Initial Stages of Hepatitis B Virus Replication
PART 1 Intracellular Trafficking of HBV Core Particle in Various Stage of DNA Replication Inside of hepatitis B virus (HBV) core particle, pgRNA is reverse transcribed by HBV DNA polymerase into minus-strand DNA and pregenomic RNA (pgRNA) is degraded concomitantly by RNase H activity of DNA polymerase. Then, plus-strand DNA is synthesized to double-stranded relaxed circular DNA. Through this HBV DNA replication, immature core particle becomes mature core particle. This mature core particle can either be transported back into nucleus to amplify the intracellular viral genome pool, or be enveloped at intracellular membrane to be secreted as a mature virion. In this study, intracellular trafficking of HBV core particles was investigated according to the of HBV DNA replication stages. To obtain the core particles, which represent the various stages of HBV DNA replication, several HBV mutants were constructed. RT YMHA, reverse transcription deficient, mutant can encapsidate pgRNA but DNA replication is completely blocked by mutation of YMDD reverse transcriptase motif to YMHA. Priming deficient TP Y65F mutant core particles, in which tyrosine 65 in TP domain of HBV DNA polymerase was changed to phenylalanine, can synthesize oligomer, the nascent minus-strand DNA. RH D750V mutant makes core particles containing pgRNA-minus strand DNA hybrid because RNase H activity is blocked by aspartic acid 750. HBV wild type and various mutants were transiently expressed in HuH7 cells and analyzed the intracellular distribution of core particles by immunofluorescence assay and confocal microscopy. From series of replicative stages of core particles, only core particle in late stages of HBV DNA replication with completed minus-strand DNA or relaxed circular DNA genome, can co-localize with microtubule, vimentin and nuclear pore complex of host cells. PART 2 Replication Competent Core Particles of Hepatitis B Virus Were Formed by Chimeric Core Protein Assembly of replication competent HBV nucleocapsid requires interaction of core protein, polymerase and pgRNA of HBV. The N-terminal portion of core protein participates in capsid particle assembly and is, by itself, assembly competent. The C-terminal portion which is rich in arginine residue, is known as nucleic acid binding domain. Although C-terminal domain of core protein is dispensable for capsid particle assembly, this domain is involved in viral replication including pregenomic RNA encapsidation. However critical amino acid residues or motif for pgRNA encapsidaion or DNA replication has not been demonstrated yet. In this study, chimeric core proteins of HBV were produced by substituting the C terminal region of HBV core protein to DHBV to identify critical site of nucleic acid binding domain for HBV replication. Chimeric core proteins were designed to contain various lengths of corresponding DHBV sequences, while N-terminal sequences of HBV core protein were unchanged. Core protein deficient mutant (C deficient) and various core protein chimeras were co-transfected into HuH7 hepatoma cell line to allow virus assembly. In this condition, core proteins were supplied in trans by core protein chimera construct, and pgRNA and other HBV proteins were provided by C deficient mutant. While some chimeric core proteins can support the HBV core particle assembly, the other cannot. The one of particle assembly competent chimeric core protein can support HBV pgRNA encapsidation and HBV DNA synthesis, but the other can support only core particle assembly. C-terminal region of HBV and DHBV core protein is about 40% homologous in amino acid sequence. Our result indicate that 40% of amino acid sequence identity of nucleic acid binding domain is sufficient to complement HBV core proteins which encapsidate HBV pgRNA and synthesize HBV DNA. PART 3 Reverse Transcriptase Activity of Hepatitis B Virus Polymerase Hepadnavirus DNA polymerase functions DNA synthesis from RNA or DNA template and acts as a primer for minus-strand DNA synthesis. From previous studies, endogenous polymerase activity in priming-deficient mutant core particles was found. This result suggest that the priming-deficient mutant polymerase has the ability to synthesize oligomers (presumably nascent minus-strand DNA) in the absence of covalent linkage between TP and the first deoxynucleotide, which means the primer independent initiation by HBV DNA polymerase. This raises a very important question that HBV DNA polymerase may have RNA polymerase feature. In this study, the RNA polymerase activity of HBV DNA polymerase was explored by testing the NTP incorporation capacities. The bulky amino acid, phenylalanine 436, at supposedly dNTP binding cleft of HBV DNA polymerase was changed to smaller amino acids, glycine or valine. NTPs incorporation capacity of HBV DNA polymerase was tested by endogenous polymerase assay with 32P-labeled ATP and cold dNTPs with isolated core particles. HBV DNA polymerase incorporates NTPs by F436 substitution to glycine which is smaller than valine. This result indicates that bulky amino acid in dNTP binding cleft acts as a steric gate in dNTP selections. Even though authentic DNA synthesis was not detected, it was found that HBV DNA polymerase incorporate NTPs by substituting F436 to G. Taken together, these results indicate that single amino acid substitution can blur property of DNA polymerase in the direction of RNA polymerase.
chimeric core protein, dNTP binding cleft, HBV polymerase, hepatitis B virus core particle, hepatitis B virus replication, intracellular trafficking, NTP incorporation, replication stages, B형 간염 바이러스의 복제, B형 간염 바이러스의 캡시드 입자, dNTP 결합부위, HBV DNA 중합효소, NTP 결합, 복제단계, 세포 내 분포, 키메라 캡시드 단백질
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