Viral “producer” cells containing replicating HCV Jc1 (Pi) are cocultured with green fluorescent protein (GFP)-expressing “target” cells (T) in the presence of E2-neutralizing mAb (AP33, 25 μg/mL) to prevent cell-free HCV transmission.24 AP33 reduces cell-free transmission by >90%, and infectivity of producer cell supernatants is minimal at the time of coculture; viral transmission thus occurs predominantly via cell-to-cell transmission in this Acalabrutinib nmr assay.2, 24 HCV cell-to-cell transmission is assessed by quantifying HCV-infected, GFP-positive target cells (Ti) by flow cytometry.2, 24 Both anti–SR-BI mAbs (10 μg/mL) efficiently blocked HCV cell-to-cell transmission (Fig. 3A

and Supporting Fig. 2A,B), indicating that these antibodies may prevent viral spread in vitro. Because these anti–SR-BI mAbs do not block HCV–SR-BI binding (Fig. 2A) but inhibit HCV entry during postbinding this website steps (Fig. 2C), these data suggest that an SR-BI postbinding function plays an important role during HCV cell-to-cell transmission. To ascertain the importance of the SR-BI postbinding function

in this process, we performed additional cell-to-cell transmission assays using mSR-BI, which in contrast to hSR-BI is unable to bind E2. Cells lacking SR-BI and robustly replicating HCV, which would be an ideal model MCE cell to study cell-to-cell transmission by mSR-BI in the absence of hSR-BI, have not been described. However, hSR-BI has been reported to be a limiting factor for HCV spread in Huh7-derived cells, as overexpression of hSR-BI increases cell-to-cell transmission.37 We

thus used Huh7.5 cells or Huh7.5 cells overexpressing either mSR-BI or hSR-BI as target cells. Cell-to-cell transmission was enhanced in Huh7.5 cells overexpressing either hSR-BI (2.04 ± 0.03 fold) or mSR-BI (1.92 ± 0.19 fold) compared with parental cells (Fig. 3B). These data indicate that E2–SR-BI binding is not essential for viral dissemination and confirm the crucial role of SR-BI postbinding function in this process. Furthermore, to assess whether anti–SR-BI mAbs prevent viral dissemination in already HCV-infected cell cultures when added postinfection, we performed a long-term analysis of HCVcc infection by culturing Luc-Jc1–infected Huh7.5.1 cells in the presence or absence of control or anti-SR-BI mAbs QQ-4G9-A6 and NK-8H5-E3 as previously described.2 When added 48 hours after infection and maintained in cell culture medium throughout the experiment, these anti–SR-BI mAbs efficiently inhibited HCV spread over 2 weeks in a dose-dependent manner without affecting cell viability (Fig. 3C,D and Supporting Fig. 2C,D). We also assessed Jc1 spread in Huh7.5.1 cells via immunostaining of infected cells as described.2 While 74.