Deletion mutants of the herpes simplex virus type 1 UL8 protein: Effect on DNA synthesis and ability to interact with and influence the intracellular localization of the UL5 and UL52 proteins
Deletion mutants of the herpes simplex virus type 1 UL8 protein: Effect on DNA synthesis and ability to interact with and influence the intracellular localization of the UL5 and UL52 proteins
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DOI：
10.1006/viro.1997.8763
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发表时间：
1997-10-13
期刊：
影响因子：
3.7
通讯作者：
Stow, ND
中科院分区：
文献类型：
--
作者：
Barnard, EC;Brown, G;Stow, ND
单纯疱疹病毒1型(HSV-1)解旋酶是病毒DNA复制机制的重要组成部分，是病毒编码的UL5、UL8和UL52蛋白的三聚体复合体。UL5和UL52亚基的组装保留了这两种酶的活性，UL8蛋白参与了这些功能的调节，促进了复合体的有效核摄取，并与其他病毒DNA复制蛋白相互作用。为了进一步了解UL8，我们构建了在人巨细胞病毒主要即刻早期启动子的控制下，在其N端或C端截短或在内部缺乏氨基酸的突变蛋白的表达载体。N端23个氨基酸或C端33个氨基酸的缺失使UL8在瞬时转染法中丧失了支持DNA复制的能力。在野生型产物存在的情况下，所测试的UL8突变体中没有一个表现出强烈的显性负表型，尽管在缺乏165个N末端或497个C末端氨基酸的突变体中观察到了一些复制抑制。免疫荧光检测了UL8突变体在共表达UL5的情况下促进UL52核高效定位的能力。用重组杆状病毒表达突变体，免疫沉淀法检测突变体与UL5和UL52的相互作用。突变体的复制能力与它们将UL52定位到细胞核的能力有关，但与它们与UL5和UL52的相互作用无关。这一性质排除了确定UL8对其假定的核功能重要的任何区域的可能性。(C)1997年学术出版社。
The herpes simplex virus type 1 (HSV-1) helicase-primase, an essential component of the viral DNA replication machinery, is a trimeric complex of the virus-coded UL5, UL8, and UL52 proteins. An assembly of the UL5 and UL52 subunits retains both enzymic activities, and the UL8 protein has been implicated in modulating these functions, facilitating efficient nuclear uptake of the complex and interacting with other viral DNA replication proteins. To further our understanding of UL8, we have constructed plasmids expressing mutant proteins, truncated at their N- or C-termini or lacking amino acids internally, under the control of the human cytomegalovirus major immediate-early promoter. Deletion of 23 amino acids from the N-terminus or 33 from the C-terminus abolished the ability of UL8 to support DNA replication in transient transfection assays. None of the UL8 mutants tested exhibited a strong dominant negative phenotype in the presence of the wild-type product, although some inhibition of replication was observed with mutants lacking 165 N-terminal or 497 C-terminal amino acids. The ability of the UL8 mutants to facilitate efficient nuclear localization of UL52 in the presence of coexpressed UL5 was examined by immunofluorescence. Selected mutants were also expressed by recombinant baculoviruses and tested for interaction with UL5 and UL52 in immunoprecipitation assays. The replicative ability of the mutants was found to correlate with their ability to localize UL52 to the nucleus, but not their interaction with UL5 and UL52. This property precluded the identification of any region of UL8 important for its presumed nuclear functions. (C) 1997 Academic Press.