Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells

Abstract

Recent evidence indicates there is a role for small membrane vesicles, including exosomes, as vehicles for intercellular communication. Exosomes secreted by most cell types can mediate transfer of proteins, mRNAs, and microRNAs, but their role in the transmission of infectious agents is less established. Recent studies have shown that hepatocyte-derived exosomes containing hepatitis C virus (HCV) RNA can activate innate immune cells, but the role of exosomes in the transmission of HCV between hepatocytes remains unknown. In this study, we investigated whether exosomes transfer HCV in the presence of neutralizing antibodies. Purified exosomes isolated from HCV-infected human hepatoma Huh7.5.1 cells were shown to contain full-length viral RNA, viral protein, and particles, as determined by RT-PCR, mass spectrometry, and transmission electron microscopy. Exosomes from HCV-infected cells were capable of transmitting infection to naive human hepatoma Huh7.5.1 cells and establishing a productive infection. Even with subgenomic replicons, lacking structural viral proteins, exosome-mediated transmission of HCV RNA was observed. Treatment with patient-derived IgGs showed a variable degree of neutralization of exosome-mediated infection compared with free virus. In conclusion, this study showed that hepatic exosomes can transmit productive HCV infection in vitro and are partially resistant to antibody neutralization. This discovery sheds light on neutralizing antibodies resistant to HCV transmission by exosomes as a potential immune evasion mechanism.

Fig. 1.

Purified exosomes of HCV-infected Huh7.5.1 cells harbor viral protein and RNA. (A) Mass spectrometric analysis of Huh7.5.1 and Huh7.5.1 Jc1-derived exosomes contain exosome-related proteins and HCV core (n = 3). (B) Electron microscopic images of purified exosomes isolated from infected Huh7.5.1 cells (a), using uranyl acetate staining. Immunogold labeling of HCV E2 envelope protein shows the presence of viral protein in exosomes derived from infected Huh7.5.1 cells (b). Immunogold and uranyl acetate staining could not be combined. Shown is one representative experiment of three.

Fig. 2.

Infected exosomes transmit infection to naive Huh7.5.1 cells and cause productive infection. (A) Schematic representation of infection experiments. (B) Immunocytochemical staining of HCV core protein Huh7.5.1 cells infected with infectious virus or exosomes (n = 4). (C) Quantitative RT-PCR analysis of HCV RNA after primary infection and (D) secondary infection. The levels are normalized HCV RNA multiplied by 1,000. Shown are the mean ± SEM of four independent experiments in triplicates.

Fig. 3.

Exosome-mediated transmission of HCV in the presence of primary neutralizing Igs (IgGs) varies between patients. (A) Huh7.5.1 cells were infected with Luc-Jc1 particles or purified exosomes from Luc-Jc1–infected cells in the presence of pooled patient-derived anti–HCV-specific IgGs from ten patients with HCV. Shown are the mean ± SEM of four (control IgG) or five (HCV IgGs) independent experiments in duplicates. (B) Infectious Jc1 particles or exosomes were incubated with Huh7.5.1 cells in the presence of patient-derived anti–HCV-specific IgGs (n = 8; Table S1) or control IgGs pooled from three healthy controls. Shown are the mean ± SEM of four independent experiments in duplicates.

Fig. 4.

Transfer of HCV RNA via exosomes independent of structural proteins. (A) Naive Huh7.5.1 cells were incubated with Jc1 containing medium or with exosomes isolated from HCV subgenomic replicon cells (Huh7-ET) or exosomes isolated from Jc1-infected Huh7.5.1 cells. Naive cells exposed to Huh7-ET exosomes have a clearly detectable amount of HCV RNA, but at a lower level than exosomes from Jc1-infected cells. Shown are the mean ± SEM of three independent experiments in triplicates. (B) Huh7-ET–derived exosomes were incubated with Huh7.5.1 cells in the presence of patient-derived anti–HCV-specific IgGs (n = 8; Table S1) or control IgG from healthy controls. No neutralization was observed; rather, enhanced exosome-mediate transmission was seen with some IgGs. Shown is the mean of triplicates ± SEM of one representative experiment of three. (C) Naive Huh7.5.1 cells were blocked with anti-CD81, anti-SRBI, and anti–Caudin-1 antibody and infected with Huh7-ET–derived exosomes. Some inhibition of HCV RNA transfer was observed for all three entry receptors compared with control antibody, but it did not reach statistical significance. Shown is the mean ± SEM of three independent experiments in duplicates.