During the revision of the manuscript, another study showing that HCV exploits the dynamic properties of CD81 for its entry has been published (Harris et al., 2012), which is in accordance with our conclusions.
EWI-2wint promotes CD81 clustering that abrogates Hepatitis C Virus entry
Version of Record online: 16 FEB 2013
© 2013 John Wiley & Sons Ltd
Volume 15, Issue 7, pages 1234–1252, July 2013
How to Cite
Potel, J., Rassam, P., Montpellier, C., Kaestner, L., Werkmeister, E., Tews, B. A., Couturier, C., Popescu, C.-I., Baumert, T. F., Rubinstein, E., Dubuisson, J., Milhiet, P.-E. and Cocquerel, L. (2013), EWI-2wint promotes CD81 clustering that abrogates Hepatitis C Virus entry. Cellular Microbiology, 15: 1234–1252. doi: 10.1111/cmi.12112
- Issue online: 13 JUN 2013
- Version of Record online: 16 FEB 2013
- Accepted manuscript online: 25 JAN 2013 11:45AM EST
- Manuscript Accepted: 10 JAN 2013
- Manuscript Revised: 14 DEC 2012
- Manuscript Received: 25 JUL 2012
- ‘Agence Nationale de Recherches sur le Sida et les hépatites virales’ ANRS
- European Social Fund. Grant Number: POSDRU/89/1.5/S/60746
Fig. S1. Cell surface distribution of CD81 and control proteins. Immunofluorescence images showing the basal membrane of living Huh-7 clones expressing EWI-2wint 1, LAL or Qcc. Experiments were performed at 37°C using TIRF microscopy. Cells were stained with Atto647N-labelled Fab fragments of TS81, SYB-1 and 11C5 mAbs to observe CD81, CD9 and CD46 respectively.
Fig. S2. EWI-2wint has no effect on CD46 dynamics. The distribution of all the apparent diffusion coefficients (ADC) was calculated for CD46 in Huh-7 clones expressing EWI-2, EWI-2wint (two clones), LAL or Qcc. Each dot represents one trajectory and 500 trajectories are represented for each cell clone. CD46 molecules were stained usingAtto647N-labelled Fab fragments of 11C5 mAb.
Fig. S3. EWI-2/EWI-2wint expression in CHO cells. CHO cells were stably transfected with hCD81 and either the empty vector (pcDNA3.1), the uncleavable EWI-2 (EWI-2, see Experimental procedures) or wild-type EWI-2 construct (EWI-2wint). It has to be noted that the cleavage of EWI-2 generating EWI-2wint naturally occurs in CHO cells. Cell surface biotinylation followed by immunoprecipitation assays were performed to control the expression of proteins. FLAG-tagged EWI-2 constructs and CD81 were immunoprecipitated with M2 and 5A6 mAbs respectively. Proteins were revealed by Western blotting with HRP-conjugated streptavidin. Asterisks indicate additional cleavage products of EWI-2wint, as previously described (Rocha-Perugini et al., 2008). Triangles indicate an unidentified partner of CD81, which is not related to EWI-2/EWI-2wint proteins.
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