Thoma Corina, Bogner Elke
Institute of Virology, Charité Universitâtsmedizin Berlin, Berlin, Germany.
Antivir Ther. 2010;15(3):391-400. doi: 10.3851/IMP1536.
In order to define the role of the human cytomegalovirus (HCMV) small terminase subunit pUL89, analysis by RNA interference was applied.
Cell lines expressing pUL89-specific short hairpin RNAs (shRNAs) were constructed by transduction of shRNAs via infection with retroviral vectors. These cell lines were infected with HCMV AD169 and were analysed for pUL89 expression, viral yield, plaque reduction, amount of viral DNA and particle formation.
After infection of the cell lines with HCMV, the expression of pUL89 was reduced by up to 86% for shRNA_A and 84% for shRNA_B at the later time points of infection. Cell lines expressing shRNA_C and the control had no effect on the pUL89 expression level. In addition, the inhibitory effect corresponded to a decrease in viral growth kinetics, viral DNA and plaque formation. Analysis by electron microscopy demonstrated that infection of cells expressing pUL89-specific shRNA_A and shRNA_B resulted in a complete inhibition of viral particle formation.
HCMV is a serious life-threatening opportunistic pathogen in immunocompromised patients. Because of multiple problems caused by the current available drugs, development of new strategies are needed. Our data clearly demonstrate that pUL89-specific shRNAs mediated the inhibition of formation of replicative infectious particles and therefore represent a new promising mechanism for antiviral therapy against HCMV infection.
为了明确人类巨细胞病毒（HCMV）小末端酶亚基pUL89的作用，采用RNA干扰进行分析。
通过逆转录病毒载体感染转导短发夹RNA（shRNA）构建表达pUL89特异性shRNA的细胞系。用HCMV AD169感染这些细胞系，并分析pUL89表达、病毒产量、蚀斑减少、病毒DNA量和病毒颗粒形成情况。
用HCMV感染细胞系后，在感染后期，shRNA_A使pUL89表达降低达86%，shRNA_B使pUL89表达降低达84%。表达shRNA_C的细胞系和对照对pUL89表达水平无影响。此外，抑制作用与病毒生长动力学、病毒DNA和蚀斑形成的降低相对应。电子显微镜分析表明，感染表达pUL89特异性shRNA_A和shRNA_B的细胞导致病毒颗粒形成完全受到抑制。
HCMV是免疫功能低下患者中严重威胁生命的机会性病原体。由于现有药物引发多种问题，需要开发新策略。我们的数据清楚地表明，pUL89特异性shRNA介导了对复制性感染性颗粒形成的抑制，因此代表了一种针对HCMV感染的抗病毒治疗新的有前景的机制。
