Human cytomegalovirus UL84 oligomerization and heterodimerization domains act as transdominant inhibitors of oriLyt-dependent DNA replication:: Evidence that IE2-UL84 and UL84-UL84 interactions are required for lytic DNA replication
Human cytomegalovirus UL84 oligomerization and heterodimerization domains act as transdominant inhibitors of oriLyt-dependent DNA replication:: Evidence that IE2-UL84 and UL84-UL84 interactions are required for lytic DNA replication
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DOI：
10.1128/jvi.78.17.9203-9214.2004
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发表时间：
2004-09-01
影响因子：
5.4
通讯作者：
Pari, GS
中科院分区：
文献类型：
--
作者：
Colletti, KS;Xu, YY;Pari, GS
人巨细胞病毒UL84编码依赖于oriLyt的DNA复制所需的75 kDa蛋白质，并在感染和转基因细胞中与IE2相互作用。UL84定位于转基因和感染细胞的细胞核，并在病毒复制间隔室中发现。在瞬时试验中，UL84可以干扰IE2介导的人巨细胞病毒UL112/113启动子的反式激活。为了确定UL84蛋白-蛋白质相互作用是否是裂解DNA合成所必需的，我们纯化了UL84蛋白，并用该蛋白制备了单抗。利用这种抗体，我们现在证明UL84在体内与自己形成了稳定的相互作用。自我相互作用点映射到蛋白质151和200氨基酸之间的区域，该区域包含一系列高电荷氨基酸残基。免疫共沉淀实验确定UL84与IE2的前215个氨基酸中存在的蛋白质结构域相互作用。我们还表明，UL84的完整亮氨酸拉链结构域是与IE2稳定相互作用所必需的，并且UL84亮氨酸拉链突变体无法补充oriLyt依赖的DNA复制。UL84亮氨酸拉链突变体不再干扰IE2介导的UL112/113启动子的反式激活，证实了亮氨酸拉链对于与IE2的功能相互作用是必不可少的。此外，在瞬时实验中，我们证明UL84的亮氨酸拉链和寡聚区都可以作为裂解复制的跨显性负抑制因子，这强烈表明DNA合成既需要IE2-UL84相互作用，也需要UL84-UL84相互作用。
Human cytomegalovirus (HCMV) UL84 encodes a 75-kDa protein required for oriLyt-dependent DNA replication and interacts with IE2 in infected and transfected cells. UL84 localizes to the nucleus of transfected and infected cells and is found in viral replication compartments. In transient assays it was shown that UL84 can interfere with the IE2-mediated transactivation of the UL112/113 promoter of HCMV. To determine whether UL84 protein-protein interactions are necessary for lytic DNA synthesis, we purified UL84 and used this protein to generate a monoclonal antibody. Using this antibody, we now show that UL84 forms a stable interaction with itself in vivo. The point of self-interaction maps to a region of the protein between amino acids 151 and 200, a domain that contains a series of highly charged amino acid residues. Coimmunoprecipitation assays determined that UL84 interacts with a protein domain present within the first 215 amino acids of IE2. We also show that an intact leucine zipper domain of UL84 is required for a stable interaction with IE2 and UL84 leucine zipper mutants fail to complement oriLyt-dependent DNA replication. UL84 leucine zipper mutants no longer interfere with IE2-mediated transactivation of the UL112/113 promoter, confirming that the leucine zipper is essential for a functional interaction with IE2. In addition, we demonstrate that both the leucine zipper and oligomerization domains of UL84 can act as transdominant-negative inhibitors of lytic replication in the transient assay, strongly suggesting that both an IE2-UL84 and a UL84-UL84 interaction are required for DNA synthesis.