To test whether PKA can modulate HBV replication, replication competent HBV wt plasmid was co-transfected with catalytic subunit of PKAα an expressing vector. Realtime RT-PCR reveal that HBV RNA expressions were not significantly affected (Fig. 10A).
Overexpression of PKAα in HBV wt transfected HuH7 cells decreased core protein and core particle formation significantly 48 hours after transfection (Fig.10B). HBV DNA synthesis was also markedly decreased 48 hours after transfection (Fig. 10C) which may be due to the reduction of core protein. To further examine the functions of PKA on HBV replication, HBV wt and PKA siRNA (siPKA) were co-transfected in HuH7 cells and core protein and HBV replicative intermediates were analyzed (Fig. 10D, second and third panels).
Knockdown of PKA by siPKA decreased core particles (middle) and DNA replication significantly (top). This result suggests that PKA might contribute to HBV replication in regulating core protein extression and/or core particle formation.
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Fig. 10. Effect of PKAa over-expression and knockdown on HBV replication. Co-transfection of HBV wt and the PKAa over-expressing vector leads to decreased core expression (A, right panel) and HBV DNA synthesis (B) in HuH7 cells. Total RNA was isolated 48 hrs after transfection, and HBV RNA was detected by realtime RT-PCR (A, left panel). HBV DNA and core protein expression were analyzed by southern and western blot,
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respectively. Transfection of HuH7 (C) and PEB8(D) cells with siRNA represses HBV replication. HuH7 cells were co-transfected with siPKA together with the HBV wt and analyzed for HBV replication. PEB8 cells were transfected with siRNA and analyzed for HBV replication. Oligonucleotides with scrambled sequences were used as negative controls (si-nontarget). HBV DNA and core particle formation were decreased in HuH7 cells 48 h after co-transfection with siRNA together with HBV wt. siPKA also represses HBV DNA synthesis in PEB8 cells.
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IV. DISCUSSION
Comparison of the amino acid sequences of the HBV, woolly monkey hepatitis B virus (WMHBV), woodchuck hepatitis B (WHV), ground squirrel hepatitis B virus (GSHV), and duck hepatitis B virus (DHBV) revealed that phosphorylation sites of C-terminal highly conserved except DHBV. The core protein of HBV is associated with protein kinase activity, capable of phosphorylating the arginine-rich, C- terminal domain of core (Roossinck, 1987;
Schlicht, 1989). In this study, HBc de/phosphorylation mutants were generated and HBV replication was investigated.
Core protein phosphorylation
Previous studies reported that phosphorylation of core protein takes place exclusively at serine residues for HBV, DHBV and GSHV (Feitelson, 1982; Liao and Ou, 1995; Kann, 1999). It has been shown that several intracellular protein kinases, such as PKC, cyclin-dependent kinase cdc, 46 kDa serine protein kinase, and two SRPK1 and 2, can phosphorylate core protein in vitro. These kinases may be responsible for the preferential phosphorylation of core protein during pgRNA encapsidation and DNA-replication in HBV-infected cells (Liao and Ou, 1995; Kann, 1999; Perlman, 2005; Basagoudanavar, 2007). In the present study demonstrated that threonine 162, serine 170, and serine 178 residues, preferentially threonine 162 and serine 178, in RRXS/T motif of core protein are phosphorylated (Fig. 2B), showing that, in addition to SRPK phosphorylation sites, PKA phosphorylation sites are also utilized by protein kinases.
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Intracellular localization of phosphorylated and non-phosphorylated core protein mutant.
Since many DNA viruses depend on the host cell machinery in the nucleus for genome replication, transcription, and mRNA processing, these DNA viruses have to target their genome and accessory proteins to the nucleus.In this study, we have examined nuclear localization of HBV core particle by phosphorylation mutant core proteins. Many reports have described that phosphorylations either up- or down-regulate nuclear transport of proteins, such as lamins (Leukel, 1995), SV-40 T antigen (Rihs, 1989), and PKC (Boulikas, 1995). The nuclear export of influenza viral RNPs also requires phosphorylation (Whittaker, 1995). As for HBV core proteins, phosphorylation of core protein may play a role in regulating nucleocytoplasmic transport. (Liao and Ou, 1995; Kann, 1999; Perlman, 2005;
Basagoudanavar, 2007). Both phosphorylated and non-phosphorylated core protein mutant were all seemed to be imported to the nucleus, almost right after their expressions. This may be due to impaired nuclear export or accelerated nuclear import of core protein. However, this may explain why these mutant can not encapsidate HBV RNA inefficiently. This discrepancy might need to be examined further with different HBV strains and other mutant core proteins.
Phosphorylation of the HBV core protein is important for DNA replication.
Although the phosphorylated mutant core proteins could package the pgRNA relatively well, these mutants were not able to fully support viral DNA replication like wt (Fig. 4, 5 and 8). These PKA phosphorylation mutants are consistent with the previous SRPK mutant experiments. Therefore the previous speculation might apply to the this result
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saying that, while the core protein phosphorylation may be important for pgRNA packaging, its dephosphorylation may be important for viral DNA replication. HBV DNA replications by individual core protein mutation on Thr162, Ser170, or Ser178 were examined and T162A mutant shows the significantly decreased replicative intermediate DNA without the reduction of encapsidation, indicating that Thr 162 residue may be important for HBV DNA synthesis (Fig. 7 and 8). Le Pogam et al. (Le Pogam, 2005) suggested that the C-termnal of core protein may function by providing the appropriate number of positive charges in the interior of core particles. These charges neutralize the negative charges associated with encapsidated nucleic acids such that a system containing the core particle with encapsidated nucleic acid is more thermodynamically stable than a system containing the core particle with no nucleic acid (Le Pogam, 2005). C-terminal of core protein may provide a thermodynamic drive for DNA synthesis by phosphorylation of C-protein. Since alanine substitutions of core protein has not putative phosphorylation site, less charges in the C-terminal of core protein could cause the pgRNA to either not be encapsidated or only be partially encapsidated. However, glutamic acid substitutions did not cause significant changes in encapsidation of pgRNA and DNA synthesis. Previous study demonstrated that serine157 is most important for pgRNA encapsidation (Lan et al, 1991) and DNA synthesis (Basagoudanavar, 2007). Since the threonine162 is located 5 amino acids downstream of serine157 and serves as the phosphorylation acceptor site determined by in vivo phosphorylation assay, it is conceivable that highly conserved threonine 162 among mammalian hepadnaviruses (Yeh et al., 1991) plays an important role in HBV replication.
Functions of PKA or related kinases in HBV replicating cells may require more
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detailed investigations to elucidate the negative or synergistic effects on HBV replication through phosphorylation or dephosphorylation of core proteins.
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V. CONCLUSION
To investigate contribution of PKA-mediated phosphoryaltion on HBV replication, phosphorylation core protein mutants by substituting the phosphorylation site of HBV core protein with alanine or glutamic acid were constructed and HBV replication were examined.
The results shown in this study indicated that the phosphorylation sites in the RRXS/T motif were important for the DNA replication. Especially, T162A and T162A containing double mutant decreased DNA synthesis significantly. In vivo phosphorylation assay also showed that T162 and S178 in the RRXS/T motif of core protein are labeled predominantly with P32 -orthophosphate. Together with results from in vivo phosphorylation assay and southern blot by phosphorylation mutants, it suggests that threonine 162 of HBV core protein may be one of phosphorylation site and modulate HBV replication.
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103 -국문요약-
PART II
B형 간염바이러스 Core 단백질의 C-말단에 위치한 인산화 부위가 genome복제에 미치는 영향
아주대학교 대학원 (의생명과학과) (지도교수: 김 경 민)
B형 간염 바이러스 core 단백질의 C-말단 도메인에 위치한 세린157, 세린164, 세린172는 세린/알지닌 특이적 인산화효소 혹은 단백질 인산화효소 C에 의해서 인산화되는 잔기로 알려져 있으며 이것이 B형 간염 바이러스의 복제에 기여한다고 알려져있다. 이와 더불어 RRXS/T motif에 위치한 트레오닌162, 세린170, 세린178는 cAMP-dependent 단백질 인산화효소 A에 의해서 인산화되는 잠정적인 잔기로 알려져 있다. In vivo 인산화 실험을 통해서 트레오닌162, 세린170, 세린178가 인산화 되는 것을 확인하였다. 인산화효소 A에 의해서 인산화되는 부위가 B형 간염 바이러스의 복제에 미치는 영향을 연구하기위해 인산화-core단백질와 비인산화-core단백질와 유사한 형태를 만들기 위해 글루타민산과 알라닌으로의 돌연변이주를 제작하였다.
T162A-104
HBc 돌연변이주에서 DNA 합성률이 크게 감소하였다. 또한 T162A와 더불어 다른조합으로 돌연변이를 제작하여 실험한 결과, T162에 A의 돌연변이가 존재할 때 DNA 합성률이 현저하게 떨어지는 것으로 나타났다. 모든 실험을 종합하여볼 때, Core 단백질의 트레오닌162, 세린170, 세린178가 인산화될 수 있으며 이 부위는 B형 간염 바이러스의 복제에 중요하다는 것을 제시하였다.
핵심어: B형 간염 바이러스, HBV core단백질의 인산화, 단백질 인산화효소 A