HCV E2 interacts with NS4B.

<p>A) 293T cells were co-transfected with indicated flag-tagged HCV protein DNA constructs and a HCV E1 construct. Input cell lysates and immunoprecipitates were blotted with antibodies against HCV E1and flag. Only NS2 robustly interacted with HCV E1. B) Lysates of 293T cells transfected with indicated flag-tagged HCV constructs together with an E2 expressing plasmid were immunoprecipitated with anti-Flag affinity resin. It was observed that p7, NS2, NS4A, and NS4B were able to individually precipitate E2. C) Mapping the domains of NS4B that interact with E2. Topology of HCV NS4B and mutants (modified from ref [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147991#pone.0147991.ref036" target="_blank">36</a>]). D) All NS4B deletion mutants except NS4B-TM robustly precipitated with E2. E) NS4B failed to precipitate soluble E2 (sE2). 293T cells were co-transfected with indicated plasmids expressing flag-NS4B or flag-NS5A or CD81 LEL fused with human Fc (hFc) and soluble E2. CD81-LEL efficiently pulled down sE2, whereas NS4B and NS5A failed to do so. The sE2 (aa 384–661 from H77 clone) lacks the entire TMD and is commonly used in binding studies as E2 substitute [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147991#pone.0147991.ref037" target="_blank">37</a>]. The CD81-LEL contains the large extracellular loop that interacts with sE2 in direct binding assays [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147991#pone.0147991.ref038" target="_blank">38</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147991#pone.0147991.ref039" target="_blank">39</a>].