CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells

Abstract

Hepatitis C virus (HCV) infects cells by the direct uptake of cell-free virus following virus engagement with specific cell receptors such as CD81. Recent data have shown that HCV is also capable of direct cell-to-cell transmission, although the role of CD81 in this process is disputed. Here, we generated cell culture infectious strain JFH1 HCV (HCVcc) genomes carrying an alanine substitution of E2 residues W529 or D535 that are critical for binding to CD81 and infectivity. Co-cultivation of these cells with naïve cells expressing enhanced green fluorescent protein (EGFP) resulted in a small number of cells co-expressing both EGFP and HCV NS5A, showing that the HCVcc mutants are capable of cell-to-cell spread. In contrast, no cell-to-cell transmission from JFH1ΔE1E2-transfected cells occurred, indicating that the HCV glycoproteins are essential for this process. The frequency of cell-to-cell transmission of JFH1W529A was unaffected by the presence of neutralizing antibodies that inhibit E2–CD81 interactions. By using cell lines that expressed little or no CD81 and that were refractive to infection with cell-free virus, we showed that the occurrence of viral cell-to-cell transmission is not influenced by the levels of CD81 on either donor or recipient cells. Thus, our results show that CD81 plays no role in the cell-to-cell spread of HCVcc and that this mode of transmission is shielded from neutralizing antibodies. These data suggest that therapeutic interventions targeting the entry of cell-free HCV may not be sufficient in controlling an ongoing chronic infection, but need to be complemented by additional strategies aimed at disrupting direct cell-to-cell viral transmission.

Impact of Intra- and Interspecies Variation of Occludin on Its Function as Coreceptor for Authentic Hepatitis C Virus Particles

“…Whether the mechanisms of cell entry during cell-to-cell spread are distinct from those during cell entry by an extracellular virion is unclear. Most notably, there is conflicting data on whether cell-to-cell spread is CD81 independent (22,38,41) or not (7). OCLN knockdown has been reported to reduce cell-to-cell transmission by 2-to 3-fold in an assay in which spread, despite the presence of neutralizing antibodies in the supernatant, was defined as occurring cell to cell (7).…”

“…Short hairpin RNA's targeting occludin was constructed in the context of the shRNA expression vector pLenti-3Ј-U6-EC-EP7, a blasticidin selectable lentiviral genome vector expressing the shRNA of interest from an internal U6 promoter (41). The target specific sequence used for the creation of an OCLN low cell line was 5Ј-GGGCATTGCTCATCCTGAAGA-3Ј, which is contained in the 5Ј-untranslated region of all known occludin transcripts in humans.…”

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Huh-7 Lunet, Huh-7.5 or Huh-7.5.1 cell lines) or genetically modified to encode a reporter system allowing easy infectivity assay or to permit receptor complementation studies. Importantly, the tools are now available to study individually the 4 main HCV receptors (CD81, SR-BI, Cldn1 and Ocln): cell lines expressing very low levels of one of the receptors and retroviral vectors to rescue the expression of this receptor or one of its homologs have been developed in several laboratories [44,101,102,104,106,116,165] (see Table 1). Thanks to HCVpp, it is also possible to extend these receptor-complementation studies in cell lines that poorly replicate HCV, since the readout of HCVpp infectivity is independent on HCV replication [113,116,119].…”

“…Although the quantified contribution of this cell-to-cell transmission and its modalities are still disputed, it can be evidenced in several systems. Usually, cell-to-cell transmission is evaluated between two co-cultured cell populations, one population of producer cells infected beforehand and one population of target cells that can be easily tracked due to a chemical [186], or geneticallyencoded fluorescent marker [101,102]. Infected events in the second populations are considered as indicator of cell-to-cell transmission if the cell-free infectivity is blocked.…”

“…Infected events in the second populations are considered as indicator of cell-to-cell transmission if the cell-free infectivity is blocked. This last condition can be 13 achieved by using saturating concentrations of neutralizing antibodies [171,186,187], particular virus mutants or target cells [101], or by culturing the cells under an agarose overlay that prevents Brownian motion of cell-free viruses [102,171]. The dual cell-based reporter system developed by Jones et al…”