Abstract
Hepatitis B virus (HBV) transcriptional regulation has been extensively studied in vitro by transient transfection using the activity of a reporter molecule as the readout for transcriptional activity (1–5). Several reporter molecules, such as chloramphenicol acetyl transferase (CAT), β-galactosidase β-gal), and luciferase (LUC), have been used for this purpose. Because of the sensitivity and ease of the assay system, LUC has become the reporter molecule most widely used. Alternatively, the levels of HBV transcripts themselves can also be measured in cell-culture systems, and, in recent years, several groups have analyzed HBV transcription in this manner (6,7). With the introduction of HBV transgenic mice, in vivo analyses of HBV transcription have allowed the application of our in vitro knowledge to the more physiologically relevant and complex environment of the liver (8). Using transcription factor knock-out mice crossed with the HBV transgenic mice, or administering compounds to the HBV transgenic mice, the effects on transcription in the context of viral replication in the liver can be assessed (9,10). This chapter will cover transcriptional analysis in in vitro systems, but the methods used for the analysis of RNA will be applicable to the in vivo system. The 3.2-kb HBV DNA genome encodes four transcripts of 3.5, 2.4, 2.1, and 0.7 kb, from which are translated the HBcore/HBe and polymerase polypeptides, the large surfac antigen, the middle and major surface-antigen polypeptides, and the X-gene polypeptide, respectively. The small HBV genome lends itself to the molecular manipulation necessary to study the regulation of synthesis of the viral transcripts. Transcriptional analysis in cell culture has shown that the levels of the 3.5-, 2.4-, 2.1-, and 0.7-kb transcripts are regulated by the nucleocapsid, large surface antigen, major surface antigen, and X-gene promoters, respectively, as well as by the two viral enhancers (4,11). In the absence of an infectious cell-culture system, the majority of the in vitro transcriptional analysis has been performed by transient transfection of plasmids containing portions of the viral DNA genome into cell lines.
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Raney, A.K. (2004). Transcriptional Control of Hepatitis B Virus. In: Hamatake, R.K., Lau, J.Y.N. (eds) Hepatitis B and D Protocols. Methods in Molecular Medicine, vol 95. Humana Press. https://doi.org/10.1385/1-59259-669-X:303
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DOI: https://doi.org/10.1385/1-59259-669-X:303
Publisher Name: Humana Press
Print ISBN: 978-1-58829-105-9
Online ISBN: 978-1-59259-669-0
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