Samaniego L A, Neiderhiser L, DeLuca N A
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
J Virol. 1998 Apr;72(4):3307-20. doi: 10.1128/JVI.72.4.3307-3320.1998.
The immediate-early (IE) proteins of herpes simplex virus (HSV) function on input genomes and affect many aspects of host cell metabolism to ensure the efficient expression and regulation of the remainder of the genome and, subsequently, the production of progeny virions. Due to the many and varied effects of IE proteins on host cell metabolism, their expression is not conducive to normal cell function and viability. This presents a major impediment to the use of HSV as a vector system. In this study, we describe a series of ICP4 mutants that are defective in different subsets of the remaining IE genes. One mutant, d109, does not express any of the IE proteins and carries a green fluorescent protein (GFP) transgene under the control of the human cytomegalovirus IE promoter (HCMVIEp). d109 was nontoxic to Vero and human embryonic lung (HEL) cells at all multiplicities of infection tested and was capable of establishing persistent infections in both of these cell types. Paradoxically, the genetic manipulations that were required to eliminate toxicity and allow the genome to persist in cells for long periods of time also dramatically lowered the level of transgene expression. Efficient expression of the HCMVIEp-GFP transgene in the absence of ICP4 was dependent on the ICP0 protein. In d109-infected cells, the level of transgene expression was very low in most cells but abundant in a small subpopulation of cells. However, expression of the transgene could be induced in cells containing quiescent d109 genomes weeks after the initial infection, demonstrating the functionality of the persisting genomes.
单纯疱疹病毒（HSV）的立即早期（IE）蛋白作用于输入的基因组，影响宿主细胞代谢的许多方面，以确保基因组其余部分的有效表达和调控，进而确保子代病毒粒子的产生。由于IE蛋白对宿主细胞代谢有诸多不同的影响，它们的表达不利于正常细胞功能和生存能力。这给将HSV用作载体系统带来了重大障碍。在本研究中，我们描述了一系列在其余IE基因的不同亚组中存在缺陷的ICP4突变体。一个突变体d109不表达任何IE蛋白，并在人巨细胞病毒IE启动子（HCMVIEp）的控制下携带绿色荧光蛋白（GFP）转基因。在所有测试的感染复数下，d109对Vero细胞和人胚肺（HEL）细胞均无毒，并且能够在这两种细胞类型中建立持续感染。矛盾的是，消除毒性并使基因组在细胞中长期存在所需的基因操作也显著降低了转基因表达水平。在没有ICP4的情况下，HCMVIEp-GFP转基因的有效表达依赖于ICP0蛋白。在感染d109的细胞中，大多数细胞中的转基因表达水平非常低，但在一小部分细胞亚群中表达丰富。然而，在初次感染数周后，含有静止d109基因组的细胞中的转基因表达可以被诱导，这证明了持续存在的基因组的功能。
