Production of hepatitis B virus in vitro by transient expression of cloned HBV DNA in a hepatoma cell line.

Abstract

Transfection of human hepatoma cell lines with cloned HBV DNA resulted in the secretion of large amounts of hepatitis B surface antigen (HBsAg) and core‐related antigens (HBc/HBeAg) if well‐differentiated cell lines were employed. Synthesis of both viral antigens was the highest in cell line HuH‐7 and continued for approximately 25 days. Particles resembling hepatitis B virions (Dane particles) by morphology, density and by the presence of the preS1 surface antigen were released from the transfected HuH‐7 cells into the culture medium. These particles produced in vitro were also indistinguishable from the naturally occurring hepatitis B virions in containing the virus‐associated DNA polymerase and mature HBV genomes. Restriction analysis of these DNA molecules was compatible with the nucleotide sequence of the transfecting HBV DNA sequence. Viral surface antigens and core proteins present in the culture medium were fractionated and characterized by immunoprecipitation and SDS‐‐PAGE after labeling with [35S]methionine. Antisera specific for X‐gene products identified in cell extracts two hitherto unknown HBV gene products. This system thus provides a new approach to open questions regarding HBV‐related gene function and HBV replication.

Melanoma Differentiation–Associated Gene 5 Senses Hepatitis B Virus and Activates Innate Immune Signaling To Suppress Virus Replication

“…Moreover, it becomes difficult to perform in vivo study of the immune response to HBV infection because HBV fails to infect mice, the main animal model widely used for studying immune response to pathogens because of the availability of reagents and the similarity of immune response to humans. Transient transfection of the HBV replicative plasmid into human hepatoma cell lines has been widely used for in vitro studies of HBV replication and of HBV interaction with host (23)(24)(25), to overcome the obstacles, whereas hydrodynamic injection to deliver the HBV replicative plasmid into mouse livers has been developed for the study of the host response to HBV infection (26)(27)(28)(29)(30). For in vitro studies, we transfected Huh7 cells with the HBV replicative plasmid and the expression plasmid for MDA5 or RIG-I and found that MDA5, but not RIG-I at a similar protein level, significantly activated downstream signaling to inhibit HBV replication.…”

Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein.

“…Briefly, 2n5i10' hepatocarcinomaderived Huh7 cells (Nakabayashi et al, 1982) were transfected with 20 µg dHBV DNA, pHBc or HBV DNA, for transient experiments, using the calcium phosphate precipitation method, as described previously (Chang et al, 1987). In order to obtain stable clones, 20 µg of either dHBV DNA or HBV DNA was co-transfected with 5 µg of a plasmid expressing the neomycin-resistance gene under the control of the SV40 early promoter.…”

Characterization of the minimal elements of the hepatitis B virus large surface antigen promoter

“…The liver cell type specificity of the large surface antigen promoter appears to be dependent upon the HNF1 transcription factor, which may contribute to the hepatotropism of the virus as the large surface antigen is an essential component of the envelope of the virus particle (Ueda et al, 1991;Bruss & Ganem, 1991). Consistent with this possibility, virus particles have been produced in transfection experiments only in highly differentiated hepatoma cell lines (Sureau et al, 1986;Tsurimoto et al, 1987;Sells et al, 1987;Chang et al, 1987;Yaginuma et al, 1987), which express the HNF1 polypeptide necessary to activate the large surface antigen promoter. Analysis of clustered point mutations in the minimal large surface antigen promoter indicates that the HNF1 and TBP binding sites are the major regulatory elements of this promoter.…”