Chan S R, Bloomer C, Chandran B
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City 66160, USA.
Virology. 1998 Jan 5;240(1):118-26. doi: 10.1006/viro.1997.8911.
Monoclonal antibody (Mab) 11D1 specific for HHV-8 showed a predominantly nuclear membrane fluorescence with about 30% of phorbol ester (TPA)-induced HHV-8-carrying BCBL-1 cells and with 2-8% of uninduced cells, but not with other herpes viruses infected cells. This Mab immunoprecipitated a 50-kDa polypeptide from BCBL-1 cells. The synthesis of this polypeptide was reduced but not inhibited by phosphonoacetic acid (PAA). A 2.3-kb cDNA insert from a cDNA library of TPA-induced BCBL-1 cells was identified by Mab 11D1. Sequence analysis shows that this cDNA is open at the 5' end and encodes two ORFs of 396AA (5' end) and 357AA (3' end). These ORFs are identical to the published HHV-8 ORFs 59 and 58, respectively in vitro transcription and translation of the cDNA resulted in the synthesis of a 50-kDa polypeptide and its partial peptide map was identical to that of the 50-kDa polypeptide detected in the TPA induced BCBL-1 cells. Riboprobe made from the cDNA insert hybridized with several viral specific RNAs from BCBL-1 cells. Levels of these RNA species were reduced, but not inhibited by PAA. These characteristics are similar to other herpes viruses genes encoding the lytic cycle associated early-late class accessory proteins that are essential for viral DNA replication. This Mab 11D1 recognizing the HHV-8 lytic cycle associated ORF 59 protein will be highly useful in monitoring the lytic replicative cycle.
针对HHV-8的单克隆抗体（Mab）11D1在约30%的佛波酯（TPA）诱导的携带HHV-8的BCBL-1细胞以及2%-8%的未诱导细胞中显示出主要为核膜荧光，但在其他感染疱疹病毒的细胞中未显示。该单克隆抗体从BCBL-1细胞中免疫沉淀出一种50 kDa的多肽。该多肽的合成被膦甲酸（PAA）减少但未被抑制。通过Mab 11D1从TPA诱导的BCBL-1细胞的cDNA文库中鉴定出一个2.3 kb的cDNA插入片段。序列分析表明，该cDNA在5'端开放，编码两个分别为396个氨基酸（5'端）和357个氨基酸（3'端）的开放阅读框（ORF）。这些ORF分别与已发表的HHV-8的ORF 59和58相同。该cDNA的体外转录和翻译导致合成了一种50 kDa的多肽，其部分肽图与在TPA诱导的BCBL-1细胞中检测到的50 kDa多肽相同。由该cDNA插入片段制备的核糖探针与来自BCBL-1细胞的几种病毒特异性RNA杂交。这些RNA种类的水平被PAA降低但未被抑制。这些特征与其他编码对病毒DNA复制至关重要的裂解周期相关早期-晚期类辅助蛋白的疱疹病毒基因相似。这种识别HHV-8裂解周期相关ORF 59蛋白的Mab 11D将在监测裂解复制周期方面非常有用。
