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Molecular biological analysis of the proteins which associated with the infection of human herpesvirus 6

人疱疹病毒6型感染相关蛋白的分子生物学分析

基本信息

批准号：
09470085
负责人：
YAMANISHI Koichi
金额：
$ 9.28万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470085/
关键词：
HHV 6 variant full sequence glycoprotein neutralizing antibody chemokine chemokine receptor signal transduction 糖蛋白 gH 中和抗体

项目摘要

Two variants of Human herpesvirus 6 (HHV-6) have been distinguished based on differences in those properties. We have determined the complete DNA sequence of HHV-6 strain HST, the causing agent of exanthem subitum, which belongs of variant B. Thus 114 potential open reading frames were identified within the 161573-bp contiguous sequence of the entire HHV-6 genome, as well as some genes with remarkable differences in amino acid identity that may lead to characteristic differences of two variants. Also, we have carried out typing of 14 different strains belonging to HHV-6A or B by PCR using variant-specific primers.The U12 gene is expressed late in infection from a spliced mRNA. The U12 gene was cloned, and the protein was expressed in K562 cells. U12 functionally encoded a calcium-mobilizing receptor for b-chemokines such as RANTES, MIP-1a, MIP-1b, and MCP-1 but not for the a-chemokine IL-8.The U83 gene is expressed late in cells infected with HHV-6B. The recombinant U83 was expressed and purified from supernatant of transfected cos-7 cells. The U83 protein induced transient calcium mobilization and exhibited an efficient chemotaxis activity for monocytic cell line THP-1 cells.HHV-6 gHs from strain U1102 and strain HST were expressed in a T7-vaccinia virus transient expression system. OHV3 reacted with HST gH, but not with U1102 gH, in an immunoprecipitation and an indirect immunofluorescence assays. In addition, OHV3 reacted with chimeric gHs, formed between U1102 gH and HST gH, containing amino acids 272 to 422 of HST gH. Sequence comparison between U1102 and HST showed seven amino acid differences in this region. Site-specific mutations were introduced into these positions and then reactivity against OHV3 was investigated. The argenine at position 389 of HST gH was shown to be a determinant of the HHV-6B-specific reactivity of OHV3.

人类疱疹病毒 6 (HHV-6) 的两个变种已根据这些特性的差异进行了区分。我们确定了引起急疹的 HHV-6 毒株 HST 的完整 DNA 序列，属于变体 B。因此，在整个 HHV-6 基因组的 161573 bp 连续序列中鉴定出 114 个潜在的开放阅读框，以及一些氨基酸一致性显着差异的基因，这些基因可能导致两个变体的特征差异。此外，我们还使用变体特异性引物通过 PCR 对属于 HHV-6A 或 B 的 14 种不同毒株进行了分型。U12 基因在感染后期通过剪接的 mRNA 表达。克隆U12基因，并在K562细胞中表达该蛋白。 U12 功能性地编码 B 趋化因子（如 RANTES、MIP-1a、MIP-1b 和 MCP-1）的钙动员受体，但不编码 a 趋化因子 IL-8。U83 基因在感染 HHV-6B 的细胞中晚期表达。从转染的cos-7细胞的上清液中表达并纯化重组U83。 U83蛋白诱导瞬时钙动员并对单核细胞系THP-1细胞表现出有效的趋化活性。来自菌株U1102和菌株HST的HHV-6 gH在T7痘苗病毒瞬时表达系统中表达。在免疫沉淀和间接免疫荧光测定中，OHV3 与 HST gH 发生反应，但不与 U1102 gH 发生反应。此外，OHV3与U1102 gH和HST gH之间形成的嵌合gH反应，所述嵌合gH包含HST gH的氨基酸272至422。 U1102 和 HST 之间的序列比较显示该区域有 7 个氨基酸差异。将位点特异性突变引入这些位置，然后研究针对 OHV3 的反应性。 HST gH 389 位的精氨酸被证明是 OHV3 的 HHV-6B 特异性反应性的决定因素。