The interaction between HCV glycoproteins and CD81 is blocked when CD81 interacts with partner protein(s).

<p>A, HCV E1E2 heterodimers immobilized onto anti-E2 coated beads interacted with CD81 in all cells lysed in Triton X-100 (TX). However, the interaction of HCV-E1E2 with CD81 was blocked in Daudi and Ramos cells lysed in Brij (Bj). Maintenance of CD81 with other tetraspanins (T) and partners (P) is disrupted by the indicated lysis conditions, as diagrammed. Anti-CD81 and irrelevant (Cont) mAbs were used in immunoprecipitations as controls. Precipitation of CD81 was revealed by western blotting with the anti-CD81 5A6 mAb. CD81-LEL corresponds to the large extracellular loop of CD81 fused to the glutathione-S transferase. B, After cell surface biotinylation, the indicated cell lines were lysed with Bj/EDTA, immunoprecipitated with 5A6 mAb and the proteins revealed by Western blotting with HRP-conjugated streptavidin. The values on the right are molecular sizes in kilodaltons. C, EWI-2wint production is directly connected to EWI-2 expression. Daudi cells interfered with negative siRNA or EWI-2 siRNA were biotinylated, lysed in Bj/EDTA, immunoprecipitated with the anti-CD81 5A6 mAb or an anti-EWI-2 mAb (8A12) and blotted sequentially with HRP-conjugated streptavidin and 5A6 mAb.