HBV Surface Antigen Expression Research Articles

Control of expression and methylation of a hepatitis B virus transgene by strain-specific modifiers.

In transgenic animals, genotype-specific modifiers exert a control over transgene methylation and expression that may or may not be position dependent. These factors belong to different classes, some of them possibly related to modifiers of position-effect variegation in Drosophila. The study of hepatitis B virus (HBV) gene expression in transgenic mice has revealed the existence of many factors influencing transcription, including hormones and tissue-specific transcription factors. We now report the effect of genotype-specific modifiers on HBV surface antigen (HBsAg) expression and transgene methylation. Compared with the C57BL/6 background, the DBA/2 and 129sv backgrounds cause enhancement of HBsAg expression, with little or not effect on transgene methylation or transcription. In contrast, a single cross with a BALB/c mouse is responsible for de novo methylation and silencing of the transgene in all offspring. Several modifiers appear to segregate in the progeny of a transgenic E36 male mouse crossed with (C57BL/6 x BALB/c) F1 females, with the emergence of a high-expressor group. Our observations suggest that different modifiers act cooperatively, at both the transcriptional and post-transcriptional levels, as part of a complex system regulating transgene expression. This transgenic model provides a system to genetically map new mouse strain-specific modifiers, some of them involved in epigenetic modification and transcription control.

Inhibition of hepatitis B virus by retroviral vectors expressing antisense RNA.

Two retroviral vectors carrying an antisense gene from the hepatitis B virus (HBV) preS/S or preC/C were constructed and used to infect the human hepatoblastoma cell line 2.2.15, which expresses HBV surface antigen (HBsAg), HBV e antigen (HBeAg) and releases HBV particles. The results showed that the inhibitory effects of antisense gene transfer, mediated by retroviral vectors on the expression of HBV antigens, appeared as early as day 3 after transduction, reached a maximum on day 5 and persisted for at least 11 days. Our data indicate that, on day 5 after introduction, antisense preS/S inhibited HBsAg and HBeAg expression by 71% and 23%, and the antisense preC/C inhibited HBsAg and HBeAg expression by 23% and 59%. HBV DNA production, in the supernatant of the 2.2.15 cells transduced with either antisense preS/S or preC/C, was also reduced on day 5, but the viability of the 2.2.15 cells was not affected. Our results demonstrate that the replication and expression of HBV can be inhibited through antisense gene transfer mediated by retroviral vectors and that the antisense-preC/C or antisense-preS/S may be potentially useful for clinical gene therapy against HBV.

Hepatitis B virus surface antigen as a reporter of promoter activity

The coding sequence for the hepatitis B virus surface antigen (HBsAg) was used as a new reporter gene for studies on eukaryotic promoter activity and upstream regulatory sequences. The data observed in transfection assays were comparable to results obtained with conventional chloramphenicol acetyltransferase (CAT) assays, as was demonstrated using various transcriptional regulation sequences. The expression of HBsAg as a reporter protein offered some advantages: 1. (i) In transient expression assays, a time course of promoter activity depending on variable culture conditions could be monitored over a period of time, since the HBsAg was secreted into the culture supernatant. 2. (ii) Evaluation of HBsAg from supernatant aliquots and quantification of the corresponding promoter activities could be performed easily, using the very sensitive and readily available diagnostic HBsAg kits. 3. (iii) In contrast to the conventional CAT assay, the cells remained available for further tests, e.g., Western blot, immunofluorescence or transcript analysis. Characteristics of several Epstein-Barr virus (EBV) promoters, depending on the virus state of EBV-positive B-cells (latency, chemical induction, lytic superinfection, trans-activation), were assayed using the HBsAg reporter system.

The hepatitis B virus enhancer modulates transcription of the hepatitis B virus surface antigen gene from an internal location.

We have used a transient expression system to examine the influence of the hepatitis B virus enhancer on the transcription of the hepatitis B virus surface antigen (HBsAg) gene. This enhancer is located 3' to HBsAg coding sequences but is contained within the mature HBsAg transcripts. Removal of the enhancer resulted in a significant reduction in HBsAg gene expression in liver cells. By using an in vitro nuclear run-on assay, this effect was determined to be at the level of transcription. We have further noted that HBsAg expression can be restored by the reinsertion of the enhancer sequences in the enhancerless vector. However, this reconstitution of HBsAg expression appears to depend on location of the enhancer with regard to the HBsAg promoters.

Expression of hepatitis B surface antigen in human cells by a recombinant BK virus DNA vector.

The construction of the first stable human cell lines that express and secrete authentic hepatitis B virus surface antigen (HBsAg), using a BK virus (BKV) episomal plasmid vector, is described. The amount of HBsAg produced by BKV vectors (up to 600 ng/10(7) cells) was comparable to other eukaryotic vector systems. The level of HBsAg expression remained the same regardless of the orientation of the HBsAg gene, substitution of the HBsAg gene promoter with the mouse metallothionein I gene promoter or the tissue origin of the human cell lines used to establish stable cellular transformants. Northern blot analysis also indicated synthesis of normal HBsAg transcripts. Surprisingly, however, the vectors were maintained at far lower than expected copy number (one to five copies/cell). Reasons for this are discussed.

Role in nude mice of interferon and natural killer cells in inhibiting the tumorigenicity of human hepatocellular carcinoma cells infected with hepatitis B virus

The human hepatoma cell line, PLC/PRF/5, which is persistently infected with hepatitis B virus (HBV), has integrated HBV-DNA, secretes HBV surface antigen (HBsAg), and does not grow readily in congenitally athymic (nu/nu) mice. The present investigation was undertaken to ascertain whether the low tumorigenicity of this cell line was governed by a host immune response and/or was related to expression of HBsAg. Subcutaneous injection of 4-5 X 10(6) cells into BALB/c nude mice produced localized encapsulated tumors with morphologic features of primary hepatocellular carcinoma in 25% of the animals within 29-40 d. No tumor growth was observed at lower cell inocula. In contrast, SK-HEP-1, an HBV-negative human hepatoma cell line, produced tumors at 1-5 X 10(6) cells inocula in 66% of the animals. Immunosuppression of mice with antilymphocyte serum (ALS) or irradiation increased tumor incidence in mice inoculated with 1 X 10(6) PLC/PRF/5 cells to almost 100% and produced local invasiveness. Immunosuppression also reduced the latency, i.e., time to tumor appearance, and increased mean tumor weight. These results suggest that tumorigenicity was limited by the host immune response. The nature of the response was delineated by treating nude mice challenged with tumor cells with sheep anti-mouse interferon globulin (anti-IFN). When 2 X 10(6) cells were injected, tumor growth occurred in 75% of anti-IFN-treated mice, whereas controls injected with the same number of cells, but not receiving anti-IFN, failed to develop tumors. The tumors in the anti-IFN-treated mice were highly invasive and the latency period until tumor appearance was reduced to 3-5 d. An inverse correlation was found between susceptibility of the hepatoma cells to natural killer (NK) activity in vitro and resistance to tumor growth in vivo. In vitro cytotoxicity for PLC/PRF/5 cells was eliminated by anti-NK 1.1 and complement, establishing the effector cell as an NK cell. NK cell activity 14 d after inoculation of mice with PLC/PRF/5 cells was augmented against PLC/PRF/5 target cells but not against SK-HEP-1 cells. Treatment of mice with ALS, irradiation, or anti-IFN abolished NK activity against PLC/PRF/5 cells. Co-cultivation of nude mouse spleen cells with PLC/PRF/5 but not with HBsAg or SK-HEP-1 cells induced secretion of murine IFNalpha. These results suggest that the IFN/NK cell system may play a role in limiting tumorigenicity and invasiveness of HBV-infected human hepatocellular carcinoma cells by a mechanism similar to that found for other cells persistently infected with viruses.

Plasmid-directed synthesis of hepatitis B surface antigen in monkey cells.

We introduced the gene encoding the hepatitis B virus surface antigen (HBsAg) into simian virus 40 (SV40)-based plasmids capable of autonomously replicating in both Escherichia coli and permissive monkey cells. After introduction into monkey cells by transfection, these plasmids directed the synthesis of high levels of HBsAg, as determined by immunofluorescence, radioimmunoassays, and identification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides comprising the antigen. Expression was dependent upon the presence of an SV40 promoter, with both the early and late promoters able to effectively initiate transcription. Using expression of HBsAg to assay promoter function, we demonstrated that an intact copy of the SV40 72-base pair repeat, which constitutes an essential element of the SV40 early promoter during the lytic SV40 cycle and which can enhance the transcriptional activity of heterologous promoters, was not required for HBsAg expression, suggesting that the hepatitis genome contains an enhancer element capable of complementing that provided by the 72-base pair repeat element of SV40. The antigen appears to be glycosylated after synthesis in transfected cells and is apparently secreted, as evidenced by the localization of [35S]cysteine-labeled antigen to the medium of transfected cultures. Using constructions in which the first ATG sequence appearing in HBsAg mRNA was that corresponding to the gene encoding the mature form of the antigen, we demonstrated that these post-translational events could occur without the involvement of a putative precursor peptide suggested by the DNA sequence of the viral genome. In view of the inability of hepatitis B virus to propagate in vitro, this strategy offers a convenient approach for further characterizing the biosynthesis of this antigen and may provide a means to identify additional polypeptides encoded by this virus.

Induction of hepatitis B surface antigen in human hepatoma-derived cell lines.

Cultures of two human hepatoma cell lines were examined for expression of hepatitis B virus surface antigen (HBsAg). The PLC/PRF/5 cells secreted HBsAg continuously into the culture medium, whereas Mahlavu cells did not secrete the antigen. However, cytoplasmic antigen was detected in a low percentage (less than 5%) of the Mahlavu cells. The expression of HBsAg also was assayed in cultures treated wtih dexamethasone (DXM), 5-iodo-2'-deoxyuridine (IdUrd), or both. The results demonstrated that: (i) DXM stimulated secretion of HBsAg by PLC/PRF/5 cells but not by Mahlavu cells; (ii) the percentage of Mahlavu cells expressing cytoplasmic HBsAg was not increased in any cultures if the medium was replaced at 24 t0 48 h intervals but was increased approx. fivefold within 4 days in cultures treated with DXM or IdUrd/DXM if the medium was not changed. However, no increase was noted in the intensity of the immunoperoxidase stain of PLC/PRF/5 cells that expressed cytoplasmic antigen in any DXM cultures; (iii) HBsAg expression was stimulated to a lesser extent in IdUrd/DXM cultures than in DXM cultures and was not enhanced in IdUrd cultures. Thus, DXM enhanced secretion of HBsAg by PLC/PRF/5 cells within 24 h and, after a delay, enhanced expression of cytoplasmic antigen by Mahlavu cells. However, antigen secretion by Mahlavu cells evidently was blocked.