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

人类疱疹病毒6型(HHV-6)的两个变种已经根据这些特性的不同而被区分开来。我们已经测定了HHV-6毒株HST的全序列，该毒株属于B变异体。在HHV-6全基因组161573个碱基的相邻序列中，确定了114个潜在的开放阅读框，以及一些氨基酸同源性显著差异的基因，这些差异可能导致两个变异体的特征差异。此外，我们还使用变种特异的引物对属于HHV-6A或B的14个不同的毒株进行了分型。U12基因在感染后通过剪接的mRNA表达。克隆了U12基因，并在K562细胞中进行了表达。U12在功能上编码RANTES、MIP-1a、MIP-1b和MCP-1等b-趋化因子的钙动员受体，但不编码a-趋化因子IL-8。U83基因在感染HHV-6B的细胞中表达较晚。从转染的Cos-7细胞上清液中表达并纯化了重组U83。U83蛋白诱导瞬时钙动员，对单核细胞系THP-1细胞具有高效的趋化活性。U1102株和HST株HHV-6GHS在T7-痘苗病毒瞬时表达系统中获得表达。免疫沉淀法和间接免疫荧光法表明，OHV3与HST-Gh反应，但不与U1102-Gh反应。此外，OHV3还与U1102 Gh和HST Gh之间形成的嵌合GHS反应，该嵌合GHS含有HST Gh的272到422个氨基酸。U1102和HST的序列比较发现，该区域有7个氨基酸差异。将定点突变引入这些位置，然后研究对OHV3的反应性。HST Gh第389位的精氨酸被证明是HHV-6B特异性OHV3反应性的决定因素。

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.