title: Spinoculation Enhances HBV Infection in NTCP-Reconstituted Hepatocytes.
abstract: Hepatitis B virus (HBV) infection and its sequelae remain a major public health burden, but both HBV basic research and the development of antiviral therapeutics have been hindered by the lack of an efficient in vitro infection system. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the HBV receptor. We herein report that we established a NTCP-complemented HepG2 cell line (HepG2-NTCP12) that supports HBV infection, albeit at a low infectivity level following the reported infection procedures. In our attempts to optimize the infection conditions, we found that the centrifugation of HepG2-NTCP12 cells during HBV inoculation (termed "spinoculation") significantly enhanced the virus infectivity. Moreover, the infection level gradually increased with accelerated speed of spinoculation up to 1,000g tested. However, the enhancement of HBV infection was not significantly dependent upon the duration of centrifugation. Furthermore, covalently closed circular (ccc) DNA was detected in infected cells under optimized infection condition by conventional Southern blot, suggesting a successful establishment of HBV infection after spinoculation. Finally, the parental HepG2 cells remained uninfected under HBV spinoculation, and HBV entry inhibitors targeting NTCP blocked HBV infection when cells were spinoculated, suggesting the authentic virus entry mechanism is unaltered under centrifugal inoculation. Our data suggest that spinoculation could serve as a standard protocol for enhancing the efficiency of HBV infection in vitro.
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana, 46202, United States of America.
authors.forename: Ran
authors.initials: R
authors.lastname: Yan
authors.affiliation: Department of Infectious Diseases, Huashan Hospital, Fudan University, 12 Wulumuqi Zhong Rd, Shanghai, 200040, China.
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana, 46202, United States of America.
authors.forename: Dawei
authors.initials: D
authors.lastname: Cai
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana, 46202, United States of America.
authors.forename: Yuanjie
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Baruch S. Blumberg Institute, Hepatitis B Foundation, 3805 Old Easton Rd, Doylestown, Pennsylvania, 18902, United States of America.
authors.forename: Andrea
authors.initials: A
authors.lastname: Cuconati
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana, 46202, United States of America.
authors.forename: Haitao
authors.initials: H
authors.lastname: Guo
mesh_terms: D015870:Gene Expression; D016679:Genome, Viral; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000502:physiology*; D022781:Hepatocytes / Q000378:metabolism* / Q000821:virology*; D006801:Humans; D066298:In Vitro Techniques; D029363:Organic Anion Transporters, Sodium-Dependent / Q000235:genetics*; D027981:Symporters / Q000235:genetics*; D014771:Virion; D053586:Virus Internalization
chemical_list: D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; C071908:sodium-bile acid cotransporter