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IV. DISCUSSION
The C-terminal of HBV core protein contains protamine-like region that is rich in arginine residue, and is known as nucleic acid binding domain. C-terminal portion of core protein is dispensable for capsid particle assembly. However many researches with series of C-terminally truncated core protein mutants showed the importance of C-terminal portion of hepadnavirus core protein in pgRNA encapsidation and DNA replication. In this study, chimeric core protein expression plasmids were constructed by substituting the C terminal region of HBV core protein to DHBV to identify critical residues or motif at C terminal region of core protein for HBV replication.
Trans-complementation of C deficient mutant by core protein and chimeric core proteins are summarized in Fig. 6. Among three chimeras, HD 192-262 was unable to assemble core particle and HD 192-220 can assemble into core particle but cannot encapsidate HBV pgRNA nor synthesize HBV DNA. HD 221-262 can trans-complement HBV core protein for both HBV pgRNA encapsidation and HBV DNA synthesis but only smaller DNA than HBV genomic DNA was detected.
HD 221-262 chimeric core protein has sequence substitutions at C-terminal domain of HBV core protein without insertion or deletion. Through alignment of substituted region of HD 221-262, C-terminal domain of HBV and DHBV core protein has higher homology with 45.2 % homology sequences, while 26.7 % of homology with whole core protein sequences (Fig. 1A). From trans-complementation
results of HD 221-262 and sequence alignment, it indicates that homologous amino acids at C-terminal domain of core protein, displayed as yellow, is crucial for HBV pgRNA encapsidation. From previous studies, C-terminally deleted core protein with 1-164 amino acids in ayw subtype could encapsidate pgRNA (Nassal, 1992; Pogam et al., 2005). This result showed that amino acid from 165 to 183 is dispensable for encapsidation in ayw subtype. This could be applied to our results (adw subtype) in which corresponding amino acids from 166 to 185 is dispensable for encapsidation.
For encapsidation, crucial amino acids may be Thr 146 to Val 149, Arg 152, Arg 154, Ser 157, Arg 161, Arg 162, Arg 163, and Pro 165.
It was suggested that amino acids from 165 to 173 residues of core protein (amino acid number of ayw subtype) may be important for full-length of HBV DNA synthesis while 174 to 183 residues is dispensable (Nassal, 1992). Recently it had been shown that Arg 172 and 173 (ayw subtype), the positively charged of amino acid, are required for full-length DNA synthesis (Pogam et al., 2005). HD 221-262 chimeric core protein trans-complement C deficient mutant for pgRNA encapsidation and DNA synthesis, while it cannot trans-complement full-length HBV DNA synthesis. This indicates that amino acid differences between HBV and DHBV sequences of HD 221-262 is crucial for synthesis of full-length HBV DNA except from 178 to 185 of adw subtype. Taken together, it can be proposed that crucial residues for full-length of HBV DNA may be Arg 168, Arg 169, Gln 171, Ser 172, and Arg 175.
Recent studies reported that core particles assembled from core protein with
truncation or insertion were unstable (Kock et al., 2004; Kock et al., 1998; Pogam et al., 2005). DHBV was defective in DNA replication when DHBV core protein had insertions for up to 25 residues at N-terminus, possibly due to earlier nucleocapsid destabilization (Kock et al., 1998). This destabilization by insertional mutation might explain the failure of trans-complementation by HD 192-220 chimeric core protein.
HD 192-220 chimeric core protein has insertion of 29 residues between amino acid 145 and 146 of HBV core protein without sequence substitution. HD 192-220 cannot rescue HBV pgRNA encapsidation and DNA synthesis except core particle assembly.
It is still possible to speculate that 29 residues insertion causes destabilization of core particle, thus encapsidated pgRNA cannot be protected from nuclease treatment for core particle isolation procedure. HD 192-262 chimera was unable to assemble into core particle even though it has same length of inserted residues like HD 192-220.
Since HD 192-262 has more extensive alterations, C-terminal substitution and 29 residues insertion, these alterations may cause the destabilization of core particles (cannot maintain particle structure) or the incompetency of core particle assembly.
It had been reported that spliced RNA of hepadnavirus was encapsidated and DNA was synthesized from this spliced pgRNA inside core particles by C-terminally truncated core protein (Kock et al., 2004; Pogam et al., 2005). It is possible that short DNA detected from HD 221-262 chimeric core particle may be synthesized from spliced RNA. This should be investigated in detail.
V. CONCLUSION
In this study, to identify the nucleic acid binding domain for HBV replication, chimeric core proteins of HBV were constructed by substituting HBV core protein C terminal region to corresponding DHBV sequences. DHBV core proteins that consist of 262 amino acids can form a three-dimensional nucleocapsids structure similar to that of HBV. Chimeric core proteins were designed to contain various lengths of corresponding DHBV sequences, while N-terminal sequences of HBV core protein were unchanged. With these chimeric core proteins, HBV pgRNA encapsidation and HBV DNA replication were examined. This chimeric core protein study showed that chimeric core protein can trans-complement HBV core proteins for C deficient mutant. This study also shows that the 45.2 % of amino acid sequence identity at core protein C-terminal region for nucleic acid binding domain is sufficient to encapsidate HBV pgRNA and synthesize HBV DNA.
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