% Inhibition of MERS pseudovirus infection. 0 h 0.5 h 1 h 2 h 4 h 6 h Time after virus addition

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Daniela Ramsey
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1 % Inhiition of MERS pseudovirus infection 1 8 h.5 h 1 h 2 h 4 h 6 h Time fter virus ddition Supplementry Figure S1. Inhiition of on MERS pseudovirus infection t the different intervls postinfection. A time-of-ddition experiment ws performed to test the inhiitory ctivity of peptide on MERS pseudovirus infection in Huh-7 cells t different intervls etween ddition of virus nd the peptide. The experiment ws performed in triplicte nd the dt re presented s men ± s.d. (error r). 1

2 % Cytotoxicity HR1P+Vero E6 HR1P+Huh-7 HR1P+293T Concentrtion ( M) % Cytotoxicity +Vero E6 +Huh T Concentrtion ( M) Supplementry Figure S2. Cytotoxicity of HR1P nd peptides. The 293T, Huh-7 nd Vero cells re used for testing the potentil toxic effect of HR1P () nd (), determined y XTT ssy. The experiment ws performed in triplicte. The dt re presented s men ± s.d. (error r) from single representtive experiment out of two repetitions. 2

3 [ ] (1-3 deg cm 2 dmol -1 ) HR1P HR1M HR1L Wvelength (nm) [ ] (1-3 deg cm 2 dmol -1 ) HR2L HR2S Wvelength (nm) Supplementry Figure S3. The secondry structures of different lengths of HR1 nd HR2 peptides. () HR1 peptides HR1L, HR1M, nd HR1P; () HR2 peptides HR2L, HR2M, nd HR2S. 3

4 1 % Inhiition of MERS-CoV S medited cell-cell fusion 8 HR1L HR1M HR1P % Inhiition of MERS-CoV S medited cell-cell fusion Peptide concentrtion ( M) HR2L HR2S Peptide concentrtion ( M) Supplementry Figure S4. Inhiitory ctivity of HR1 () nd HR2 () peptides on MERS-CoV S medited cell-cell fusion. Experiments were performed in triplicte nd the dt re expressed s mens ± s.d. (error r). The experiment ws repeted twice nd similr results were otined. 4

5 Supplementry Figure S5. Model of the MERS-CoV S protein S2 suunit-medited memrne fusion nd the mechnism of ction of the fusion inhiitory peptide. In the ntive stte, prt of the HR1 domin in the S2 suunit, possily in rndom coil conformtion, is shielded y the S1 suunit, nd HR2 forms the stem of the spikes with prtilly helicl structure. The entirety of S proteins on the virl surfce my tke n oligomeric form through self-ssocition. In the receptor inding stte, the S1 suunit inds with receptor DPP4 on the trget cell surfce. In the pre-hirpin stte, the S1 suunit my e dissocited from the S2 suunit, which undergoes series of conformtionl chnges. The fusion peptide (FP) t the N-terminus of the S2 suunit inserts into the trget cell memrne, resulting in exposure of the HR2-trimer nd HR1- trimer. HR1-trimer t this stge serves s trget of the peptides derived from the HR2 domin. In the fusion stte, the HR1 nd HR2 helices ssocite with ech other to form the 6-HB fusion core, drwing the virl nd cellulr memrnes into close proximity for fusion nd resulting in the formtion of fusion pore through which the virl genetic mterils re relesed into the trget cell., the peptide derived from the HR2 domin, cn ind to the HR1-trimer to form heterologous 6-HB nd lock virl fusion core formtion, resulting in inhiition of MERS-CoV fusion with the trget cell memrne. The 6-HB formtion cn occur on the cell plsm memrne or endosoml memrne when the virus enters into the cell through plsm memrne fusion or endocytosis pthwy, respectively. 5

6 SARS- CoV S2 MERS- CoV S2 Supplementry Figure S6. The emerging hydrogen ond on HR1 in MERS-CoV. Residues G928 nd D932 in HR1 domin in S2 suunit of SARS-CoV () correspond to Q1 nd D124 in HR1 domin in S2 suunit of MERS-CoV (), respectively. In the HR1 domin of MERS-CoV, the distnce of the intrmoleculr hydrogen ond etween Q1 nd D124 is 2.55Å. The corresponding residues in the HR1 domin of SARS- CoV S2 could not form such hydrogen ond. 6

7 SARS - CoV S2 MERS-CoV S2 c d Supplementry Figure S7. Enhnced interctions inside the HR2 region in MERS- CoV. In the HR2 domin of SARS-CoV S2, N1159 inds to the min chin of Q1161 with distnce of 3.3Å (). In the corresponding sites in the HR2 domin of MERS-CoV S2, D1261 inds to T1263 through their side chins with distnce of 2.76Å (). K1172 nd E1176 in the HR2 domin of SARS-CoV S2 show no interction (c), while the corresponding residues (K1174 nd E1178) in the HR2 domin of MERS-CoV S2 form hydrogen ond with distnce of 2.Å (d). 7

8 SARS - CoV S2 MERS-CoV S2 Supplementry Figure S8. Enhnced interction etween HR1 nd HR2 in MERS- CoV. Q917 in HR1 nd L1178 in HR2 of SARS-CoV S2 do not disply n interction ond (), wheres the corresponding residues Q19 in HR1 nd Y128 in HR2 of MERS-CoV S exhiit hydrogen ond etween HR1 nd HR2 with distnce of 2.7Å (). 8

9 Blnk HR1P HR1P/ Blnk Blnk HR1P HR1P/B Blnk Mrkers 6-HB 116kD 66.2kD 45kD 35kD 25kD 18.4kD 14.4kD Supplementry Figure S9. Imges of the full gels presented in the min pper. () The imge showing 6-HB formtion etween HR1P nd y N-PAGE in Fig. 4; () The imge showing the moleculr mss determined y gel electrophoresis in Fig. 4. 9

Supplementary Figure S1

Supplementary Figure S1 Supplementry Figure S Connexin4 TroponinI Merge Plsm memrne Met Intrcellulr Met Supplementry Figure S H9c rt crdiomyolsts cell line. () Immunofluorescence of crdic mrkers: Connexin4 (green) nd TroponinI