The purified virus particles from LEE011 fraction 3 were then subjected to western blot for CD59 detection. Two CD59 blockers, BRIC229 and rILYd4, were used in the current study to abrogate CD59 function. BRIC229, a mouse antihuman CD59 monoclonal Ab (mAb) (IBGRL, Bristol, UK), is a widely used CD59-blocking Ab, whereas rILYd4 is a recombinant form of the fourth domain of intermedilysin (ILY), and has been identified as a high-affinity inhibitor of human CD59.6 Microplates were coated with rabbit antihuman CD59 polyclonal Abs (pAbs, Santa Cruz, Santa Cruz,

CA) or control IgG at 1 μg/mL in phosphate-buffered saline (PBS) overnight at 4°C. After washing and blocking, plates were used for CD59 measurement or HCV capture. To measure CD59 levels, the wells were incubated with Triton

X-100-pretreated supernatant from JFH-1-infected or uninfected Huh7.5.1 cells (100 μL/well) at 37°C for 1 hour. After incubation and washing, the bound CD59 was detected Selleck PS 341 by incubating with BRIC229 or control IgG, followed by incubating with HRP-labeled secondary Ab. Cell-free supernatant samples containing HIV-1 particles from two HIV-1-infected human monocytic cell lines, THP-1 (CD59-positive) and U1 (CD59-negative), were used as positive and negative controls, respectively, as HIV-1 particles derived from CD59-expressing THP-1 cells contain CD59, whereas HIV-1 particles derived from CD59-deficient U1 cells are CD59-negative.6 Cell-free supernatant from Ad5 (adenovirus serotype 5)-infected MCE公司 Huh7.5.1 cells were also included in the ELISA detection to rule out the possibility of cell-derived CD59. Ad5, a nonenveloped (naked) DNA virus, causes cytolytic infection and has no Env for CD59 incorporation. Therefore, any CD59 detected in these supernatant samples would be derived from dead cells and cell debris due to Ad5 cytolytic infection rather than Ad5 virions. To detect HCV capture, the wells were incubated with untreated cell-free supernatant from JFH-1-infected, uninfected Huh7.5.1 cells (100 μL/well), or purified virus fraction 3 at 37°C for 1 hour.

After incubation, free virus was removed by washing with PBS, and the remaining bound HCV was lysed with 200 μL of TRIzol (Invitrogen) for isolation of viral RNA, which was subjected to qPCR for measuring HCV RNA copy numbers as described in our Supporting Material. Cell-free supernatant samples from HIV-1-infected and uninfected THP-1 cells were used as positive and negative controls, respectively, as antihuman CD59 Abs have been reported to efficiently capture intact HIV-1 virions.5 PHHs and Huh7.5.1 cells treated or untreated with PI-PLC were lysed in 1× cell lysis buffer (Cell Signaling Technology, Danvers, MA), and then subjected to protein extraction and western blot as described in our Supporting Material. HCV particles purified from cell-free supernatant of JFH-1-infected Huh7.5.