The natural product rocaglamide A (Roc-A) has been shown to directly bind to PHB1 and 2 and blocks PHB-CRaf-MEK-ERK signaling (Polier et al., 2012). at 4?C. The cleared lysates were used for immunoprecipitation using a 1:1 mixture of Streptavidin beads (Pierce). Beads were washed three times with RIPA buffer, and bound Norgestrel proteins were eluted by boiling the samples in SDS-PAGE sample buffer and then resolved on 9% SDS-PAGE. Biotinylated proteins were detected by anti-PHB1 and anti-PHB2 antibodies. 2.4. Cytotoxicity/Cell Viability Assay PHHs (105 per well) were treated with Roc-A or DMSO at various concentrations for 48?h in 48-well plates. The numbers of viable cells in culture were determined using the CellTiter-Glo Cell Viability Luminescent Assay kit according to the manufacturer’s instruction (Promega). 2.5. Statistical Analysis Bar graphs were plotted to show mean??standard deviation (SD) of at least two independent experiments. Statistical analyses were performed using Graphpad Prism 5. A p value of Norgestrel (+)-Roc-A were synthesized using the same protocol followed by amide formation (Gerard et al., 2006). 3.?Results 3.1. PHB1 and 2 Interact with HCV E2 We have previously conducted a comparative proteomics analysis of the HCV-infected human hepatoma cell line Huh7.5.1 in order to identify HCV E2-interacting proteins. PHB1 and 2 were found to be the most abundant proteins in the E2 complex as detected by mass spectrometry. To validate the result, we performed immunoprecipitation using lysates from cells infected with the Flag-E2 JFH1 virus and confirmed that PHB1 and 2 co-precipitated with HCV E2 (Fig. S1A). The PHB-E2 association does not require the presence of other viral components Norgestrel as demonstrated in co-immunoprecipitation (Co-IP) studies (Fig. S1B). 3.2. PHB1 and 2 are Required for HCV Entry PHB1 is a ubiquitously expressed protein displaying antiproliferative activity (McClung et al., 1989). PHB2, also named repressor of estrogen receptor activity (REA), suppresses estrogen receptor (ER)-dependent gene activation (Montano et al., 1999). Interestingly, PHB has been implicated in the entry process of dengue and chikungunya virus (CHIKV) and also binds to HIV-1 glycoprotein and envelope proteins of white spot syndrome virus (Lan et al., 2013, Wintachai et al., 2012, Kuadkitkan et al., 2010, Emerson et al., 2010). To explore the role of PHB in modulating HCV infection, we transfected Huh7.5.1 cells with siRNA targeting PHB1 and PHB2, respectively. Reduction of endogenous PHB1 or 2 significantly Rabbit Polyclonal to CYB5 inhibited cell culture grown HCV (HCVcc) as measured by either luciferase assays or real-time PCR quantification of viral RNA (Fig. 1A and B). By contrast, PHB knockdown had no effect at viral RNA levels if the infection took place first (Fig. S1C), suggesting that PHBs are required at an early stage of HCV infection. Notably, PHB1 and PHB2 knockdown also decreased the protein levels of each other (Fig. S1D). Open in a separate window Fig. 1 Endogenous PHB1 and PHB2 are required for HCV infection. (ACB) Endogenous PHB1 and 2 were knocked down by siRNA transfection.