduction by measuring the activity of virus-encoded luciferase in cell lysates. Transduction mediated by MERS-S WT was set as 100%. The averages from three individual experiments performed with quadruplicate samples are shown; error bars indicate SEMs. (B) 293T cells transfected to express the indicated viral proteins or empty expression vector (control) were detached and incubated with human Fc-tagged, soluble DPP4 (solDPP4-Fc) and an Alexa Fluor 488-conjugated anti-human antibody before DPP4 binding was quantified by flow cytometry. For normalization, binding of solDPP4-Fc to MERS-S WT was set as 100%. The results of a single representative experiment carried out with triplicate samples are shown and were confirmed in a separate experiment. Error bars indicate SDs. (C) Total cellular RNA was extracted from the indicated cell lines, reverse transcribed to cDNA and DPP4 transcript levels were analyzed by quantitative PCR in combination with the 2^−ΔΔCT method using ACTB as the housekeeping gene control and 293T cells as a reference (DPP4 level was set as 1). The results of a single representative experiment performed with triplicate samples are shown. Error bars indicate SDs. (D) Caco-2, Vero E6, and 293T cells (either untransfected or transfected with expression plasmid for DPP4) were analyzed for DPP4 cell surface expression by flow cytometry using anti-DPP4 and Alexa Fluor 488-conjugated anti-mouse antibodies. For normalization, DPP4 surface expression in untransfected 293T cells was set as 1. The results of a single, representative experiment performed with duplicate samples are shown; error bars indicate SDs. Similar results were obtained in two separate experiments. Statistical significance was analyzed by paired (A) or unpaired (B to D) two-tailed Student t tests.

FIG 4

RBD polymorphisms do not modulate sialic acid dependence of MERS-S-driven host cell entry. Caco-2 cells were preincubated with recombinant neuraminidase or were left untreated (control) before being inoculated with rhabdoviral particles harboring MERS-S WT, the indicated S protein mutants, influenza A virus (WSN, subtype H1N1) hemagglutinin and neuraminidase (WSN-HA/NA), or VSV-G. At 18 h posttransduction, transduction efficiency was quantified by measuring the activity of virus-encoded luciferase in cell lysates. For normalization, transduction efficiency of untreated cells was set as 100%. The combined data from three independent experiments performed with quadruplicate samples are presented. Error bars indicate SEMs, and statistical significance was analyzed by paired two-tailed Student t tests.

FIG 5

RBD polymorphisms do not impact MERS-S proteolytic activation. (A) 293T cells were transfected with expression plasmid for DPP4 alone (gray bars) or in combination with expression plasmid for TMPRSS2 (blue bars). At 24 h posttransfection, the cells were preincubated with dimethyl sulfoxide (DMSO; control [filled bars]) or cathepsin L inhibitor (MDL28170 [bars with checkerboard filling]) before being inoculated with rhabdoviral particles harboring MERS-S WT, the indicated S protein mutants, or VSV-G. Transduction efficiency was quantified by measuring the activity of virus-encoded luciferase in cell lysates at 18 h posttransduction. For normalization, luciferase activity measured for 293T cells overexpressing DPP4 and incubated with DMSO (filled gray bars) was set as 100%. (B) Caco-2 cells, for which MERS-S-driven entry relies on TMPRSS2 instead of cysteine proteases, were preincubated with medium containing water (control) or increasing concentrations of a TMPRSS2 inhibitor, camostat mesylate, before being inoculated with rhabdoviral pseudotypes harboring MERS-S WT, the indicated S protein mutants, or VSV-G. Transduction efficiency was quantified as described above. For normalization, transduction levels in the absence of inhibitor were set as 100%. The averages of four (A) or three (B) individual experiments performed with quadruplicate samples are shown. Error bars indicate SEMs. Statistical significance of differences of the indicated data pairs (A) or between WT and mutant S proteins (B) was analyzed by paired two-tailed Student t tests.

FIG 6

RBD polymorphisms do not change MERS-S sensitivity toward interferon-induced transmembrane proteins. 293T cells stably expressing interferon-induced transmembrane proteins (IFITM1 to -3) or chloramphenicol acetyltransferase (CAT; control) were transduced with rhabdoviral particles harboring MERS-S WT, the indicated S protein mutants, WSN-HA/NA, Machupo virus glycoprotein (MACV-GPC), or no viral glycoprotein (negative control; data not shown). At 18 h posttransduction, transduction efficiency was quantified by measuring the virus-encoded luciferase activity in cell lysates. For normalization, transduction of control 293T-CAT cells was set as 100%. The averages from three individual experiments performed with quadruplicate samples are shown. Error bars indicate SEMs, and statistical significance was analyzed by paired two-tailed Student t tests.

FIG 7

RBD polymorphisms increase resistance of MERS-S against antibody-mediated neutralization. Rhabdoviral particles harboring MERS-S WT, the indicated MERS-S mutants, or VSV-G were preincubated with increasing concentrations of MERS-S-specific monoclonal antibodies (mAb) that target different epitopes in the RBD (F11 or JC57-14) (A) or different dilutions of serum from a MERS patient (CSS53) (B) or a single dilution (1:200) of sera from three different MERS patients (CSS53 [data taken from panel B for direct comparison], CSS16, and CSS23) (C) before being inoculated onto Caco-2 cells. Cells transduced in the absence of mAb/serum served as controls. Transduction efficiency was quantified by measuring the activity of virus-encoded luciferase in cell lysates at 18 h posttransduction. For normalization, transduction in the absence of mAb/serum was set as 100%. The averages from three independent experiments performed with quadruplicate samples are presented in panel A; error bars indicate SEMs. Statistical significance of differences between transduction mediated by MERS-S WT and the S protein mutants was analyzed by paired two-tailed Student t tests. The results of a single representative experiment performed with triplicate samples are shown in panels B and C and were confirmed in a separate experiment. Error bars indicate SDs.