Schröder H C, Falke D, Weise K, Bachmann M, Carmo-Fonseca M, Zaubitzer T, Müller W E
Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Mainz, F.R.G.
Virus Res. 1989 May;13(1):61-78. doi: 10.1016/0168-1702(89)90087-7.
A monoclonal antibody (MAb) raised against the pore-complex lamina fraction from CV-1 cells was used to study alterations of gene expression in herpes simplex virus type 1 (HSV-1)-infected cells. This MAb, which recognized only one cellular polypeptide of 60,000 Da, selectively stained the nucleus in immunofluorescence microscopy, showing a punctuated pattern either at the nuclear surface or at the nuclear rim. By immunoelectron microscopy, the p60 antigen could be localized in the nuclear pore complex structure. Infection of CV-1 cells with HSV-1 resulted in a drastic change of the nuclear staining pattern. Four hours p.i., a clustering of the p60 antigen and, 12 h p.i., a formation of finger-like holes, penetrating the nucleus, occurred. Later in infection (22 h p.i.) the antigen was found to be almost absent. By RNA blot hybridization it was demonstrated that, after HSV-1 infection, the level of cellular mRNA (beta-tubulin) gradually decreased, while the level of HSV major DNA binding protein (DBP) mRNA increased, reaching maximal level 3-6 h p.i. Interestingly, the level of beta-tubulin gene transcripts changed differentially in the polysomal and in the nuclear fraction during the initial phase of infection, in contrast to the viral DBP transcripts, indicating that, after HSV infection, host cell transcripts accumulate in the nucleus. Evidence is presented indicating that this change is not due to altered nucleocytoplasmic mRNA transport but is due to an impaired splicing of host cell transcripts in HSV-infected cells. The MAb, directed against the nuclear pore p60 antigen, strongly inhibited the ATP-dependent efflux of both cellular and viral mRNA from isolated nuclei. The ATP-dependence of the efflux did not change during viral infection. However, the inhibitory potency of the MAb was found to be lost at the final stage of HSV infection, paralleling the loss of p60 antigen.
一种针对CV-1细胞孔复合体层板组分产生的单克隆抗体（MAb）被用于研究单纯疱疹病毒1型（HSV-1）感染细胞中基因表达的变化。这种MAb仅识别一种60,000道尔顿的细胞多肽，在免疫荧光显微镜下选择性地对细胞核进行染色，在核表面或核边缘呈现出点状模式。通过免疫电子显微镜观察，p60抗原可定位于核孔复合体结构中。用HSV-1感染CV-1细胞导致核染色模式发生剧烈变化。感染后4小时，p60抗原聚集，感染后12小时，出现穿透细胞核的指状孔。在感染后期（感染后22小时），几乎检测不到该抗原。通过RNA印迹杂交证明，HSV-1感染后，细胞mRNA（β-微管蛋白）水平逐渐下降，而HSV主要DNA结合蛋白（DBP）mRNA水平升高，在感染后3 - 6小时达到最高水平。有趣的是，与病毒DBP转录本不同，在感染初期，β-微管蛋白基因转录本在多核糖体和细胞核组分中的变化存在差异，这表明HSV感染后，宿主细胞转录本在细胞核中积累。有证据表明，这种变化不是由于核质mRNA转运改变，而是由于HSV感染细胞中宿主细胞转录本剪接受损。针对核孔p60抗原的MAb强烈抑制从分离细胞核中ATP依赖的细胞和病毒mRNA外流。在病毒感染期间，外流的ATP依赖性没有改变。然而，发现MAb的抑制效力在HSV感染的最后阶段丧失，这与p60抗原的丧失平行。
