F. Residues R169 and R175 are important for HBV replication
V. CONCLUSION
To identify residues and motifs critical for hepatitis B virus (HBV) replication in the carboxyl-terminal nucleic acid binding domain of HBV core protein, chimeric HBV core proteins were generated by substituting varying lengths of the carboxyl-terminus of duck hepatitis B virus (DHBV) core protein for the corresponding regions of HBV core protein.
core protein with carboxyl-terminal amino acids 221–262 of DHBV core protein substituted for residues 146–185 of the HBV core protein supported HBV pgRNA encapsidation and DNA synthesis. A chimeric core protein with residues 221–241 and 251–262 of DHBV core substituted for HBV core residues 146–166 and 176–185, respectively, could rescue full-length DNA synthesis. However, a reciprocal core chimera with residues 242–250 of DHBV core (242RAGSPLPRS250) introduced in place of 167–175 of HBV core (167RRRSQSPRR175) significantly decreased pgRNA encapsidation and DNA synthesis, and full-length DNA was not detected, demonstrating that the 167RRRSQSPRR175 motif may be critical to efficient viral replication. Five amino acids differing between viral species (underlined above) were tested for replication rescue; R169 and R175 were found to be necessary.
45
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51 -국문요약-
B형 간염 바이러스 복제에 미치는 Core 단백질의 기능적 역할
PART I
B형 간염 바이러스 Core 단백질 C-말단의
167RRRSQSPRR
175이 복제에 미치는 영향
아주대학교 대학원 (의생명과학과) (지도교수: 김 경 민)
B형 간염 바이러스(HBV) core 단백질 C-말단의 핵산 결합 도메인이 HBV 복제에 미치는 영향을 알아보기 위하여, HBV의 core 단백질 C-말단의 일부를 오리 B형 간염바이러스 (DHBV)의 core 단백질과 치환하여 키메라 캡시드 단백질을 제작하였다. HBV의 core 단백질의 146-185 아미노산을 DHBV core 단백질의 221-262 아미노산과 치환해주면 HBV pgRNA의 인캡시데이션과 DNA 복제를 할수 있는것으로 확인되었다. Core 단백질의 C-말단중 40%의 아마노산 동일성 혹은 45%의 아미노산 상동성만으로도 HBV pgRNA의
52
인캡시데이션과 DNA 복제를 하는데 충분한 것으로 확인되었다. DHBV 221-241 과 251-262 아미노산을 HBV의 146-166과 176-185 아미노산으로 치환하여 만든 키메라 core 단백질은 완전체 길이의 DNA를 합성할 수
있는것으로 확인 되었지만 DHBV의 (242RAGSPLPRS250) 를 HBV의
(167RRRSQSPRR175) 로 치환하게 되면 HBV pgRNA의 인캡시데이션과 DNA 복제가 상당히 감소하는 것으로 나타났다. 이 결과를 통해서 C-말단의 (167RRRSQSPRR175) 도메인이 바이러스의 복제에 중요할 것이라고 추측할수 있다.
이 도메인에서 두 바이러스간의 다른 아미노산을 분석하여 추가의 키매라 단백질을 제작하여 확인해본 결과, 알지닌169 와 알지닌175가 중요한 것으로 확인 되었다.
핵심어: B형 간염 바이러스, 키메라 Core 단백질, Core 단백질의 C-말단, 인캡시데이션, B형 간염 바이러스 복제
PART II
Phosphorylation of the C-Terminal Domain of HBV Core Protein modulates
Genome Replication
53
I. INTRODUCTION
Hepadnaviruses are small, enveloped DNA viruses that replicate preferentially in liver cells and are involved in acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Hepatitis B virus (HBV), a prototype hepadnavirus, has a partially duplexed relaxed circular DNA genome, which is replicated by reverse transcription of a pregenomic (pg) RNA within cytoplasmic core particle composed of viral core protein (Ganem, 2001).
HBV replication cycle starts when N-terminal domain of the large envelope protein (L protein) of the virus binds to the unidentified receptor of hepatocyte (Ganem, 2001;
Hourioux et al, 2002). Fusion of the viral envelope with a cellular membrane liberates the subviral core particle into the cell cytoplasm. Core particle releases the partially double-stranded relaxed circular (RC) DNA to the nucleus and RC DNA is then converted to covalently closed circular DNA (cccDNA). This cccDNA serves as a template for transcription via host RNA polymerase II (Yao et al, 2000). The viral mRNAs are exported to the cytoplasm. pgRNA serves as mRNA for core and polymerase (P) proteins. After translation, core proteins form core particles via dimeric intermediates (core particle assembly) and package its pgRNA and P protein into core particles (encapsidation). To accomplish encapsidation, pgRNA having an epsilon (ε) sequence is associated with viral P protein to generate RNA and P protein complex (RNP complex). After encapsidation, when hydroxyl group of tyrosine 63 (for ayw or 65 for adw) of P protein, a protein primer, is covalently linked to newly synthesized nucleotide without a nucleic acid primer,
minus-54
strand DNA synthesis is initiated at a bulge region of epsilon (ε), the encapsidation signal, synthesizing the TGAA or GAA, the short oligomer (Tavis et al, 1994; Wang et al, 1994;
Nassal and Rieger, 1996; Kim et al, 2004). The short, nascent DNA then switches template to a complementary sequence near the 3’-end of the pgRNA where minus-strand DNA synthesis resumes (template switch) (Loeb et al, 1991). The specificity of this template switch is determined by base pairing between nucleotides in ε and nucleotides flanking the acceptor site (Staprans et al, 1991). Subsequently, minus-strand DNA synthesis continues until the 5’-end of pgRNA, while RNA template is degraded by the RNase H activity of HBV P protein except the short stretch of RNA (Loeb et al, 1991). In a majority of cases, this RNA fragment undergoes a template switch from the 3’-end of the minus strand to the near the 5’-end, termed DR2, anneal to a complementary 11-nucleotide sequence and serves as primer for plus-strand DNA synthesis at DR2 (primer translocation) (Seeger and Maragos, 1989; Loeb et al, 1991). Once synthesizing plus-strand DNA has reached at the 5’-end of minus-strand DNA, a final translocation to the 3’-end of minus-strand DNA occurs (circularization), resulting in partially double-stranded RC DNA genome. In a minority of capsids, primer translocation does not occur, and plus-strand DNA primes from DR1 (in situ priming) to produce a duplex-linear genome (DL) DNA (Staprans et al, 1991).
The HBV core protein consists of 183 or 185 amino acids. The amino-terminus of core protein (amino acids 1–144) participates in core particle assembly through protein-protein interaction and is, by itself, assembly competent (Gallina et al, 1989) and . The carboxyl-terminus of core protein contains a protamine-like nucleic acid binding domain rich in arginine. Fourteen of the sixteen arginines are grouped into four clusters with three or
55
four arginines (Lewellyn and Loeb, 2011a) and are conserved among mammalian hepadnaviruses, suggesting the importance in HBV replication. In addition to arginine-rich domain, many studies have reported phosphosphorylation sites located in the arginine-rich carboxyl-terminal domain of the core protein (Machida, 1991; Yeh, 1991; Lan et al, 1999;
Köck et al, 2004; Pogam et al, 2005). Three phosphorylation sites, serine 155, serine 162, and serine 170 residues in the SPRRR motif of subtype ayw (equivalent to serine 157, serine 164, and serine 172 of subtype adw) in the arginine-rich carboxyl-terminal domain of core protein have been identified to be phosphorylated by serine/arginine rich protein kinase (SRPK) and/or other intracellular protein kinases (Lan, 1999; Gazina et al, 2000; Melegari et al, 2005). In addition to serine 157, serine 164, and serine 172 (in adw subtype) residues, threonine 162, serine 170, and serine 178 residues (adw) in the conserved RRXS/T motif have been suggested to be the putative phosphorylation sites. Enomoto and his colleague have demonstrated that serine 170 and serine 178 residues are phosphorylated by cAMP-dependent protein kinase A (PKA) in vitro (Enomoto, 2006).
Phosphorylation of core protein may play a major role in regulating nucleocytoplasmic transport of the core protein. (Liao and Ou, 1995; Kann, 1999; Perlman, 2005; Basagoudanavar, 2007). While agreeing on the importance of phosphorylation, previous studies are controversial in detail: mutational analysis of phosphorylation target sites in the transfected cells indicated the inhibitory roles by phosphorylation (Liao and Ou, 1995), while nuclear localization signal (NLS) activation through phosphorylation was suggested by the nuclear transport of core particles (Kann, 1999; Kann 2003). Unlike Ser157, Ser164, and Ser172 (in adw subtype) residues that contribute pgRNA encapsidation and
56
HBV DNA replication, the importance of putative PKA phosphorylation site for HBV replication has never been explored.
The goals of the present study are to understand whether the putative PKA phosphorylation sites are indeed phosphorylated and can contribute to HBV replication. First, in vivo phosphorylation assay were performed. Then, the role of phosphorylation at the PKA phosphorylation sites for virus replication were examined by analyzing the phenotypes of a series of glutamic acid or alanine substitutions in the putative phosphorylation sites of core protein. HBV RNA encapsidation, DNA synthesis, endogenous polymerase activity, and intracellular localization of the core protein were analyzed. Finally, HBV replication was investigated by phosphorylation and dephosphorylation by PKA. HBV DNA synthesis was decreased when PKA up and down-regulated by PKA over-expressing vector or siRNA transfection. Further studies are required to examine how PKA regulates HBV replication. In addition to the SRPK phosphorylation and dephosphorylation of core protein, putative phosphorylation and dephosphorylation by PKA may modulate HBV replication.
57
II. MATERIALS AND METHODS
A. DNA construction
The partially redundant HBV wild type (wt) construct designated pPB from the 1.2 length of the HBV subtype adw R9 had been subcloned into pcDNA3 (Invitrogentm) to express HBV under the cytomegalovirus immediate early (CMV IE) promoter (Kim et al, 2004). P protein-deficient mutant was prepared, in which polymerase expression was abolished by modifying AUG start codon to ACG with frame-shifting the polymerase ORF with a silent mutation in core ORF. Phosphorylation site point and combination mutants of HBV core protein were generated from P-deficient mutant background by PCR mediated mutagenesis. To supply HBV polymerase in trans, we constructed the HBV P protein supplier, HBV pol DPS by deleting the 5’end signal, from which HBV polymerase (P) protein was provided but encapsidation were prohibited. The P gene of HBV was amplified by PCR from plasmid pPB, which contains full length HBV genome, and cloned into HindIII / BstEII sites in of pPB. To generate the RT-YMHA pol DPS mutant, the EcoRI- and EcoRV-digested DNA fragment from a reverse transcriptase (RT) reaction deficient RT-YMHA mutant, wherein the conserved YMDD motif of the RT active site was modified to YMHA (Kim et al, 2004), was cloned into the pHBV pol construct. A plasmid encoding PKAa was created by inserting a fragment of the PKAa into the multicloning region of the pcDNA3 vector (Promega).