RNA-BINDING AND DNA-BINDING ACTIVITIES IN HEPATITIS-B VIRUS CAPSID PROTEIN - A MODEL FOR THEIR ROLES IN VIRAL REPLICATION
RNA-BINDING AND DNA-BINDING ACTIVITIES IN HEPATITIS-B VIRUS CAPSID PROTEIN - A MODEL FOR THEIR ROLES IN VIRAL REPLICATION
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DOI：
10.1128/jvi.66.9.5232-5241.1992
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发表时间：
1992-09-01
影响因子：
5.4
通讯作者：
STANDRING, DN
中科院分区：
文献类型：
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作者：
HATTON, T;ZHOU, SL;STANDRING, DN
乙肝病毒衣壳或核心蛋白(p21.5)通过羧基末端鱼精蛋白区域与核酸结合，该区域包含组织成四个重复(I至IV)的核酸结合基序。利用在大肠杆菌中表达的羧基末端截短的蛋白，我们检测到重复序列中既有RNA结合活性，也有DNA结合活性。通过在琼脂糖凝胶上分解纯化的大肠杆菌衣壳并用溴化乙锭揭示其RNA含量来评估RNA结合和包装活性，只需要近端重复序列I(RRRDRGRS)。引人注目的是，用四个Arg残基取代Repeat I的突变体能够包装RNA，证明Arg残基驱动RNA结合。相反，用P-32-核酸探测固定的核心蛋白的活性显示：(I)需要更多的鱼精蛋白区域(重复序列I和II)，(Ii)似乎比RNA更好地结合DNA，(Iii)明显受来自非洲爪哇卵母细胞的p21.5的磷酸化调节。缺失分析表明，这种活性可能依赖于重复序列II中的SPXX类型的DNA结合基序。在重复序列III和IV中发现的类似基序也可能具有结合DNA的功能。在这些观察的基础上，结合对最近表明衣壳蛋白突变导致病毒基因组复制缺陷的研究的重新解释，我们提出了一个模型，该模型认为肝炎病毒衣壳蛋白直接参与了RNA在衣壳内反转录成DNA的过程。在这个模型中，重复序列I与RNA结合，而远端重复序列则逐渐招募来结合延长的DNA链。后一种基序可能是复制在能量上可行所必需的。
The hepatitis B virus capsid or core protein (p21.5) binds nucleic acid through a carboxy-terminal protamine region that contains nucleic acid-binding motifs organized into four repeats (I to IV). Using carboxy-terminally truncated proteins expressed in Escherichia coli, we detected both RNA- and DNA-binding activities within the repeats. RNA-binding and packaging activity, assessed by resolving purified E. coli capsids on agarose gels and disclosing their RNA content with ethidium bromide, required only the proximal repeat I (RRRDRGRS). Strikingly, a mutant in which four Arg residues replaced repeat I was competent to package RNA, demonstrating that Arg residues drive RNA binding. In contrast, probing immobilized core proteins with P-32-nucleic acid revealed an activity which (i) required more of the protamine region (repeats I and II), (ii) appeared to bind DNA better than RNA, and (iii) was apparently modulated by phosphorylation in p21.5 derived from Xenopus oocytes. Deletion analysis suggested that this activity may depend on an SPXX-type DNA-binding motif in repeat II. Similar motifs found in repeats III and IV may also function to bind DNA. On the basis of these observations, together with a reinterpretation of recent studies showing that capsid protein mutants cause defects in viral genome replication, we propose a model suggesting that hepadnavirus capsid proteins participate directly in the intracapsid reverse transcription of RNA into DNA. In this model, repeat I binds RNA whereas the distal repeats are progressively recruited to bind elongating DNA strands. The latter motifs may be required for replication to be energetically feasible.