The Effect of CMV ppUL82 Driven by its Natural Promoter on the Viral Gene Expression and Viral yield

• Main Authors: Yu-Ting Hung, 洪于婷
• Other Authors: Szu-Hao Kung
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/11615352571786675079

碩士 === 國立陽明大學 === 醫學生物技術研究所 === 94 === Human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality among immunocompromised patients, especially recipients of transplants and patients with acquired immunodeficiency syndrome (AIDS). Laboratory diagnosis of HCMV has thus become one of the most frequent tests conducted in clinical virology laboratories. However, cell culturing and isolation of HCMV, a gold standard method for laboratory diagnosis of HCMV, often become difficult when early-passaged clinical isolates are encountered. On the other hand, development of the anti-HCMV agents has promoted the emergence of drug-resistant HCMV strains that require the determination of antiviral susceptibility. Herein we propose to develop the genetically modified cell lines to hopefully resolve the problems. An HCMV protein, ppUL82 protein, has been shown to enhance the yield of progeny virus, possibly via transactivation of the immediate early (IE) promoter. Thus, a stable cell line, designated U373MG-pp65p-Flag-UL82-IG, was established to permit the ppUL82 and the green fluorescent protein (GFP) to be expressed following the expression kinetics of the UL82 gene. Another stable cell line, U373MG-pUL94-SIG, was developed by stable transfection of cells with an expression cassette that contains the secreted alkaline phosphatase (SEAP) reporter gene governed by the promoter of a HCMV late gene, the UL94 gene. Upon infection of U373MG-pp65p-Flag-UL82-IG cells with CMV strains, the infected cells displayed GFP as early as 1 dpi (for prototype AD169 and three CMV strains) or 2 dpi (for two early-passaged clinical isolates). On the other hand, GFP was not seen until 3 dpi or later for the infected-UL82-null control cells. As the infection progressed, GFP could be seen at the periphery of the initially infected U373MG-pp65p-Flag-UL82-IG cells (as GFP clusters) 6 dpi. This was in marked contrast with the infected UL82-null control cells where GFP was restrained in the originally-infected cells (restricted GFP pattern) at the same time point. The temporal regulation of the ectopic ppUL82 followed the expression kinetics of its natural promoter. Representative HCMV IE, E and L genes were transcriptionally up-regulated significantly in the infected U373MG-pp65p-Flag-UL82-IG compared with the UL82-null control cells as revealed by the real-time PCR analysis. In line with the finding, the copy number of viral genome was also significantly enhanced up to 4.98 fold in the presence of the exogenous ppUL82. The U373MG-pUL94-SIG cell line was assessed for the suitability for determination of antiviral susceptibility. For the cell line infected by AD169, a drug-sensitive strain, the induced SEAP activity from U373MG-pUL94-SIG cell line was progressively inhibited as a function of the concentration of PAA in the culture medium. Moreover, evaluations of the stable line with representative drug-resistant HCMV strains demonstrated that their PAA susceptibilities could be determined. The U373MG-pp65p-Flag-UL82-IG evidently increases virus replication and yield, permitting early diagnosis and isolation of low-passage HCMV cultures. U373MG-pUL94-SIG cell line can be utilized to develop a rapid screening method for determination of anti-HCMV susceptibilities, with the potential to the development of an automated assay.