Zou N, Lin B Y, Duan F, Lee K Y, Jin G, Guan R, Yao G, Lefkowitz E J, Broker T R, Chow L T
Department of Biochemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA.
J Virol. 2000 Apr;74(8):3761-70. doi: 10.1128/jvi.74.8.3761-3770.2000.
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.
乳头瘤病毒的E2蛋白是一种位点特异性DNA结合核蛋白。它作为主要的复制起始点识别蛋白发挥作用，并协助预起始复合物的组装。它还有助于调节天然病毒启动子的转录。E2蛋白由一个氨基末端（N）反式作用结构域、一个中央铰链（H）结构域和一个羧基末端（C）蛋白二聚化及DNA结合结构域组成。铰链在乳头瘤病毒中高度不同，对其功能了解甚少。我们将增强型绿色荧光蛋白（GFP）与全长人乳头瘤病毒11型（HPV-11）E2蛋白融合，结果表明所得融合蛋白（称为gfpE2）保持了野生型蛋白的转录和复制功能，并形成了类似的核内亚结构。使用一系列GFP融合蛋白，我们发现铰链赋予了强大的核定位能力，而N或C结构域同时存在于细胞质和细胞核中。生化分级分离表明，N结构域和铰链而非C结构域独立地与核基质相关联。突变分析表明，许多嗜黏膜乳头瘤病毒中保守的一簇碱性氨基酸残基是有效核定位和核基质关联所必需的。这种突变不再抑制HPV-11上游调控区控制的报告基因表达。然而，该突变体中极小一部分可能通过搭便车机制在细胞核中与E1共定位，并能够支持瞬时复制。我们提出，铰链对于HPV-11 E2蛋白在mRNA转录和病毒DNA复制过程中的多种调控功能至关重要。
