探討B型肝炎病毒核殼蛋白的磷酸化在病毒生活史扮演之角色

Phosphorylation of Hepatitis B Virus Nucleocapsid Protein in Regulating The Viral Life Cycle

吳宜瑾(Yi-Jin Wu)

指導教授 ： 葉秀慧

國立臺灣大學/醫學院/微生物學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.03374

摘要

B型肝炎病毒(HBV)核殼蛋白(nucleocapsid protein)共183個胺基酸，其中1-149胺基酸為assembly domain，150-183胺基酸為nucleic acid-binding domain。多篇研究指出，核殼蛋白對於HBV在進行複製時扮演著重要的角色，藉由C端Serine位點(S155、S162、S170)磷酸化修飾的有無，可能影響複製過程中的包殼(Encapsidation)及反轉錄 (Reverse transcription)步驟之進行。由於先前的文獻都是利用點突變方式將Serine變成Alanine，導致特定位點無法進行磷酸化，以探討核殼蛋白之磷酸化修飾對HBV生活史之影響。然而突變成Alanine的核殼蛋白可能會造成結構改變，無法直接證明其C端特定Serine位點磷酸化修飾之重要性。我們因此提出利用製備可專一辨識核殼蛋白C端特定Serine位點Phosphorylated form之抗體，探討核殼蛋白C端Serine位點磷酸化修飾於生活史中不同階段之變化，並用於尋找細胞中負責此磷酸化修飾之磷酸酶(Kinase)，以釐清及其對HBV生活史中Encapsidation和Reverse transcription之影響。
為此，我們針對HBV核殼蛋白C端特定Serine位點，利用Synthetic peptide共製備了四支抗體。Dot-blot結果指出P4-P抗體有可能辨認核殼蛋白未經磷酸化修飾的S162位點。進一步利用點突變HBc質體分析P4-P抗體所辨認的HBc之磷酸化修飾位點，將表現Wild type及針對C端特定Serine位點進行點突變之核殼蛋白表現質體送入細胞中，利用P4-P抗體進行Western blot分析，發現此抗體傾向辨認經CIP處理之核殼蛋白；且除S162A外，其S170A之點突變亦會造成其對核殼蛋白較高之辨識力。我們因此提出一假說，認為HBV核殼蛋白C端之磷酸化修飾，有可能影響其結構之改變，決定Arginine rich之序列與病毒RNA之結合。而P4-P抗體對S162A及S170A突變之較強訊號，反應出其可能辨識未經磷酸化修飾的核殼蛋白C端結構，而此結構會降低核殼蛋白進行RNA Encapsidation之能力。
利用P4-P抗體的特性，我們接著利用kinase inhibitor處理細胞之策略，指出PKA有可能為參與調控核殼蛋白C端結構之磷酸酶。進一步針對核殼蛋白C端序列中可能為PKA標的之S168及S176位點，分別進行點突變，發現PKA有可能經由其對S168位點之磷酸化修飾，影響核殼蛋白C端結構。
本研究藉由一傾向辨認去磷酸化核殼蛋白之抗體之分析結果，提出HBV核殼蛋C端之磷酸化修飾有可能透過改變其結構，而影響其對病毒RNA進行Encapsidation能力之假說，並指出PKA有可能為細胞中調控此結構之磷酸酶。未來將針對此假說做深入之研究，並將進一步探討此磷酸化修飾對HBV生活史中各複製過程之影響。

關鍵字

B型肝炎病毒 ； 核殼蛋白 ； 磷酸化 ； 磷酸酶Serine（S） ； Alanine（A） ； Aspartic acid（D）

並列摘要

The nucleocapsid protein of Hepatitis B virus (HBV), namely HBc, contains 183 amino acids. The N terminal 1-149 a.a. is sufficient for the viral assembly; and the 150-183 a.a. at C terminal domain (CTD) is responsible for nucleic acid binding activity . It has long been documented that HBc is a phospho-protein, with the major phosphorylation sites at the serine residues within CTD. Aided by the site directed mutagenesis, previous studies demonstrated that the phosphorylation at serine residues of S155, S162, and S170 could be critical for regulating the viral RNA encapsidation and the reverse transcription in viral life cycle. However, the possible structure changes introduced by the site directed mutagenesis make these findings inconclusive. Besides, the putative kinases responsible for the phosphorylation of these serine residues are still remained to be identified.
We propose to address this critical issue by synthesizing the Abs which can recognize the phosphorylated form of specific Ser at CTD of HBc. Four Abs were generated using the synthetic peptides containing phosphorylated Ser residues. The results from dot-blot analysis revealed that P4-4 Ab could recognize the de-phosphorylated form of S162 of HBc. To further validate this, we transfected the wild type and also the HBc mutant constructs, containing specific mutations at each Ser residues of S155, S162, and S170, into Huh-7 cells for the Western blot analysis. The results showed that P4-P Ab has higher affinity for the CIP-treated HBc protein. Moreover, not only showing higher affinity for S162A, the P4-P also showed the higher affinity for S170A. It thus raised a possiblity for the presence of a phosphorylation dependent conformation at the CTD of HBc. P4-P Ab can recognize this conformation when it tends to be de-phosphorylated at S162 and S170. The RNA encapsidation was previously found defective in HBV replcion with S162A and S170A mutations. Therefore, the conformation of HBc recognized by P4-P Ab could be in a “closed form”, which block the accessibility of the arginine rich motif within CTD for interacting with the viral RNA.
By treating the HBc protein expressing cells with a panel of kinase inhibitors, PKA was identified as a putative kinase involved in regulating the conformation recognized by P4-P Ab. Two serine residues at CTD of HBc as the PKA consensus sequence, S168 and S176, were mutated to alanine and aspartic acid for evaluating the recognition by P4-P Ab. In comparison with WT, the S168A significantly decreased the signal recognized by P4-P Ab, suggesting that PKA could through this residue to regulate the conformation at HBc CTD.
Our current study proposed an interesting possibility for the presence of a phosphorylation dependent structure at the CTD of HBc, which could potentially involved in regulating the viral RNA encapsidation process. Such a possibility and also the effect of PKA mediated regulation of this structure in viral life cycle is worthy to be further investigated.

並列關鍵字

Hepatitis B virus ； nucleocapsid ； Phosphorylation ； Kinase ； Serine (S) ； Alanine(A) ； Aspartic acid (D)

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