IFITM proteins inhibit entry driven by the MERS-coronavirus spike protein: evidence for cholesterol-independent mechanisms

Abstract

The interferon-inducible transmembrane (IFITM) proteins 1, 2 and 3 inhibit the host cell entry of several enveloped viruses, potentially by promoting the accumulation of cholesterol in endosomal compartments. IFITM3 is essential for control of influenza virus infection in mice and humans. In contrast, the role of IFITM proteins in coronavirus infection is less well defined. Employing a retroviral vector system for analysis of coronavirus entry, we investigated the susceptibility of human-adapted and emerging coronaviruses to inhibition by IFITM proteins. We found that entry of the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is sensitive to inhibition by IFITM proteins. In 293T cells, IFITM-mediated inhibition of cellular entry of the emerging MERS- and SARS-CoV was less efficient than blockade of entry of the globally circulating human coronaviruses 229E and NL63. Similar differences were not observed in A549 cells, suggesting that cellular context and/or IFITM expression levels can impact inhibition efficiency. The differential IFITM-sensitivity of coronaviruses observed in 293T cells afforded the opportunity to investigate whether efficiency of entry inhibition by IFITMs and endosomal cholesterol accumulation correlate. No such correlation was observed. Furthermore, entry mediated by the influenza virus hemagglutinin was robustly inhibited by IFITM3 but was insensitive to accumulation of endosomal cholesterol, indicating that modulation of cholesterol synthesis/transport did not account for the antiviral activity of IFITM3. Collectively, these results show that the emerging MERS-CoV is a target of the antiviral activity of IFITM proteins and demonstrate that mechanisms other than accumulation of endosomal cholesterol can contribute to viral entry inhibition by IFITMs.

Figure 1

IFITM expression and antiviral activity. (A) 293T cells were transiently transduced with retroviral vectors encoding IFITM 1, 2 or 3 with a C-terminal c-myc tag or chloramphenicol acetyltransferase (cat) as control. Expression of IFITM proteins in cell lysates was determined by Western blot analysis, employing a myc-specific antibody. Expression of beta-actin was assessed as a loading control. Comparable results were obtained in a separate experiment; (B) The experiment was carried out as described for panel (A) but cells were transduced with vectors encoding IFITM proteins without antigenic tag, and IFITM expression was analyzed with an IFITM1-specific monoclonal antibody and an antiserum raised against IFITM2, which is cross-reactive with IFITM3. Similar results were obtained in an independent experiment (C) 293T cells, transiently transduced to express IFITM1, 2 or 3, or cat as described for panel (B), were transduced with infectivity-normalized MLV vectors encoding firefly luciferase and bearing the entry proteins of murine leukemia virus (MLV), Lassa virus (LASV), Machupo virus (MACV), vesicular stomatitis virus (VSV) or influenza A virus (FLUAV). At 72 h post inoculation, the transduction efficiency was determined by measuring luciferase activities in cell lysates. The average of four independent experiments, each carried out with triplicate samples, is shown. Error bars indicate standard error of the mean (SEM).

Figure 2

Inhibition of S protein-driven cell entry by IFITM proteins. (A) 293T cells, transfected to express the viral receptors and transduced to express IFITM1, 2, 3, or cat as control, were transduced with infectivity-normalized retroviral vectors bearing the S proteins of the globally circulating human coronaviruses NL63 and 229E as well as the S proteins of the emerging SARS- and MERS-CoV. Transduction efficiency was determined at 72 h post inoculation by measuring luciferase activity in cell lysates. Transduction of control cells was set as 100%. The average of three independent experiments carried out with triplicate samples is shown, error bars indicate SEM. Statistical significance was calculated using one tailed, paired t-test. * p ≤ 0.05; ** p ≤ 0.01; (B) A549 wild type cells (control) and A549 cells transduced to stably express IFITM3 were transfected with siRNA directed against IFITM3. Scrambled siRNA were used as a control. Knockdown of IFITM3 expression was analyzed by Western blot. Detection of β-actin served as a loading control; (C) A549 control cells or A549-IFITM3 cells were transfected with siRNA directed against IFITM3 or scrambled siRNA as control. Cells were then transduced with the retroviral vectors described in (A). Transduction efficiency was analyzed at 72 h post transduction. Transduction of cells transfected with the scrambled siRNA was set as 1. The average of three independent experiments performed with triplicate samples is shown; error bars indicate SEM. The Welch-Test for independent samples was used to determine whether the effects of the siRNAs on transduction of A549 control and A549-IFITM3 cells were significantly different. * p ≤ 0.05; ** p ≤ 0.01.

Figure 3

Sensitivity of S protein-driven entry to inhibition by IFITMs and U18666A do not correlate. (A) 293T cells expressing the viral receptors were treated with the indicated concentrations of U18666A, which increases endosomal cholesterol levels, and then transduced with retroviral particles bearing the indicated glycoproteins. Transduction efficiency was analyzed at 72 h post inoculation by determining luciferase activities in cell lysates. The average of two (NL63-S, 229E-S) to four (SARS-S, MERS-S) separate experiments carried out with triplicate samples are shown. Error bars indicate SEM. (B) Cytotoxicity of the indicated concentrations of U18666A or equal volumes of the solvent DMSO were measured under the conditions specified in (A) using an MTT reduction assay. The result of a single experiment carried out with triplicate samples is shown; error bars indicate standard deviation (SD). Similar results were obtained in two separate experiments.

Figure 4

The relative sensitivities of coronavirus S protein- and FLUAV-HA-driven entry to inhibition by IFITMs and the cholesterol trafficking inhibitors clomiphene and terconazole do not correlate. 293T cells transfected to express the viral receptors were treated with the indicated concentrations of clomiphene (A) or terconazole (B), which increase endosomal cholesterol levels, and then transduced with retroviral particles bearing the indicated glycoproteins. Transduction efficiency was analyzed at 72 h post inoculation by determining luciferase activities in cell lysates. The average of three (clomiphene) or four (terconazole) independent experiments carried out with triplicate samples is shown. Error bars indicate SEM.

Figure 5

Cholesterol trafficking inhibitors induce accumulation of intracellular cholesterol. Cos7 cells were treated with either DMSO (mock) or the indicated inhibitors for 21 h at a concentration of 10 µM (terconazole, clomiphene) or 2.5 µM (U18666A). Cells were then fixed, stained with filipin III (blue) and staining analyzed by fluorescence microscopy using equal exposure times.