Neurovirulent Murine Coronavirus JHM.SD Uses Cellular Zinc Metalloproteases for Virus Entry and Cell-Cell Fusion

Abstract

The coronavirus (CoV) S protein requires cleavage by host cell proteases to mediate virus-cell and cell-cell fusion. Many strains of the murine coronavirus mouse hepatitis virus (MHV) have distinct, S-dependent organ and tissue tropisms despite using a common receptor, suggesting that they employ different cellular proteases for fusion. In support of this hypothesis, we found that inhibition of endosomal acidification only modestly decreased entry, and overexpression of the cell surface protease TMPRSS2 greatly enhanced entry, of the highly neurovirulent MHV strain JHM.SD relative to their effects on the reference strain, A59. However, TMPRSS2 overexpression decreased MHV structural protein expression, release of infectious particles, and syncytium formation, and endogenous serine protease activity did not contribute greatly to infection. We therefore investigated the importance of other classes of cellular proteases and found that inhibition of matrix metalloproteinase (MMP)- and a disintegrin and metalloprotease (ADAM)-family zinc metalloproteases markedly decreased both entry and cell-cell fusion. Suppression of virus by metalloprotease inhibition varied among tested cell lines and MHV S proteins, suggesting a role for metalloprotease use in strain-dependent tropism. We conclude that zinc metalloproteases must be considered potential contributors to coronavirus fusion.IMPORTANCE The family Coronaviridae includes viruses that cause two emerging diseases of humans, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), as well as a number of important animal pathogens. Because coronaviruses depend on host protease-mediated cleavage of their S proteins for entry, a number of protease inhibitors have been proposed as antiviral agents. However, it is unclear which proteases mediate in vivo infection. For example, SARS-CoV infection of cultured cells depends on endosomal acid pH-dependent proteases rather than on the cell surface acid pH-independent serine protease TMPRSS2, but Zhou et al. (Antiviral Res 116:76-84, 2015, doi:10.1016/j.antiviral.2015.01.011) found that a serine protease inhibitor was more protective than a cathepsin inhibitor in SARS-CoV-infected mice. This paper explores the contributions of endosomal acidification and various proteases to coronavirus infection and identifies an unexpected class of proteases, the matrix metalloproteinase and ADAM families, as potential targets for anticoronavirus therapy.

Keywords: TMPRSS2; coronavirus; membrane fusion; metalloprotease; virus entry.

Copyright © 2017 American Society for Microbiology.

Figures

FIG 1

JHM.SD is less sensitive than A59 to bafilomycin A. Pretreated L2 cells were infected with rJHM.SD-fluc or rA59-fluc at an approximate multiplicity of infection (MOI) of 0.5 and then assayed for luciferase activity 7 hpi as described in Materials and Methods. In parallel, cells were infected and then treated with DMSO-bafilomycin A beginning 1 h postinfection, and the pretreatment effect (relative to DMSO alone for each virus) was divided by the corresponding posttreatment effect to correct for posttreatment effects. (Top and middle) An asterisk indicates significant difference between the 0 and 10 nM or 100 nM treatment within each virus (2-way ANOVA with Dunnett's multiple comparisons of simple effects within columns). (Bottom) After correction, the effect of bafilomycin A was significantly smaller for JHM.SD than for A59 (n = 5; P < 0.0001 for the bafilomycin A effect, P < 0.0001 for the virus strain effect, and P < 0.0008 for the interaction, all by 2-way ANOVA). Symbols: *, significant difference (Tukey's multiple comparisons between all cell means) within each MHV strain between the bafilomycin A treatment and the 0 nM bafilomycin A control; #, significant difference between JHM.SD and A59 at the indicated bafilomycin A concentration (Tukey's multiple comparisons between all cell means). Data shown are representative of 3 independent experiments with n = 5 technical replicates.

FIG 2

TMPRSS2 activity directly mediates bafilomycin A-independent MHV infection. (A) JHM.SD is more sensitive than A59 to TMPRSS2 transfection. HEK-293T cells cotransfected with pCAGGS-mCeacam1a-4L and pCAGGS-hTMPRSS2-FLAG were infected with the indicated virus. Using two-way ANOVA (n = 5), P values were <0.0001 for the effects of TMPRSS2 and the virus strain and their interaction. Asterisks indicate the TMPRSS2 transfection levels at which the 2 viruses were significantly different from each other by Tukey's multiple comparison. (B) Camostat abrogates the effect of TMPRSS2 on JHM.SD infection. Transfected HEK-293T cells were treated with DMSO or camostat (final DMSO concentration, 1.5%) prior to infection. Using two-way ANOVA, P values were <0.0001 for TMPRSS2 transfection, camostat treatment, and their interaction. Number signs indicate significant differences from the no-TMPRSS2 control within the DMSO group using Dunnett's multiple comparisons (no significant difference from the no-TMPRSS2 control at any level of TMPRSS2 transfection within the camostat group). (C) TMPRSS2 enhances JHM.SD infection in the presence of bafilomycin A. Transfected HEK-293T cells were treated with DMSO or bafilomycin A (final DMSO concentration, 0.5%) prior to infection with JHM.SD-fluc. Using two-way ANOVA, P values were <0.0001 for the effects of TMPRSS2 and bafilomycin A and their interaction. Symbols: #, significant difference from the no-TMPRSS2 control within the DMSO group; †, significant difference from the no-TMPRSS2 control within the bafilomycin A group (determined by Dunnett's multiple-comparison test). (D) TMPRSS2 overcomes bafilomycin A inhibition of A59 infection. Transfected HEK-293T cells were treated with DMSO or bafilomycin A (final DMSO concentration, 0.5%) prior to infection with rA59-fluc. Using two-way ANOVA, P values were <0.0001 for the effects of TMPRSS2 and bafilomycin A and their interaction. Symbols: #, the TMPRSS2 transfection levels at which A59 infection differed from the no-TMPRSS2 control within the DMSO group; †, the TMPRSS2 transfection levels at which A59 infection differed from the no-TMPRSS2 control within the bafilomycin A group (determined by Dunnett's multiple-comparison test). All data are representative of at least 2 independent experiments with n = 5 technical replicates.

FIG 3

TMPRSS2 overexpression decreases productive MHV infection. (A) TMPRSS2 decreases CEACAM1a protein levels. HEK-293T cells cotransfected with pCAGGS-mCeacam1a-4L and pCAGGS-hTMPRSS2-FLAG or pCAGGS-hTMPRSS2-S441A-FLAG were infected with rA59/SJHM.SD-EGFP and lysed for immunoblotting at 18 hpi. (B) TMPRSS2 decreases cell-associated MHV protein levels. HEK-293T cells cotransfected with pTK-mCeacam1a-4L and pCAGGS-hTMPRSS2-FLAG or pCAGGS-hTMPRSS2-S441A-FLAG were infected as indicated and lysed for immunoblotting 18 hpi. Goat polyclonal anti-S antibody AO4 was used to detect the S protein and mouse anti-N monoclonal antibody 1-16-1 to detect N protein. The vertical lines indicate boundaries between nonadjacent lanes (rA59/SJHM.SD-EGFP and rA59-EGFP were run on the same gel, but their positions were exchanged for consistency with other panels; rA59/SMHV-2-EGFP and the mock-infected cells were run in parallel on a separate gel). (C) TMPRSS2 cleavage of S may be nonproductive. Probing the lysates shown in panel C with anti-S2 MAb 5B19.2, previously mapped to the fusion peptide, detected an ∼150-kDa fragment (black box) inconsistent with S2′ cleavage. (D) TMPRSS2 decreases MHV titer. HEK-293T cells were cotransfected with pTK-mCeacam1a-4L and 200 ng of pCAGGS-hTMPRSS2-FLAG or pCAGGS-hTMPRSS2-S441A-FLAG and infected with the indicated viruses; cell supernatants were collected at 18 hpi and titers determined. Both active and inactive TMPRSS2 significantly decreased the MHV titer (using 2-way ANOVA with Dunnett's multiple comparisons of each TMPRSS2 level with the 0-ng control within each virus, P < 0.0001 for the effect of the virus, P < 0.0001 for the effect of TMPRSS2 transfection, and P < 0.0045 for the interaction; *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 for the multiple comparisons). Data are representative of 2 independent experiments performed in triplicate. (E) TMPRSS2 activity decreases syncytium size. HEK-293β5 cells were cotransfected with pTK-mCeacam1a-4L and pCAGGS-hTMPRSS2-FLAG or pCAGGS-hTMPRSS2-S441A-FLAG and infected as described for panels B to D, treated at 2 hpi with DMSO or camostat as indicated (final DMSO concentration of 0.1% for all treatments), and fixed for microscopy at 18 hpi.

FIG 4

Metalloprotease inhibitor batimastat reduces JHM.SD and A59 infection of L2 cells. L2 cells were treated with DMSO or the indicated inhibitors as described in Materials and Methods (1.5% DMSO final concentration) and infected with rJHM.SD-fluc (left) or rA59-fluc (right), and luciferase activity was measured at 8 hpi. For each treatment, the effect of pretreatment relative to DMSO alone was divided by the effect of posttreatment relative to DMSO alone to correct for postentry effects, and the results were analyzed using 2-way ANOVA with Dunnett's multiple comparisons tests, comparing each protease inhibitor alone or with bafilomycin A to DMSO alone or with bafilomycin A, respectively. Both bafilomycin A and protease inhibitor treatment had significant and interactive effects on both JHM.SD and A59 infection (P < 0.0001 for protease inhibition, bafilomycin A, and the interaction for both JHM.SD and A59). Asterisks indicate the level of significance of the results of Dunnett's multiple-comparison tests of simple effects within columns (protease inhibitor versus DMSO control within each bafilomycin A treatment group; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Data shown are representative of 3 independent experiments with n = 5 technical replicates.

FIG 5

Cell-penetrating and extracellular metalloprotease inhibitors are nontoxic and decrease JHM.SD infection in multiple cell lines. Cells were pretreated with batimastat or TAPI-1 and/or bafilomycin A (final DMSO concentration, 1.5% for all treatments in all cells), infected with JHM.SD-fluc (MOI of 0.5) (A) or mock infected (B), washed, and incubated for an additional 6 h at 37°C and 30 min at room temperature in the presence of inhibitor before cell viability (A) and viral luciferase activity (B) were assessed. Representative data from two independent experiments with n = 5 technical replicates are shown. (A) Both batimastat and TAPI-1 were essentially nontoxic under the tested conditions. (B) Both batimastat and TAPI-1 decreased infection in all cell types. Asterisks show the results of two-way ANOVA with Dunnett's multiple-comparison test of simple effects within columns (metalloprotease inhibitor versus DMSO) for L2 and 17Cl1 cells and Tukey's multiple-comparison test between all cell means (not all results are shown) for DBT cells (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (C) Tabular results of two-way ANOVA on the data from panel B.

FIG 6

Batimastat inhibits syncytium formation but not S1/S2 cleavage. (A) L2 cells were infected (MOI of 0.01) as indicated. Five hours postinfection, the medium was replaced with fresh medium containing the indicated inhibitor (final DMSO concentration of 1%). Fifteen hours postinfection, the cells were fixed and infection analyzed by bright-field and fluorescence microscopy. (B) L2 cells were infected (MOI of 0.1) as indicated. At 5 hpi, the medium was replaced with fresh medium containing the indicated inhibitor. At 16 hpi, the supernatant was removed and the cells lysed and subjected to immunoblotting with a polyclonal anti-S antibody (AO4). β-Tubulin was detected as a loading control.

FIG 7

TMPRSS2 is an alternative to metalloprotease for JHM.SD entry. (A) TMPRSS2 rescues blockade of JHM.SD entry by bafilomycin A, batimastat, or both. HEK-293T cells cotransfected with pTK-mCeacam1a-4L and pCAGGS-hTMPRSS2-FLAG were pretreated with batimastat and/or bafilomycin A (final DMSO concentration, 1.5%) and infected with JHM.SD-fluc (MOI, 0.05 PFU/cell), and the treatment was maintained until the luciferase activity was assayed at 7.5 hpi. Using two-way ANOVA (n = 5), P values were <0.0001 for drug treatment, TMPRSS2 level, and the interaction between them; *, transfection levels at which Dunnett's multiple-comparisons tests showed significant differences from the baseline for all treatments. Data are representative of two independent experiments with n = 5 technical replicates. (B) TMPRSS2 inefficiently rescues MHV cell-to-cell spread in the presence of batimastat. HEK-293β5 cells cotransfected with pTK-mCeacam1a-4L and pCAGGS-hTMPRSS2-FLAG or pCAGGS-hTMPRSS2-S441A-FLAG were infected with rA59/SJHM.SD-EGFP (MOI, 0.1); at 2 hpi, the medium was replaced with medium containing DMSO, batimastat, camostat, or both, as indicated (final DMSO concentration, 0.1% for all treatments), and syncytium formation was assessed at 18 h.

FIG 8

Effect of metalloprotease inhibition on MHV infection is S strain specific. L2 cells were treated with batimastat (50 μM) and/or bafilomycin A (100 nM) or DMSO as described in Materials and Methods (1.5% DMSO final concentration for all conditions) and infected with isogenic chimeric viruses bearing the S protein from JHM.SD, A59, or MHV-2 (MOI, 0.5). Two-way ANOVA showed significant effects of treatment, strain, and interaction (P < 0.0001 for all); the asterisks represent the results of Tukey's multiple-comparison tests between each pair of viruses under each condition (level of significance for at least 2 of the 3 comparisons; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Data are representative of 2 independent experiments with n = 5 technical replicates.

FIG 9

Independent and sequential cleavage models of MHV entry. (A) In the independent pathway model, JHM.SD is more efficiently cleaved by cell surface acid-independent proteases (vertical stripes) such as metalloprotease or TMPRSS2 and fuses at the plasma membrane, whereas A59 better survives endocytosis and/or is more efficiently cleaved by acid-dependent endosomal proteases (horizontal stripes). In the sequential cleavage model (B), cleavage by acid-independent proteases produces a metastable intermediate that is more readily cleaved by endosomal proteases, and JHM.SD S is more efficiently cleaved by acid-independent proteases but less efficiently cleaved by endosomal proteases.