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Expression of the Hepatitis B Virus Surface Antigen Gene in Cell Proc. Nati. Acad. Sci. USA Vol. 78, No. 4, pp. 2606-2610, April 1981 Microbiology Expression of the hepatitis B virus surface antigen gene in cell culture by using a simian virus 40 vector (recombinant DNA/animal virus/gene expression/antigen characterization) ANN M. MORIARTY*, BILL H. HOYER*, J. WAI-KUO SHIH*, JOHN L. GERIN*, AND DEAN H. HAMERtf *Division of Molecular Virology and Immunology, Georgetown University Medical Center, Rockville, Maryland 20852; and tDNA Recombinant Unit, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20205 Communicated by Robert M. Chanock, December 29, 1980 ABSTRACT We have constructed a simian virus 40 recom- rying a 1350-bp fragment ofHBV DNA that includes the struc- binant carrying a fragment of DNA from hepatitis B virus. Cul- tural sequences for surface antigen. We show that monkey kid- tured monkey kidney cells infected with this recombinant produce ney cells infected with this recombinant synthesize surface hepatitis B surface antigen. The antigen is excreted into the cul- antigen that is excreted into the culture medium as 22-nm par- ture medium as 22-nm particles with the same physical properties, ticles. These results set an upper limit on the amount of HBV antigenic composition, and constituent polypeptides as those found genetic information required for 22-nm particle formation and in the sera of patients with type B hepatitis. demonstrate the feasibility ofusing SV40 recombinants to study HBV gene expression in cultured primate cells. At least half of the world population shows evidence of past or present infection by hepatitis B virus (HBV), and the approxi- mately 200 million carriers in the world are at serious risk of MATERIALS AND METHODS chronic liver disease and, possibly, primary liver cancer. The The following methods have been described: general proce- classic marker for chronic infection by this virus is the surface dures for the construction ofrecombinant plasmids and viruses antigen HBsAg which circulates in the serum of HBV carriers (10); growth of African green monkey kidney cells and propa- in three forms: 22-nm spherical particles, 22-nm filaments of gation of virus stocks (11); preparation of plasmid (12) and in- various lengths, and the 42-nm spherical form known as the tracellular SV40 DNA (13); analysis of DNA by restriction en- Dane particle. The 22-nm particles and filaments are subviral donuclease cleavage (11) and agarose gel electrophoresis (14, forms containing two predominant polypeptides, with apparent 15); and transformation of EK2 Escherichia coli strain HB101 molecular weights of about 23,000 and 29,000, together with (16). Restriction endonucleases and T4 DNA ligase were pur- several minor polypeptides of larger size (1, 2). The two pre- chased from Bethesda Research Laboratories (Rockville, MD) dominant species, which are probably identical except that the and reaction conditions were according to the supplier. larger is glycosylated, carry both the group (a) and the subtype The source of HBV DNA was plasma, subtype adw, from an (d/y) antigenic determinants of HBsAg (3). The Dane particle, HBsAg-positive donor. Dane particles were purified by the which represents the infectious virion, consists ofa lipoprotein method ofRobinson (17) and incubated in the endogenous DNA coat (HBsAg) surrounding an internal core particle which con- polymerase reaction (18) with all four deoxynucleotide triphos- tains a DNA polymerase and the 3200-base pair (bp) circular phates prior to DNA extraction. DNA genome. The 22-nm particle is the predominant form in The 22-nm form of HBsAg was purified from the plasma of the sera of chronic carriers and circulates at concentrations as chronic carriers as described (19). Hyperimmune guinea pig high as 100-200 ,ug/ml. antiserum to HBsAg/ad (V801-502-058) was from Research Re- Characterization ofthe life cycle and biology ofHBV has been sources Branch (National Institute of Allergy and Infectious hampered by its narrow host range, which is restricted to hu- Diseases) and monospecific antibodies to the HBs/a and HBs/ mans and a few other primates, and by its inability to grow in d determinants were prepared from this serum by affinity chro- cultured cells. Recently, however, several groups have suc- matography (3). Fluorescein isothiocyanate-conjugated rabbit ceeded in cloning the viral genome in Escherichia coli phage anti-guinea pig IgG was obtained from Cappel Laboratories A (4) and plasmid vectors (5, 6) and in determining its primary (Cochranville, PA). Radioimmunoassays for hepatitis B core structure (7-9). This has allowed the identification of a contin- antigen (20), 8 antigen (21), and e antigen (HBeAg test kit, Ab- uous 892-bp sequence that could encode surface antigen (7), a bott Laboratories) have been described. HBsAg was detected 549-bp sequence that may specify the core antigen (8), and sev- by the Ausria II radioimmunoassay (Abbott Laboratories) and eral additional open sequences of unknown function (9). quantitated by a parallel-line assay with a known standard (BoB Although the DNA sequence provides crucial structural in- HBsAg/adw vaccine, reference lot 1, 40 jig/ml). The d/y sub- formation, it clearly is not sufficient to establish all of the HBV type of HBsAg was determined by the competition radioim- gene products or to indicate how these products interact during munoassay method of Hoofnagle (22). infection of the target cell. For this purpose it would be useful All experiments requiring physical containment level P3 to develop a system for introducing defined portions ofthe viral were performed at the certified P3 facility of the Georgetown genome into cultured cells. Simian virus 40 (SV40), a small DNA Division of Molecular and tumor virus that can lytically infect cultured monkey cells, pro- University Virology Immunology vides a useful vector for this In this we describe purpose. paper Abbreviations: SV40, simian virus 40; HBV, hepatitis B virus; HBsAg, the construction and propagation of a SV40 recombinant car- hepatitis B surface antigen; SVHBV, SV40-hepatitis B virus recombi- nant; SVHBV-HBsAg, hepatitis B surface antigen produced by cells The publication costs ofthis article were defrayed in part by page charge infected with SVHBV; bp, base pair(s). payment. This article must therefore be hereby marked "advertise- t Present address: Laboratory of Biochemistry, National Cancer Insti- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. tute, National Institutes of Health, Bethesda, MD 20205. 2606 Downloaded by guest on September 26, 2021 Microbiology: Moriarty et al. Proc. Natl. Acad. Sci. USA 78 (1981) 2607 (Rockville, MD) according to the National Institutes of Health recombinant DNA research guidelines. SV40 RESULTS Construction and Propagation of the SV40-HBV Recombi- Step B Inant. The SV40-HBV recombinant described here carries a 1350-bp fragment of HBV DNA, representing about 40% ofthe HBV genome, inserted into the late gene region of SV40. The first step in the construction of this recombinant was to amplify the HBV genome by cloning it in an E. coli plasmid vector (Fig. 'BR322-SV40 1). Dane particles were purified from the serum of a chronic HBsAg carrier, subtype adw, and the partially single-stranded viral genome was repaired by an endogenous DNA polymerase reaction. Two fragments, 1350 and 1850 bp, were obtained after cleavage of this DNA with BamHI. Partial digestion with Step C BamHI generated a full HBV genome which was ligated to BamHI-cleaved plasmid pBR322 DNA and cloned in E. coli. From the published sequence data (7-9) we anticipated that the HBsAg coding sequence would be located within the 1350- bp BamHI fragment. This fragment was purified by electro- phoresis, subcloned in pBR322, isolated, ligated to a BamHI- cleaved pBR322-SV40 vector plasmid, and recloned in E. coli. Hae II digestion of the resultant pBR322-SV40-HBV "double recombinant" plasmid which retained only the 1350-bp HBV fragment removed all but 143 bp ofthe pBR322 DNA and yielded ahomogenous preparation of4950-bp SV40-HBV linear recombinant molecules. These molecules retained the SV40 origin of DNA replication and the complete SV40 early gene region but lacked most of the SV40 late gene region and hence were defective. Nevertheless, they could be packaged into SV40 coats and propagated as virions by making a mixed DNA infection ofmonkey kidney cells with an SV40 temperature-sen- sitive early gene mutant (SV40tsA239) as helper. This mixed in- fection was performed at the nonpermissive temperature (39°C) to ensure that progeny virions would be produced only by cells Cells doubly infected with the SV40-HBV recombinant, which sup- plies functional SV40 early gene products, and with the helper, Bam HI 5200 34,00 1350 Lanes supplies all ofthe required SV40 late gene products (11, 25, 26). SlS~~1 To determine if the SV40-HBV recombinant was encapsi- 1 dated into SV40 virions, we took advantage ofthe fact that only a}~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. 2 those genomes incorporated into viral particles during the orig- inal DNA infection will be transferred and replicated in a sub- + 3 sequent viral infection (27). Accordingly, we infected a fresh culture of monkey cells with the virus stock from the DNA in- fection, prepared intracellular viral DNA 3 days later, and ex- 5 amined it by restriction endonuclease cleavage and agarose gel t 5200l 4750 electrophoresis (Fig. 1). This showed that the stock contained approximately
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