Ripalti A, Boccuni M C, Campanini F, Landini M P
Istituto di Microbiologia, Facoltà di Medicina e Chirurgia, Policlinico S. Orsola, Bologna, Italy.
J Virol. 1995 Apr;69(4):2047-57. doi: 10.1128/JVI.69.4.2047-2057.1995.
We have used an antisense RNA approach in the analysis of gene function in human cytomegalovirus (HCMV). An astrocytoma cell line (U373-MG) that is permissive for virus replication was permanently transfected with a construct bearing sequence from HCMV UL44 (coding for the major late DNA-binding protein, ppUL44, also known as pp52 or ICP36) in an antisense orientation and under the control of the immediate-early enhancer-promoter element. Upon HCMV infection at a high multiplicity, we found a marked reduction in UL44 protein products (the ICP36 family of proteins) in established cell transfectants and a strong inhibition of virus yield in infected-cell supernatants at two weeks postinfection, while herpes simplex virus replication was not affected. In infected cells, viral DNA replication was strongly inhibited. While gene products such as pUS22 and pUL32 were also inhibited, pUL123 and pUL82 accumulated in the infected cells over time. Our data suggest an essential role for the UL44 family of proteins in HCMV replication and represent a model of virus inhibition by virus-induced antisense RNA synthesis in genetically modified cells.
我们已采用反义RNA方法来分析人巨细胞病毒（HCMV）的基因功能。一种对病毒复制敏感的星形细胞瘤细胞系（U373-MG）被永久转染了一个构建体，该构建体带有来自HCMV UL44的序列（编码主要晚期DNA结合蛋白，ppUL44，也称为pp52或ICP36），呈反义方向，并受立即早期增强子-启动子元件的控制。在高感染复数下进行HCMV感染后，我们发现在已建立的细胞转染子中UL44蛋白产物（ICP36蛋白家族）显著减少，并且在感染后两周时感染细胞上清液中的病毒产量受到强烈抑制，而单纯疱疹病毒的复制未受影响。在感染的细胞中，病毒DNA复制受到强烈抑制。虽然诸如pUS22和pUL32等基因产物也受到抑制，但随着时间的推移，pUL123和pUL82在感染细胞中积累。我们的数据表明UL44蛋白家族在HCMV复制中起重要作用，并代表了在基因修饰细胞中通过病毒诱导的反义RNA合成来抑制病毒的一种模型。
