Babiss L E, Ginsberg H S
J Virol. 1984 Apr;50(1):202-12. doi: 10.1128/JVI.50.1.202-212.1984.
To determine the role adenovirus 5 early region 1b-encoded 21- and 55-kilodalton proteins play in adenovirus productive infection, mutants have been isolated which were engineered to contain small deletions or insertions at 5.8, 7.9, or 9.6 map units. By using an overlap recombination procedure involving H5dl314 (delta 3.7 to 4.6 map units) DNA cleaved at 2.6 map units with ClaI and the adenovirus 5 XhoI-C (0 to 15.5 map units) fragment containing the desired mutation, viral mutants were isolated by their ability to produce plaques on KB cell line 18, which constitutively expresses only viral early region 1b functions (Babiss et al., J. Virol. 46:454-465, 1983). DNA sequence analysis of the viral mutants isolated (H5dl118, H5dl110, H5in127, and H5dl163) indicates that all of the viruses contain mutations which affect the 55-kilodalton protein, whereas dl118 should also produce a truncated form of the 21-kilodalton protein. When analyzed for their replication characteristics in HeLa cells, all of the mutant viruses exhibited extended eclipse periods and effected yields that were reduced to 10% or less of that produced by H5sub309 (parent virus of the mutants which is phenotypically identical to wild-type adenovirus 5). When compared with characteristics of sub309, the early and late transcription and DNA replication of the mutants were similar, but synthesis of late polypeptides and late cytoplasmic mRNAs was greatly reduced. Quantitation of mutant virus-specific late mRNAs associated with polysomes revealed a threefold reduction when compared with that of sub309. Analysis of infected cell extracts further revealed that these mutants were incapable of efficiently shutting off host cell protein synthesis, suggesting that the 55-kilodalton protein plays a role in this process. These data suggest that early region 1b products may function by interacting with additional viral or host cell macromolecules to modulate host cell shutoff or that some late viral mRNA or polypeptide may potentiate this reaction.
为确定腺病毒5型早期区域1b编码的21千道尔顿和55千道尔顿蛋白在腺病毒增殖性感染中所起的作用，已分离出一些突变体，这些突变体经基因工程改造，在5.8、7.9或9.6个图距单位处含有小的缺失或插入。通过使用一种重叠重组程序，该程序涉及用ClaI在2.6个图距单位处切割的H5dl314（3.7至4.6个图距单位缺失）DNA和含有所需突变的腺病毒5型XhoI-C（0至15.5个图距单位）片段，通过它们在KB细胞系18上产生噬斑的能力分离出病毒突变体，KB细胞系18仅组成性表达病毒早期区域1b的功能（巴比斯等人，《病毒学杂志》46:454 - 465，1983年）。对分离出的病毒突变体（H5dl118、H5dl110、H5in127和H5dl163）进行DNA序列分析表明，所有病毒都含有影响55千道尔顿蛋白的突变，而dl118还应产生一种截短形式的21千道尔顿蛋白。当分析它们在HeLa细胞中的复制特性时，所有突变病毒都表现出延长的隐蔽期，并且产生的产量降低到H5sub309（突变体的亲本病毒，其表型与野生型腺病毒5相同）产生产量的10%或更低。与sub309的特性相比，突变体的早期和晚期转录以及DNA复制相似，但晚期多肽和晚期细胞质mRNA的合成大大减少。对与多核糖体相关的突变病毒特异性晚期mRNA进行定量分析发现，与sub309相比减少了三倍。对感染细胞提取物的分析进一步表明，这些突变体无法有效关闭宿主细胞蛋白质合成，这表明55千道尔顿蛋白在这一过程中起作用。这些数据表明，早期区域1b产物可能通过与其他病毒或宿主细胞大分子相互作用来调节宿主细胞关闭，或者某些晚期病毒mRNA或多肽可能增强这一反应。
