Transmission of HBV DNA Mediated by Ceramide-Triggered Extracellular Vesicles

Takahiro Sanada; Yuichi Hirata; Yutaka Naito; Naoki Yamamoto; Yoshiaki Kikkawa; Yuji Ishida; Chihiro Yamasaki; Chise Tateno; Takahiro Ochiya; Michinori Kohara

doi:10.1016/j.jcmgh.2016.10.003

Transmission of HBV DNA Mediated by Ceramide-Triggered Extracellular Vesicles

Cell Mol Gastroenterol Hepatol. 2016 Oct 24;3(2):272-283. doi: 10.1016/j.jcmgh.2016.10.003. eCollection 2017 Mar.

Authors

Affiliations

¹ Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.
² Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan.
³ Mammalian Genetics Project, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.
⁴ PhoenixBio Co, Ltd, Higashi-Hiroshima, Hiroshima, Japan.

Abstract

Background & aims: An extracellular vesicle (EV) is a nanovesicle that shuttles proteins, nucleic acids, and lipids, thereby influencing cell behavior. A recent crop of reports have shown that EVs are involved in infectious biology, influencing host immunity and playing a role in the viral life cycle. In the present work, we investigated the EV-mediated transmission of hepatitis B virus (HBV) infection.

Methods: We investigated the EV-mediated transmission of HBV infection by using a HBV infectious culture system that uses primary human hepatocytes derived from humanized chimeric mice (PXB-cells). Purified EVs were isolated by ultracentrifugation. To analyze the EVs and virions, we used stimulated emission depletion microscopy.

Results: Purified EVs from HBV-infected PXB-cells were shown to contain HBV DNA and to be capable of transmitting HBV DNA to naive PXB-cells. These HBV-DNA-transmitting EVs were shown to be generated through a ceramide-triggered EV production pathway. Furthermore, we showed that these HBV-DNA-transmitting EVs were resistant to antibody neutralization; stimulated emission depletion microscopy showed that EVs lacked hepatitis B surface antigen, the target of neutralizing antibodies.

Conclusions: These findings suggest that EVs harbor a DNA cargo capable of transmitting viral DNA into hepatocytes during HBV infection, representing an additional antibody-neutralization-resistant route of HBV infection.

Keywords: BSA, bovine serum albumin; ESCRT, endosomal sorting complexes required for transport; EV, extracellular vesicle; Extracellular Vesicles; GEq, genome equivalent; HA, hemagglutinin; HBIG, hepatitis B immune globulin; HBV; HBV, hepatitis B virus; HBc, hepatitis B core; HBcAg, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; MVB, multivesicular body; PBS, phosphate-buffered saline; PXB-cells, primary human hepatocytes derived from chimeric mice with human liver; STED, stimulated emission depletion; Transmission Pathway; anti-HBs, antibody to hepatitis B surface antigen; mRNA, messenger RNA; nSMase, neutral sphingomyelinase; nts, nucleotides; qPCR, quantitative real-time polymerase chain reaction.