The UL97 gene product of human cytomegalovirus is an early-late protein with a nuclear localization but is not a nucleoside kinase
The UL97 gene product of human cytomegalovirus is an early-late protein with a nuclear localization but is not a nucleoside kinase
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DOI：
10.1128/jvi.70.9.6340-6346.1996
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发表时间：
1996-09
影响因子：
5.4
通讯作者：
D. Michel;I. Pavić;A. Zimmermann;E. Haupt;K. Wunderlich;M. Heuschmid;T. Mertens
中科院分区：
文献类型：
--
作者：
D. Michel;I. Pavić;A. Zimmermann;E. Haupt;K. Wunderlich;M. Heuschmid;T. Mertens
用针对UL97蛋白47kda片段的多克隆抗血清分析了UL97基因产物在人巨细胞病毒（HCMV）感染人包皮成纤维细胞（HFF）时的时间表达和该蛋白的亚细胞定位。Western blot（免疫印迹）分析感染16 h后可检测到UL97蛋白。由于UL97的表达在两种DNA复制抑制剂磷酸乙酸和更昔洛韦的存在下才会降低，因此我们得出结论，UL97是一个早晚期基因，需要DNA复制才能最大限度地表达。间接免疫荧光法可在感染后22 h在病毒感染的HFF细胞核中观察到该蛋白。在含有UL97开放阅读框（ORF）的重组痘苗病毒感染的胸苷激酶缺陷143B细胞中，以及在HCMV主要立即早期启动子控制下瞬时表达UL97开放阅读框的HFF中，也检测到UL97蛋白的核定位。然而，UL97 ORF的瞬时表达的5'端缺失突变体还显示了UL97蛋白的细胞质定位，证实了该蛋白n端区域存在核定位位点。我们的高压液相色谱分析证实了UL97蛋白对更昔洛韦的磷酸化作用，但没有观察到天然核苷的特异性磷酸化，表明UL97蛋白不是核苷激酶。在重组ul97缺陷HCMV的空斑纯化过程中，该病毒生长缺陷；因此，我们推测UL97可能对病毒生命周期至关重要。
The temporal expression of the UL97 gene product during human cytomegalovirus (HCMV) infection of human foreskin fibroblasts (HFF) and subcellular localization of this protein were analyzed by using a polyclonal antiserum raised against a truncated UL97 protein of 47 kDa. The UL97 protein was detectable 16 h after infection by Western blot (immunoblot) analysis. Since only reduced UL97 expression occurred in the presence of two inhibitors of DNA replication, phosphonoacetic acid and ganciclovir, we conclude that UL97 is an early-late gene, requiring DNA replication for maximum expression. By indirect immunofluorescence, the protein could be visualized in the nuclei of virus-infected HFF 22 h after infection. Nuclear localization of the UL97 protein was also detected in thymidine kinase-deficient 143B cells infected with a recombinant vaccinia virus containing the entire UL97 open reading frame (ORF), as well as in HFF transiently expressing the entire UL97 ORF under the control of HCMV major immediate-early promoter. However, transiently expressed 5'-terminal deletion mutants of the UL97 ORF in addition showed a cytoplasmic localization of the UL97 protein, confirming the presence of a nuclear localization site in the N-terminal region of the protein. Our high-pressure liquid chromatography analyses confirmed the ganciclovir phosphorylation by the UL97 protein, but no specific phosphorylation of natural nucleosides was observed, indicating that the UL97 protein is not a nucleoside kinase. During plaque purification of recombinant UL97-deficient HCMV, this virus was growth defective; hence, we presume that UL97 may be essential for the viral life cycle.