Hepatitis B virus surface antigen as a reporter of promoter activity

Marschall, Manfred; Motz, Manfred; Leser, Ulrike; Schwarzmann, Fritz; Oker, Barbara; Wolf, Hans

doi:10.1016/0378-1119(89)90341-7

Cited by 13 publications

(13 citation statements)

References 24 publications

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“…1, were stably introduced into cell culture: pEBV 57 was based on the artificial linkage of the EBV ori Lyt element, upstream to the HBsAg coding sequence. Transcriptional activity ofori Lyt was monitored by HBsAg reporter expression [22]. pEBV 59 encoded the EBV BZLF 1 transactivator (Zta) under constitutive control of the CMV immediate early promoter-enhancer.…”

Section: Resultsmentioning

confidence: 99%

“…The cells were cultured separately or in coculture at an initial cell density of 107 per 10ml medium. Transfections were done with 10gg of DNA per 10 7 cells according to the DEAE dextran protocol [22,24]. R59Z cell lysates were produced by sonication and were incubated in a 5 fold excess (lysate of 5 x 107 R59Z cells per 107 R7-57 cells).…”

Section: Resultsmentioning

confidence: 99%

“…The specific DNA sequences were introduced into the polylinker of p 220.2, using restriction sites Barn HI/Hin d III (pEBV 57) and Barn HI/Sal I (pEBV 59), respectively. Subcloning of the original viral sequences was described elsewhere: HBsAg coding sequence [22], BZLF 1 Lytic transition of Epstein-Barr virus imitated by recombinant B-cells 25 coding sequence [23] and CMV immediate early promoter-enhancer [4]. The ori Lyt regulatory element was subcloned as a 1060 bp fragment from the EBV Barn HI H clone [1,30] using Hin cII and Apa LI restriction sites.…”

Section: Construction Of Eukaryotic Vectorsmentioning

confidence: 99%

“…Raji cells (Burkitt's lymphoma) were transfected by the DEAE dextran technique [24-] with simple modifications [22]. Positive clones were selected for hygromycin B resistance, encoded by pEBV 57 and pEBV 59.…”

Section: Selection Of Recombinant Cellsmentioning

confidence: 99%

“…Radioimmunoassay was used to quantitate the activity of promoter elements, as described for the HBsAg (hepatitis B virus surface antigen) reporter system [22]. In brief, recombinant HBsAg, used as reporter protein, was secreted from transfected cells.…”

Section: Radioimmunoassay and Enzyme-linked Immunoassaymentioning

confidence: 99%

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The lytic transition of Epstein-Barr virus is imitated by recombinant B-cells

Marschall

Alliger

Schwarzmann

et al. 1993

Archives of Virology

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Summary. Lytic transition of Epstein-Barr virus (EBV) is initiated by distinctimmediate early regulators of the viral cycle, in synchronization to temporary, permissive conditions during host cell differentiation. We developed eukaryotic vectors suitable to imitate the processes involved in lyric transition in cell culture systems. Two stable B cell lines were established: R59Z activator cells were used to induce lytic EBV expression in a constitutive manner by the production of the BZLF 1 trans-activator (Zta). R7-57 reporter cells, on the other hand, signaled induced activity of the lyric origin of EBV replication (ori Lyt). Different modes, like chemical induction, lyric superinfection with EBV and single gene trans-activation converted the recombinant ori Lyt element in R7-57 reporter cells. BZLF 1, transiently expressed in R7-57 reporter cells, was the only EBV trans-activator found, sufficient in inducing the viral lytic cycle. Basing on these experiments, trans-cellular activation of EBV was tested by cocultivation of BZLF 1-expressing R59Z activator cells with the R7-57 reporter line. No lyric effect on the reporter cells could be measured, neither by cocultivation of activator cells nor by coincubation of BZLF 1-containing cell lysates. Latency breaking activity, however, was transferred from activator to reporter cells when active, exogenous virus was added. The cell system described in these experiments provides a tool for the detection of EBV reactivation and demonstrates the potential of the lytic regulatory gene BZLF 1.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Construction Of Eukaryotic Vectorsmentioning

confidence: 99%

Section: Selection Of Recombinant Cellsmentioning

confidence: 99%

Section: Radioimmunoassay and Enzyme-linked Immunoassaymentioning

confidence: 99%

See 3 more Smart Citations

The lytic transition of Epstein-Barr virus is imitated by recombinant B-cells

Marschall

Alliger

Schwarzmann

et al. 1993

Archives of Virology

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Replication of hepatitis B virus in differentiated adult rat hepatocytes transfected with cloned viral DNA

Diot

Gripon

Rissel

et al. 1992

Journal of Medical Virology

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The possibility of obtaining expression of human hepatitis B virus (HBV) genes and production of virus particles in normal liver cells from heterologous species like normal adult rat hepatocytes, by transfecting the complete HBV genome, was investigated. Various techniques for hepatocyte transfection were assayed including the usual calcium-phosphate coprecipitation technique, the Pasco and Fagan modified calcium-phosphate procedure, and the lipofection technique. Transfection efficiency was determined by measuring the production of HBV surface antigen under various culture conditions. Transfection was the most efficient when assayed 1 or 2 days after hepatocyte plating at low density. Few variations in the efficiency were observed between the different transfection procedures. We show that under these culture conditions, replication of HBV can be achieved in differentiated adult rat hepatocytes. Synthesis of relaxed circular and single-stranded DNA forms and of viral transcripts including pregenome RNA occurred in the cells whereas viral antigens and mature and immature viral particles were released into the culture medium. The production of viral proteins was always higher in hepatocytes cocultivated with rat liver epithelial cells and maintained at a low density. In contrast, viral replication was not obtained by transfecting undifferentiated rat liver epithelial cells. These results demonstrate that replication of HBV can occur in hepatocytes from mammalian species non-closely related to primates and strongly support the idea that attachment of the virus and its penetration into the cells are critical steps in the host-specificity of the infection process and that hepatic-specific regulating factors could be essential for viral replication.(ABSTRACT TRUNCATED AT 250 WORDS)

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