Development of novel methods for diagnosis of cytomegalovirus and HHV-6 reactivation.

为了开发诊断人巨细胞病毒（HCMV）和人疱疹病毒6 （HHV-6）再激活的敏感和特异性方法，我们研究了我们之前发现的它们的潜伏期相关转录物。因此，我们发现人类疱疹病毒6 （h6lt）的潜伏期相关转录本在体内和体外潜伏期和再激活之间的一个相当稳定的中间阶段最大限度地表达。h6lt在这一阶段作为即时早期（IE）蛋白1的来源，其上调了病毒的再激活。我们还分析了在H6LT中编码的HHV-6 IE2蛋白的功能。通过酵母双杂交筛选，发现异质核核糖核蛋白K （hnRNP K）和酪蛋白激酶2 （CK2β）亚基与HHV-6 IE2特异性相互作用。这些发现表明，在H6LT中编码的HHV-6 IE2蛋白可能影响病毒和细胞RNA的转录和翻译。为了分析h6lt的基因调控，我们建立了HHV-6重组系统。重组HHV-6具有增强型绿色荧光蛋白-嘌呤霉素基因盒，内含巨细胞病毒主即刻早期启动子。在HHV-6潜伏期，巨细胞病毒启动子使用了巨细胞病毒潜伏期的转录起始位点，这表明HHV-6潜伏期的转录控制可能与HCMV潜伏期有一些共同的机制。为了确定HHV-6再激活的因素，我们研究了健康成人HHV-6再激活与工作疲劳的关系。用半定量PCR法检测HHV-6病毒DNA的再激活。结果，88%（35/40）的健康成人在疲劳期间唾液中会脱落重新激活的HHV-6, 23%（9/40）的健康成人在假期后（大约1周）会脱落HHV-6。假日过后，HHV-6 DNA拷贝数也有所减少。这些发现表明HHV-6在工作引起的疲劳中被重新激活。少

To develop the sensitive and specific method for diagnosis of human cytomegalovirus (HCMV) and human herpesvinas 6 (HHV-6) reactivation, we have investigated their latency-associated transcripts that we have previously identified. As a result, we have revealed that the latency-associated transcripts of human herpesvinis 6 (H6LTs) were maximally expressed at a fairly stable intermediate stage between latency and reactivation both in vivo and in vitro., and that H6LTs functioned as sources of immediate-early (IE) protein 1 at this stage, which up-regulated the viral reactivation. We have also analyzed the function of HHV-6 IE2 protein that is encoded in the H6LT. We used yeast two-hybrid screening, and found heterogeneous nuclear ribonucleoprotein K (hnRNP K) and the beta subunit of casein kinase 2 (CK2β) specifically interacted with HHV-6 IE2. These findings indicate that the HHV-6 IE2 protein encoded in the H6LT may affect viral and cellular RNA transcription and translation in viral l … More atency and reactivation.To analyze the gene regulation of H6LTs, we have established the recombination system of HHV-6. The recombinant HHV-6 had an enhanced green fluorescent protein-puromycin gene cassette containing the cytomegalovirus major immediate-early promoter. During HHV-6 latency, the cytomegalovinas promoter used the transcription start sites employed in cytomegalovinis latency, which suggests that the transcriptional control of HHV-6 latency may share some common mechanism with HCMV latency.To identify the factor(s) of HHV 6 reactivation, we have studied the association with HHV-6 reactivation and the work-induced fatigue in healthy adults. Reactivation of HHV-6 was examined for viral DNA by semi-quantitative PCR method. As a result, 88% (35/40) of healthy adults shed the reactivated HHV-6 in the saliva during the fatigue, and 23% (9/40) shed HHV-6 after holidays (approximately 1 week). The copy number of HHV-6 DNA was also reduced after holidays. These findings suggest that HHV-6 is reactivated during the work-induced fatigue. Less