Biochemical activities of highly purified, catalytically active human APOBEC3G: Correlation with antiviral effect
Biochemical activities of highly purified, catalytically active human APOBEC3G: Correlation with antiviral effect
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DOI：
10.1128/jvi.02680-05
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发表时间：
2006-06-01
影响因子：
5.4
通讯作者：
Levin, Judith G.
中科院分区：
文献类型：
--
作者：
Iwatani, Yasumasa;Takeuchi, Hiroaki;Levin, Judith G.
APOBEC 3G（APO 3G）是一种具有两个锌指结构域的胞苷脱氨酶，在不存在Vif的情况下抑制人类免疫缺陷病毒I型复制。在这里，我们使用仅含有一种蛋白质组分的纯系统，即，在杆状病毒系统中表达的高度纯化的催化活性酶。我们证明，载脂蛋白3G脱氨基胞嘧啶单链DNA（ssDNA），而它有效地结合到ssDNA和ssRNA，约一半的DNA/RNA杂交，和差双链DNA和RNA。此外，ssDNA的脱氨基和结合的碱基特异性不相关。在电泳迁移率变动试验中检测APO 3G与ssDNA寡核苷酸结合所需的最小长度为16个核苷酸。有趣的是，如果核衣壳蛋白和APO 3G存在于同一反应中，我们发现它们不会干扰彼此与RNA的结合，并且形成含有RNA和两种蛋白质的复合物。最后，我们还确定了每个锌指结构域的功能活动。因此，尽管两个锌指结构域都具有结合核酸的能力，但第一个锌指结构域比第二个锌指结构域更有助于结合和APO 3G掺入病毒体。相反，脱氨作用仅与第二锌指相关。此外，对于抗病毒作用，第二个锌指比第一个锌指更重要，这表明脱氨酶与抗病毒活性之间存在相关性。
APOBEC3G (APO3G), a cytidine deaminase with two zinc finger domains, inhibits human immunodeficiency virus type I replication in the absence of Vif. Here, we provide a comprehensive molecular analysis of the deaminase and nucleic acid binding activities of human APO3G using a pure system containing only one protein component, i.e., highly purified, catalytically active enzyme expressed in a baculovirus system. We demonstrate that APO3G deaminates cytosines in single-stranded DNA (ssDNA) only, whereas it binds efficiently to ssDNA and ssRNA, about half as well to a DNA/RNA hybrid, and poorly to double-stranded DNA and RNA. In addition, the base specificities for deamination and binding of ssDNA are not correlated. The minimum length required for detection of APO3G binding to an ssDNA oligonucleotide in an electrophoretic mobility shift assay is 16 nucleotides. Interestingly, if nucleocapsid protein and APO3G are present in the same reaction, we find that they do not interfere with each other's binding to RNA and a complex containing the RNA and both proteins is formed. Finally, we also identify the functional activities of each zinc finger domain. Thus, although both zinc finger domains have the ability to bind nucleic acids, the first zinc finger contributes more to binding and APO3G encapsidation into virions than finger two. In contrast, deamination is associated exclusively with the second zinc finger. Moreover, zinc finger two is more important than finger one for the antiviral effect, demonstrating a correlation between deaminase and antiviral activities.