The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association
The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association
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DOI：
10.1128/jvi.74.8.3761-3770.2000
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发表时间：
2000-04-01
影响因子：
5.4
通讯作者：
Chow, LT
中科院分区：
文献类型：
--
作者：
Zou, NX;Lin, BY;Chow, LT
乳头瘤病毒的E2蛋白是一种位点特异性的DNA结合核蛋白，它是主要的复制起始识别蛋白，并协助起始前复合物的组装。它也有助于调节原生病毒启动子的转录。E2蛋白由氨基末端(N)反式作用域、中心铰链(H)结构域和羧基末端(C)蛋白二聚化和DNA结合结构域组成。这种铰链在乳头瘤病毒中高度分化，对其功能知之甚少。我们将增强的绿色荧光蛋白（GFP）与全长人乳头瘤病毒11型（HPV-11） E2蛋白融合，结果表明，这种称为gfpE2的融合维持了野生型蛋白的转录和复制功能，并形成了类似的亚核灶。使用一系列的GFP融合蛋白，我们发现铰链具有很强的核定位，而N或C结构域同时存在于细胞质和细胞核中。生化分离表明，N结构域和铰链，而不是C结构域，独立与核基质相关。突变分析表明，在许多嗜粘性乳头瘤病毒中保守的一簇碱性氨基酸残基是有效的核定位和核基质结合所必需的。该突变不再抑制HPV-11上游调控区域控制的报告基因表达。然而，该突变体的一小部分与细胞核中的E1共定位，可能是通过一种背带机制，并能够支持瞬时复制。我们认为，在mRNA转录和病毒DNA复制过程中，该铰链对hpv - 11e2蛋白的多种调控功能至关重要。
The E2 protein of papillomaviruses is a site-specific DNA binding nuclear protein, It functions as the primary replication origin recognition protein and assists in the assembly of the preinitiation complex. It also helps regulate transcription from the native viral promoter. The E2 protein consists of an amino-terminal (N) trans-acting domain, a central hinge (H) domain, and a carboxyl-terminal (C) protein dimerization and DNA binding domain. The hinge is highly divergent among papillomaviruses, and little is known about its functions. We fused the enhanced green fluorescent protein (GFP) with the full-length human papillomavirus type 11 (HPV-11) E2 protein and showed that the resultant fusion, called gfpE2, maintained transcription and replication functions of the wild-type protein and formed similar subnuclear foci. Using a series of GFP fusion proteins, we showed that the hinge conferred strong nuclear localization, whereas the N or C domain was present in both cytoplasm and nucleus. Biochemical fractionation demonstrated that the N domain and hinge, but not the C domain, independently associated with the nuclear matrix. Mutational analyses showed that a cluster of basic amino acid residues, which is conserved among many mucosotropic papillomaviruses, was required for efficient nuclear localization and nuclear matrix association. This mutation no longer repressed the HPV-11 upstream regulatory region-controlled reporter expression. However, a very small fraction of this mutant colocalized with E1 in the nucleus, perhaps by a piggyback mechanism, and was able to support transient replication. We propose that the hinge is critical for the diverse regulatory functions of the HPV-11 E2 protein during mRNA transcription and viral DNA replication.