Prolonged gene expression and cell survival after infection by a herpes simplex virus mutant defective in the immediate-early genes encoding ICP4, ICP27, and ICP22
Prolonged gene expression and cell survival after infection by a herpes simplex virus mutant defective in the immediate-early genes encoding ICP4, ICP27, and ICP22
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DOI：
10.1128/jvi.70.9.6358-6369.1996
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发表时间：
1996-09-01
影响因子：
5.4
通讯作者：
DeLuca, NA
中科院分区：
文献类型：
--
作者：
Wu, NX;Watkins, SC;DeLuca, NA
在感染的早期，单纯疱疹病毒(HSV)表达四种即刻早期(IE)调节蛋白：ICP4、ICP0、ICP22和ICP27。顺式病毒中IE蛋白组的系统失活，以及随后对产生的突变体的表型分析，应该有助于深入了解这些蛋白在HSV生命周期中的功能，以及感染细胞中被感染早期阻断的病毒株感染的特定大分子事件的改变或扰动。这一方法也可能为评估HSV突变体用于基因转移实验的有效性和安全性提供合理的基础。在这项研究中，我们产生并检测了一株同时突变于HSV ICP4、ICP27和ICP22基因的HSV突变体的表型。与ICP4(D120)、ICP4和ICP27(D92)以及ICP4和ICP22(D96)缺陷突变不同，ICP4、ICP27和ICP22(D95)缺陷突变对Vero和人胚胎肺细胞的毒性明显降低。以每个细胞10pfu的复数感染D95的细胞在感染后至少3天内保持相对正常的形态，并表达来自病毒和细胞基因组的基因。其他突变背景的毒性太大，不能在感染后1天后进行基因表达检测。然而，当通过感染细胞形成集落的能力来衡量细胞存活率时，d95抑制集落形成的作用类似于d92。在感染d120、d92、d96和d95的细胞中，宿主细胞的DNA合成受到抑制，这一明显的悖论得到了证实。此外，所有突变体在电子显微镜下都表现出明显和独特的核形态变化。D95感染细胞的外观与未感染细胞的外观不同，在细胞核内形成了大的圆形结构。D95感染细胞大量表达ICP0，ICP0在感染后早期聚集在细胞核的细小点状结构中，并融合或生长成电子显微镜下可见的大的圆形物体。因此，虽然在没有ICP4、ICP22和ICP27的情况下ICP0的大量积累可能允许延长基因的表达，但细胞存活受到损害，部分原因是抑制了细胞DNA的合成。
Very early in infection, herpes simplex virus (HSV) expresses four immediate-early (IE) regulatory proteins, ICP4, ICP0, ICP22, and ICP27. The systematic inactivation of sets of the IE proteins in cis, and the subsequent phenotypic analysis of the resulting mutants, should provide insights into how these proteins function in the HSV life cycle and also into the specific macromolecular events that are altered or perturbed in cells infected with virus strains blocked very early in infection. This approach may also provide a rational basis to assess the efficacy and safety of HSV mutants for use in gene transfer experiments. In this study, we generated and examined the phenotype of an HSV mutant simultaneously mutated in the ICP4, ICP27, and ICP22 genes of HSV. Unlike mutants deficient in ICP4 (d120), ICP4 and ICP27 (d92), and ICP4 and ICP22 (d96), mutants defective in ICP4, ICP27, and ICP22 (d95) were visually much less toxic to Vero and human embryonic lung cells. Cells infected with d95 at a multiplicity of infection of 10 PFU per cell retained a relatively normal morphology and expressed genes from the viral and cellular genomes for at least 3 days postinfection. The other mutant backgrounds were too toxic to allow examination of gene expression past 1 day postinfection. However, when cell survival was measured by the capacity of the infected cells to form colonies, d95 inhibited colony formation similarly to d92. This apparent paradox was reconciled by the observation that host cell DNA synthesis was inhibited in cells infected with d120, d92, d96, and d95. In addition, all of the mutants exhibited pronounced and distinctive alterations in nuclear morphology, as determined by electron microscopy. The appearance of d95-infected cells deviated from that of uninfected cells in that large circular structures formed in the nucleus. d95-infected cells abundantly expressed ICP0, which accumulated in fine punctate structures in the nucleus at early times postinfection and coalesced or grew to the large circular objects that were revealed by electron microscopy. Therefore, while the abundant accumulation of ICP0 in the absence of ICP4, ICP22, and ICP27 may allow for prolonged gene expression, cell survival is impaired, in part, as a result of the inhibition of cellular DNA synthesis.