Max-Planck-Institut für Biochemie1 and 3Am Klopferspitz 18a and D-82152 Martinsried, Germany
Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Science, Shanghai, China2
Author for correspondence: Ludwig Weiss. Fax +49 89 7092 9559. e-mail Ludwig-Weiss{at}t-online.de
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Abstract |
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The remarkable liver tropism of HBV can be attributed to at least two mechanisms: (i) the preferential attachment and entry of the virus into the host cell via an as yet unknown hepatocyte-specific receptor(s), and (ii) liver-specific virus regulatory elements in the HBV genome. One such regulatory element is the HBV enhancer II/core promoter region (EII/Cp), which is located immediately upstream from the core ORF and regulates the transcription of a set of viral RNAs of about 3·5 kb in length. These 3·5 kb RNAs encode not only the nucleocapsid proteins and the viral polymerase, but the shortest form of this 3·5 kb set of RNAs also serves as the pregenomic RNA, which is encapsidated and reverse transcribed during HBV replication and, thus, represents the template for HBV DNA synthesis (Summers & Mason, 1982 ; Will et al., 1987
). Furthermore, another 3·5 kb RNA is translated to generate HBeAg. Although its biological function remains largely uncertain, HBeAg is an important diagnostic marker: clinical studies have shown that the level of HBeAg correlates with viral DNA replication and virus propagation (Hollinger, 1996
).
Transcription of the 3·5 kb RNAs occurs in well-differentiated hepatocytes but not in either dedifferentiated hepatocytes or cells of nonliver origin. This liver-specific regulation of the EII/Cp region is contributed to by the binding of several liver-specific transcription factors within this regulatory region (Lopez-Cabrera et al., 1990 , 1991
; Yuh & Ting, 1991
; Guo et al., 1993
; Zhang et al., 1993
; Li et al., 1995
; Raney et al., 1995
). HBV EII has been mapped to a 148 bp DNA fragment ranging from nt 1497 to 1644 (HBV subtype adr4) (Wang et al., 1990
), which is located upstream from and partly overlaps Cp. Because of different binding regions for trans-acting factors, EII was initially divided into two fragments, A (nt 14971556) and B (nt 15571644), with almost 70% of EII activity retained by fragment B. Therefore, fragment B was thought to be the major functional unit of EII and, upon further characterization, was subdivided into three elements, B1 (nt 15571575), B2 (nt 15761606) and B3 (nt 16071644) (based on HBV subtype adr1; Wu et al., 1992
).
Since its identification, many efforts have been made to investigate the interaction between EII and liver-enriched (Lopez-Cabrera et al., 1991 ; Yuh & Ting, 1991
; Guo et al., 1993
; Li et al., 1995
; Raney et al., 1995
) as well as ubiquitous transcription factors (Zhang et al., 1993
). In our previous report (Li et al., 1998
), a novel liver-specific transcription activator, which is an orphan nuclear receptor named B1F, was identified. In transfection assays with nonhepatic cells, i.e. HeLa cells, B1F could significantly stimulate EII/Cp activity, which was barely detectable without B1F stimulation. B1F has been shown to bind specifically to the B1 element of EII. In order to gain further insight into how this interaction may contribute to virus biological functions at the molecular level, mutation analysis of the B1F-binding site was performed and its influence on virus transcription in HepG2 cells was investigated.
To test whether the B1F-binding site within the B1 element is important for the activity of HBV EII, four point mutations were introduced into the B1 element by PCR-based site-directed mutagenesis. Firstly, bandshift assays were performed as described previously (Li et al., 1998 ) to verify that the mutated fragment was no longer capable of binding B1F. As shown in Fig. 1(a
), fragment B1m4 (AACTACAGATCTCGAGGCA, lane 2), in contrast to the wild-type B1F-binding site (lane 1), was no longer able to bind B1F. Next, the functional significance of such a mutation was investigated in CAT assays. For these CAT assays, two each of wild-type and B1m4 CAT reporter plasmids were constructed (Fig. 1b
). These reporter plasmids contain the HBV EII region in one of two locations, either upstream or downstream from an HBV CpCAT gene construct. In general, there was a significant decline in the activity of EII if the wild-type B1 sequence was replaced by B1m4 (Fig. 1c
). Interestingly, there was an approximately 15-fold decline in CAT activity when reporter plasmids pENII_B1CpCAT and pENII_B1m4CpCAT were compared, whereas the reduction in CAT expression was only about threefold if EII was located downstream from the CAT gene. These data indicated that the stimulatory effect of B1F on the transcription of the CAT gene is much more significant if EII is located upstream from Cp, which is the natural arrangement in the viral genome. Therefore, the location of EII upstream from Cp is an important positional factor for B1F to fully exert its stimulatory effect. It is very likely that transcription from Cp as well as its tissue specificity is carefully co-ordinated through factors binding to several sites in EII. The position of EII and Cp relative to each other should greatly influence the transcription of pregenomic RNAs. Since HBV EII partly overlaps Cp, it is quite difficult to clearly separate one from the other either functionally or physically. In the case of the B1F-binding site, its location-dependent activity may also reflect its role as an upstream regulatory element for Cp, rather than that of a classical enhancer element. Taken together, these observations indicate that, especially in the natural order of the EII/Cp region, the interaction between B1F and its target binding sequence is critical for the activity of the HBV EII/Cp element.
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In conclusion, binding of B1F to its target sequence within the B1 element of the HBV EII region is crucial for the activity of EII and also plays an important role in liver-specific transcription initiated from HBV Cp with respect to virus antigen expression (especially HBeAg) and pregenomic RNA generation.
In the past few years, several liver-specific and liver-enriched factors such as HNF1, HNF3, HNF4 and C/EBP, as well as ubiquitous factors such as SP1, have been shown to bind to the EII region (Lopez-Cabrera et al., 1991 ; Yuh & Ting, 1991
; Guo et al., 1993
; Zhang et al., 1993
; Li et al., 1995
; Raney et al., 1995
). The action of a combination of transcription factors binding to the EII/Cp region and other regulatory regions of the HBV genome permits modulation of HBV transcription in such a way that all of the virus transcripts are synthesized in a balanced ratio, as required for normal HBV propagation. The data presented in this report indicate that B1F plays a critical role in this interplay of different transcription regulators. Undoubtedly, the investigation of such an interplay among various transcription factors is not only critical for a detailed understanding of EII function, but is also of great importance for understanding the cell-specific transcription machinery (Raney et al., 1997
).
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References |
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Received 24 August 2000;
accepted 14 November 2000.