Abstract
It is near a half century since hepatitis B virus (HBV) was identified. HBV receptor molecules and the entry mechanism of HBV into hepatocytes have not been elucidated completely, though there are some reports on infection systems and on the receptor molecules. Thus, we still have not reached finding a real HBV receptor and there have been no useful and convenient infection system in vitro and in vivo for HBV, which makes it impossible for us to understand a precise HBV life cycle and HBV involved related diseases. An HBV infection system is really needed to explore ways and means of treatment of HBV related diseases based on evidence as well. Here, we designed and tried to generate an HBV pseudotype, which has a viral particle containing a retrovirus capsid and a genome inside surrounded by HBV membrane proteins. We proved successful generation of this pseudotype by immunoprecipitation with anti-HBVs antibodies and by CsCl density gradient ultracentrifugation, followed by RT-PCR targeting a retroviral gene, an EGFP gene in this case, respectively. Though our established system is constructed on growth dependent integration of retroviral genomes and thus was very hard to observe its infection in a primary human hepatocytes culture system, successful generation of the HBV pseudotype will make it possible for us to perform a biological assay to clone an HBV receptor based on infectivity and will facilitate its separation and identification
Highlights
It has been near half a century, since hepatitis B virus (HBV) was identified by Blumberg [1]
Establishment of HBV psuedotype packaging cells We utilized a murine leukemia virus (MLV) packaging system since considering in vitro use for biological cloning of an HBV receptor in cultured cell lines based on infectivity and for checking infectivity of HBV with ease afterwards, even though mature hepatocytes have a limited growth activity in vitro
Retroviral genomes are integrated into host genome after reverse-transcribed only in growing cells except those of lentiviruses, which were utilized in a previous report [24]
Summary
It has been near half a century, since hepatitis B virus (HBV) was identified by Blumberg [1]. Though treatment of chronic hepatitis B with interferons has been continued and nucleotide analogs, most of which were explored as anti-HIV agents, and prevention against HBV infection by vaccination has been developed, HBV infected patients yet reach three hundred and fifty million people worldwide [2]. HBV shows highly species specific spectrum for its infection and can infect only primates and amplify mainly in parenchymal hepatocytes, but never infects the other animals including mice [2,4]. Even human hepatocyte-originated hepatocellular carcinoma cell lines such as HepG2, HuH7, HuH6, Hep3B and so on never permit HBV infection, or extremely limited even if possible, though there are some reports about in vitro infection of HBV to HepG2 [5,6]
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