Overlap of interaction domains indicates a central role of the P protein in assembly and regulation of the Borna disease virus polymerase complex
Overlap of interaction domains indicates a central role of the P protein in assembly and regulation of the Borna disease virus polymerase complex
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DOI：
10.1074/jbc.m408913200
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发表时间：
2004-12-31
影响因子：
4.8
通讯作者：
Staeheli, P
中科院分区：
文献类型：
--
作者：
Schneider, U;Blechschmidt, K;Staeheli, P
博纳病病毒的活性聚合酶复合体由病毒蛋白N、P和l组成，病毒蛋白X（负调节因子）是聚合酶活性的调节因子。先前研究了P与N和X的相互作用，但与L的相互作用定义不清。利用哺乳动物双杂交系统，我们观察到L特异性地与P相互作用，但不与N、X或自身相互作用。对P分子中L结合结构域的定位显示，它与多聚和与N相互作用所需的两个相邻结构域重叠。竞争实验表明，当N存在时，L和P之间的相互作用是低效的，这表明L可能优先与感染细胞中的游离P相互作用。有趣的是，一个多聚缺陷的P突变体保持了与L、N和X相互作用的能力，但未能支持人工博纳病病毒小基因组的报告基因表达。此外，只有当使用具有完整多聚结构域的P突变体时，才会观察到对小基因组活性的显性负面影响，这表明P多聚体而不是单体具有生物活性。当野生型P作为桥接分子可用时，缺乏L或N功能相互作用结构域的P突变体仍然与这些病毒蛋白形成复合物，这表明P多聚体具有作为RNA聚合酶复合物组装的支架的潜力。
The active polymerase complex of Borna disease virus is composed of the viral proteins N, P, and L. The viral X ( negative regulatory factor) protein acts as a regulator of polymerase activity. Interactions of P with N and X were previously studied, but interactions with L were poorly defined. Using a mammalian two-hybrid system, we observed that L specifically interacts with P but not with N, X, or itself. Mapping of the L-binding domain in the P molecule revealed that it overlaps with two adjacent domains required for multimerization and interaction with N. Competition experiments showed that the interaction between L and P was inefficient when N was present, indicating that L may preferentially interact with free P in infected cells. Interestingly, a multimerization-defective P mutant maintained the ability to interact with L, N, and X but failed to support reporter gene expression from an artificial Borna disease virus minigenome. Furthermore, dominant negative effects on minigenome activity were only observed when P mutants with an intact multimerization domain were used, suggesting that P multimers, rather than monomers, exhibit biological activity. P mutants lacking functional interaction domains for L or N still formed complexes with these viral proteins when wild-type P was available as a bridging molecule, indicating that P multimers have the potential to act as scaffolds on which the RNA polymerase complex is assembled.