Timpe, J. M., Z. Stamataki, A. Jennings, K. Hu, M. J. Farquhar, H. J. Harris, A. Schwarz, I. Desombere, G. L. Roels, P. Balfe, and J. A. McKeating. Hepatology 47:17-24.2008.
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In this study we demonstrated that HCV can transmit to naïve cells in the presence of antibodies (Abs) or agarose that limit or neutralize cell-free virus infectivity. Intracellular sources of virus remain sensitive to the neutralizing activity of Abs, confirming that the transmitting viruses are not resistant to the neutralizing Abs used. The frequency of infected naïve target cells in the infectious middle assay was minimally affected by inhibiting extracellular routes of virus transmission. These data suggest that HCV can transmit by at least two routes in vitro, cell-free virus infection of naïve targets and direct transfer between cells. The latter route offers a mechanism to evade nAbs that may partially explain the ineffectiveness of antibodies to control HCV replication during the chronic phase of disease in addition to more the more familiar genetic escape mechanisms. If such routes of transmission occur in vivo, one may question whether therapeutic vaccination or immunoprophylaxis will be an effective control of persistent HCV replication.
Hepatitis C virus (HCV) infection of Huh-7.5 hepatoma cells results in focal areas of infection where transmission is potentiated by cell-cell contact. To define route(s) of transmission HCV was allowed to infect hepatoma cells in the presence or absence of antibodies that neutralize cell-free virus infectivity. Neutralizing antibodies (nAbs) reduced cell-free virus infectivity by >95% and had minimal effect(s) on the frequency of infected cells in the culture. To assess whether cell-cell transfer of viral infectivity occurs, HCV infected cells were co-cultured with fluorescently labeled naïve cells in the presence or absence of nAbs. Enumeration by flow cytometry demonstrated cell-cell transfer of infectivity in the presence or absence of nAbs and immunoglobulins from HCV+ patients. The host cell molecules CD81 and the tight junction protein Claudin 1 (CLDN1) are critical factors defining HCV entry. Soluble CD81 and anti-CD81 abrogated cell-free infection of Huh-7.5 and partially inhibited cell-cell transfer of infection. CD81 negative HepG2 hepatoma cells were resistant to cell-free virus infection but became infected after co-culture with JFH infected cells in the presence of nAb, confirming that CD81 independent routes of cell-cell transmission exist. Further experiments with 293T and 293T-CLDN1 targets suggest that cell-cell transmission is dependent upon CLDN1 expression. In conclusion, these data suggest that HCV can transmit in vitro by at least two routes, cell-free virus infection and direct transfer between cells, with the latter offering a novel route to evade nAbs.
HCV JFH transmits cell to cell. JFH infected Huh-7.5 producers were incubated for 15 minutes with (A) DMEM, (B) DMEM + 10µg/ml DEN3 (an irrelevant antibody), (C) DMEM + 10µg/ml C1 (anti-HCV antibody), or (D) DMEM + 100µg/ml P70 (HCV-neutralizing antibodies prepared from an infected patient). The producers were cultured with naive CMFDA-labeled Huh-7.5 targets (green). After 24h, infected cells were detected by staining for NS5A (Alexa 594, red). Newly infected target cells, positive for both NS5A and CMFDA, appear orange. The scale bar shown is 50 µm. Infectivity of cell free virus in the presence of C1 or P70 was reduced by >95% compared to DEN3 or untreated cultures.
Quantification of cell-cell transmission. JFH infected Huh-7.5 producers were co-cultured with CMFDA ("Cell Tracker") labeled targets at a ratio of 1:4 in the presence or absence of Abs (upper image). After 24h, the cells were fixed and stained for NS5A (R-PE, red) and infected cells quantified by flow cytometry. In the FACS plot the lower quadrants (left = unlabelled, right = CMFDA+, green) contain uninfected cells; the upper left quadrant (red) represents infected producers and the upper right (green + red = orange) newly infected targets.