DISCUSSION

In this study, polyclonal antibodies against each domains of HBV DNA polymerase were generated. Immunofluoresence assay demonstrated that TP-specific antibody is useful to detect HBV DNA polymerase expression in cells although antibodies against spacer, RT, or RNase H could not detect HBV DNA polymerase in cells. Previous reports suggested that the difficulty of generating antibody against HBV DNA polymerase may be due to the impurities of polymerase antigens during the purification procedure because, instead of the polymerase antigen, contaminants were turned out to be more immunogenic than polymerase antigen that used for immunizations (zu Putlitz et al., 1999). Also several attempts to obtain HBV DNA polymerase antibodies from animals had been unsuccessful. It might be speculated that those animals had been immunized fewer times for short periods of time. The specificity of polyclonal antiserum against TP domain may be enhanced by many boost immunization for long periods of time. The failure to generate polyclonal antibody against spacer, RT or RNase H domain might be due to a rapid degradation of polymerase fragment from its C terminus, which had been demonstrated by Rehermann et al. Also, TP specific antibody can detect polymerase-surface (PS) fusion protein, the protein product of HBV RNA splicing (demonstrated in Part II).

It had been difficult to detect HBV DNA polymerase in HBV replicating cells.

Since HBV pgRNA has unusual bicistronic structure, HBV DNA polymerase is synthesized by inefficient internal initiation of translation. Also, it had been known

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that HBV DNA polymerase was degraded rapidly. Recent reports have shown that DNA polymerase of DHBV and HBV are accumulated at a detectable level in the cytoplasm (Yao et al., 2000; Cao and Tavis, 2004). Putlits et al (zu Putlitz et al., 1999) reported that HBV polymerase was predominantly localized in the cytoplasm of transfected cells regardless of the presence of other HBV proteins. Tavis groups (Cao and Tavis, 2004) demonstrated that non-encapsidated duck hepatitis B virus DNA polymerase are bound to undefined cytoplasmic structure but not to endoplasmic reticulum (Yao et al., 2000; Cao and Tavis, 2004). In this study, HBV DNA polymerase is distributed as granular patterns in the cytoplasm without evident co-localization with ER, Golgi apparatus and peroxisome (Fig. 6). These results were consistent with previously reported data which shown with DHBV and HBV. Also the detection sensitivity and the signal intensity of HBV DNA polymerase were greatly reduced in HBV replicating HuH7 cells (Fig. 6) (Cao and Tavis, 2004). These results led us to speculate that HBV DNA polymerase, possibly together with other HBV proteins, may have additional roles in pathogenesis besides for the replication of virus.

In addition to the hardship of the detection system establishment, the rapid degradation of HBV DNA polymerase makes it difficult to analyze the function or the nature of it. The known functional motif for viral replication of HBV DNA polymerase such as GLY motif for DNA priming and YMDD motif for DNA polymerase activity are also present in sequence of DHBV DNA polymerase, but previous reports demonstrated that HBV DNA polymerase cannot be replaced by

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DHBV DNA polymerase structurally and enzymatically. In this study chimeric DNA polymerase has been employed to elucidate which domains of HBV DNA polymerase are important for encapsidation and replication. Loeb et al. demonstrated that chimeric avian hepadnavirus is useful to identify the requirements for avian hepadnavirus encapsidation (Ostrow and Loeb, 2002, 2004). Although HBV has simpler cis-acting requirements for encapsidation than DHBV, the domains of HBV DNA polymerase that bind with these identified cis-acting sequences remain unknown. However chimeric DNA polymerase with HBV and DHBV did not support the encapsidation and replication of HBV. According to these results, several possibilities are (1) chimeric polymerase protein cannot bind to critical cis-acting element of pgRNA for HBV encapsidation or replication, or assembled capsid particle, (2) the chimeric polymerase proteins are not expressed in co-transfected HuH7 cells, (3) the primary sequence of chimeric polymerase may influence the other HBV transcripts or HBV proteins. Therefore the expressions of chimeric DNA polymerases were tested and were not visible by immunofluorescence assay. These results suspected that the stringency of encapsidation requirement for trans-acting factor might more influence than the stringency of cis-acting element in HBV life cycle even proper translation of chimeras was not achieved. It is possible that either chimeric DNA polymerase expression is extremely low to detect easily or chimeric DNA polymerase is not expressed in hepatoma cells. If later possibility is true, then other implications may arise such that the RNA of chimeric DNA polymerase may influence the HBV expressions such as core particle formations.

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If core particle formation is independent to chimeric DNA polymerase RNA level, core particle formation abilities should be the same between the P deficient mutant transfected cells and the P deficient mutant and chimeric DNA polymerase constructs co-transfected cells, since core proteins for core particle are solely provided from P deficient mutant pgRNA. However Figure 7 results show that core particle formation is prevented or strongly inhibited, if any, in DTC and DHN co-transfected cells and is facilitated in DRN co-co-transfected cells. These differences in the core particle formation led us to postulate that (1) RNA of the chimeric DNA polymerase may directly influence the core proteins to assemble into the core particles, (2) it may indirectly influence the translation of core protein, or (3) it may modulate the level of transcripts, such as pgRNA, subgenomic RNA and/or spliced RNA expression. The first and the second possibilities are very difficult to discuss at this moment since the core protein expression levels are not directly compared between the various chimera co-transfected cells. The third possibility can be tested from the RNAs of the P deficient mutant and chimeric DNA polymerase constructs co-transfected cells.

Although HBV, the mammalian hepadnaviruses, and DHBV, the avian hepadnaviruses, have similar genetic organizations and use same replication mechanism, mammalian and avian hepadnaviruses are distantly related in primary sequences with little homologies. It had been known that avian hepadnavirus could not rescue the replication of mammalian hepadnaviruses, and the vice versa (Hu et al., 2004). When the P deficient mutant, and chimeric DNA polymerase of HBV and

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DHBV were co-transfected to HuH7 cells. To map the essential domain of DNA polymerase for HBV encapsiation or replication, chimeric polymerases could not rescue the replication of P deficient mutant. This data indicate that HBV encapsidation and replication can not tolerate the small changes in the DNA polymerase since the chimeric DNA polymerase retained the most of the original sequences of HBV and maintained the already known essential motif of DNA polymerase because DHBV and HBV DNA polymerase share the essential motifs for replication.

Unexpectedly, the sequences of chimeric DNA polymerases, possibly through the RNA transcripts, seemed to modulate the core particle formations, even though further studies are necessary. It may not be too outrageous to postulate that chimeric DNA polymerase transcripts may have some effects on HBV life cycles.

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