1 Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, PR China
2 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, PR China
Correspondence
Yu-Mei Wen
ymwen{at}shmu.edu.cn
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ABSTRACT |
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INTRODUCTION |
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In a previous study with two naturally occurring HBV isolates, we showed that a mutation at residue 306 of RT that changed proline to serine (rtP306S) could change the replicative competency of an HBV isolate with a low level of replication (#2-18) to one with a high level (Lin et al., 2001b). Because rt306 is located close to the C terminus of RT and does not overlap with other open reading frames of HBV, the change in replicative competency of the mutant was not related to changes in other genes and thus was relatively simple to analyse. Based on homologous modelling studies of HBV RT, we suggested that rt306 was located at the connecting loop or hinge region between two
-helices in the thumb subdomain that interact with the DNA template-primer. In this case, substitution of rtP306S was likely to affect the precise conformation of the two
-helices and their interactions with the DNA template-primer and, consequently, impair the polymerase activity. To analyse the impact of the amino acid residue at rt306 further, here we constructed 11 HBV mutants in which the proline residue at rt306 (rtP306) was substituted with different amino acids using site-directed mutagenesis. The replicative competencies of these mutants were studied and analysed.
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METHODS |
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Cell culture, transfection and extraction of HBV DNA from intracellular particles.
HepG2 cells were cultured, and recombinant plasmids used for transfection were extracted and purified using the Qiagen Maxiprep kits as reported previously (Lin et al., 2001b). Wild-type and mutated HBV DNA was released from the recombinant plasmids by digestion with SapI [1 U (µg DNA)1] for 18 h, followed by extraction and purification. HBV DNA (25 µg) from each clone was used to transfect HepG2 cells in 60 mm plates using the calcium phosphate precipitation method as reported previously (Lin et al., 2001a
). Duplicate plates were used for all samples and 10 µg reporter plasmid DNA expressing secreted alkaline phosphate was cotransfected into each cell culture as an internal control to normalize the transfection efficiency between plates. Cells were collected at 72 h after transfection and vector pUC18 DNA was used as the mock transfection control. Transfection experiments were done three times on separate days. Wild-type recombinant DNA (p56) was used as the reference when transfection experiments were carried out on different days and the final results of the mutated recombinant plasmids used for transfection were compared with the reference result and normalized.
Cells were washed twice with chilled PBS and lysed with 600 µl lysis buffer (10 mM Tris/HCl, pH 8·0, 1 mM EDTA, 1 % NP-40, 8 % sucrose) as reported by Günther et al. (1995) with some modifications. After centrifugation at 12 000 g for 2 min, clear supernatants were collected and 6 µl 1 M magnesium acetate, 12 µl DNase I (5 mg ml1) and 3 µl RNase A (20 mg ml1) were added, followed by incubation at 37 °C for 30 min to digest the remaining DNA and RNA. After centrifugation at 12 000 g for 1 min, the supernatants were collected, 16 µl 0·5 M EDTA and 130 µl 35 % PEG 8000 in 1·75 M NaCl were added and the mixture was kept on ice for 1 h to precipitate the core particles. After spinning at 9000 g for 5 min, the pellets were resuspended and again digested with DNase I [0·1 µg (µg DNA)1] and incubated at 37 °C for 10 min to ensure full digestion of the remaining DNA used for transfection. The resuspended core particles were then digested with proteinase K and nucleic acids were extracted and precipitated. At this stage, DNA was further digested with HpaII. Full-length HBV DNA (#56 or the various mutants) released from plasmid originating from E. coli is digested by HpaII into seven fragments at nt 509, 1572, 2035, 2332, 3006 and 3042, respectively, while due to DNA methylation at sequences of CCGG, viral DNA derived from cells is resistant to this enzyme (Reyna-Lopez et al., 1997
). A pair of primers flanking one of the six restriction sites of HpaII was used for PCR (Fig. 1
) to ensure that the amplified DNA product was derived from the viral core particles within the transfected cells and not from remaining contaminating plasmid HBV DNA used for transfection.
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HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) assay.
Supernatants were collected 72 h after cell transfection and were assayed for the HBsAg and HBeAg using diagnostic ELISA kits (Kehua Co.).
Cloning of the enhancer I fragments and CAT assay.
Five different enhancer I fragments spanning nt 9571318 (Bock et al., 2000), according to the nucleotide sequence of wild-type HBV, were amplified separately by PCR for the wild-type HBV (rtP306) and four constructed polymerase mutants with mutations at rt306 (rtP306G, rtP306S, rtP306D and rtP306E). PCR products were cloned at the restriction sites KpnI and BglII of reporter vector pCAT3-promoter (Promega). The primers used were: E1, 5'-GGGGTACCCTTCCTGTTAACAGGCCTAT-3' (starting at nt 957), and E2, 5'-GAAGATCTTTTCCGCGAGAGGACGACAGA-3' (terminating at nt 1318).
Huh-7 cells were cultured in Dulbecco's modified Eagle's minimal essential medium supplemented with 10 % fetal calf serum. Two micrograms of the enhancer I constructs was used to transfect cells using Lipofectamine 2000 (Invitrogen) with 1·0 µg vector pCMV (Promega) expressing
-galactosidase, used as the internal standard. Cells were harvested and lysed 48 h after transfection and, after normalization by comparison with intracellular
-galactosidase, the CAT activity of the lysates was assayed using a CAT ELISA kit following the manufacturer's instructions (Roche). Experiments were carried out three times and data are shown as means±SD.
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RESULTS |
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Transcriptional activities of the enhancer I mutant
As shown in Fig. 3, compared with the empty vector, all cloned enhancer I fragments showed transcriptional function. More importantly, there was no difference in the level of transcriptional activities of enhancer I fragments with mutations corresponding to the substituted residues 306 of RT, irrespective of whether these mutants showed enhanced (rtP306D and rtP306E) or decreased (rtP306S and rtP306G) replication efficiency.
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DISCUSSION |
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Apart from substitutions of the amino acid at rt306, the remaining nucleotide sequences of the full-length HBV genome were identical in all mutants. The higher level of HBsAg detected by the two mutants with enhanced replication competency (glutamic acid and leucine) and lower levels of HBsAg detected with the mutants with reduced replication competency further supported the conclusion that changes of the amino acid at rt306 modulated HBV replication. It should be noted that the codon corresponding to rt306 overlaps the enhancer I region (nt 10471049) of the HBV genome (Bock et al., 2000). Thus, one could speculate that the varying replication efficiencies of the HBV mutants were due to alterations in the activities of the enhancer I mutants. To study the possible effects of mutated enhancer I at codon rt306, we cloned five different enhancer I fragments, representing wild-type and mutants with both enhanced replicative efficiency (rtP306D and rtP306E) and decreased replication efficiency (rtP306S and rtP306G). The results indicated that the mutation at codon 306 did not affect the transcriptional activities of enhancer I (Fig. 2
); thus, the effect of mutated sequences of HBV enhancer I on HBV replication could be excluded.
Although the low replication competency of rtP306G could be explained by the simple structure of glycine, rendering higher flexibility at this hinge region and resulting in impaired binding of the -helices with the DNA template-primer, this mechanism is difficult to correlate with the reduced replication competency of the rtP306T mutant. Therefore, the impact on the structure of substitutions of rtP306 appears to be relatively complex and cannot be explained solely by the current structural model of HBV RT, which contains only the catalytic domain of HBV RT. Because rtP306 is located at the interface between the thumb and the palm subdomain and the RNase H domain is located at the C terminus of the thumb subdomain, a change of rtP306 to other amino acids could alter the interactions of the thumb subdomain with the palm subdomain and/or the RNase H domain via either hydrophilic or hydrophobic interactions of the side chains of the amino acids. It could be assumed that enhanced replication of the rtP306E mutant is due to interaction of its hydrophilic side chain with the side chains of other amino acids located in RNase H. A change in these interactions could potentially interfere with interactions between the enzyme and the DNA template-primer and consequently affect the enzymic activity of HBV polymerase. Alternatively, the residue at the thumbpalm interface may play an additional role(s) in virus replication, such as involvement in modulating the conformational changes of the polymerase during the various steps of the replication process. Recently, Fisher et al. (2003)
reported the biochemical basis of how HIV RT copies both RNA and DNA templates. They found that mutations proximal to the minor groove binding track in the thumb region of HIV RT (N265D or N255D) led to loss of processing polymerization on viral DNA or both RNA and DNA templates, and the mechanism was described as being due to either a loss of template-strand-specific hydrogen bonding or a local change in conformation (Das et al., 2001
). However, detailed three-dimensional structural information about HBV RT is not yet available. In addition, the HepG2 cell transfection system (Sells et al., 1984
) used in this study reflects only part of the virus replication cycle (i.e. DNA synthesis); therefore, an appropriate cell-culture system and genome-based structural and functional analysis of RT from more HBV isolates are necessary to elucidate the regulatory elements of this enzyme.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 3 September 2004;
accepted 6 September 2004.
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