DETECTION OF ADENOVIRUS TYPE 2-INDUCED EARLY POLYPEPTIDES USING CYCLOHEXIMIDE PRETREATMENT TO ENHANCE VIRAL PROTEIN-SYNTHESIS
DETECTION OF ADENOVIRUS TYPE 2-INDUCED EARLY POLYPEPTIDES USING CYCLOHEXIMIDE PRETREATMENT TO ENHANCE VIRAL PROTEIN-SYNTHESIS
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DOI：
10.1128/jvi.19.1.232-242.1976
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发表时间：
1976-01-01
影响因子：
5.4
通讯作者：
GREEN, M
中科院分区：
文献类型：
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作者：
HARTER, ML;SHANMUGAM, G;GREEN, M
通过聚丙烯酰胺梯度凝胶电泳和放射自显影法分析了由2型腺病毒感染的细胞[KB人口腔癌]合成的[35 S]甲硫氨酸标记的多肽。将环己酰亚胺（CH）加入感染的培养物中，以积累相对于宿主细胞mRNA的早期病毒mRNA。这使得病毒蛋白质的合成量增加，相对于宿主蛋白质后，去除CH和脉冲标记与[35 S]蛋氨酸。在标记期间，加入阿拉伯糖基胞嘧啶以防止晚期病毒蛋白的合成。该方法便于检测6种早期病毒诱导的多肽，命名为EP 1至EP 6（早期蛋白），表观分子量为75，000（75 K）、42 K、21 K、18 K、15 K和11 K。通过分别对来自感染和未感染细胞的[35 S]和[3 H]甲硫氨酸标记的多肽进行共电泳获得了支持性数据。这些早期多肽中有三种以前没有报道过。CH预处理使EP 4和EP 6的合成速率提高了20- 30倍，使其他的合成速率提高了约2倍。病毒诱导的蛋白质的合成的最大速率变化，以不同的方式，随着时间postinfection和CH预处理。由于CH预处理似乎增加了早期病毒蛋白的水平，这可能是一个有用的程序，以帮助他们的分离和功能表征。
[35S]methionine-labeled polypeptides synthesized by adenovirus type 2-infected cells [KB human oral carcinoma] were analyzed by polyacrylamide gradient gel electrophoresis and autoradiography. Cycloheximide (CH) was added to infected cultures to accumulate early viral mRNA relative to host cell mRNA. This allowed viral proteins to be synthesized in increased amounts relative to host proteins after removal of CH and pulse-labeling with [35S]methionine. During the labeling period arabinosyl cytosine was added to prevent the synthesis of late viral proteins. This procedure facilitated the detection of 6 early viral-induced polypeptides, designated EP1 through EP6 (early protein), with apparent MW''s of 75,000 (75K), 42K, 21K, 18K, 15K and 11K. Supportive data were obtained by coelectrophoresis of [35S]- and [3H]methionine-labeled polypeptides from infected and uninfected cells, respectively. Three of these early polypeptides were not previously reported. CH pretreatment enhanced the rates of synthesis of EP4 and EP6 20- to 30-fold and enhanced that of the others approximately 2-fold. The maximal rates of synthesis of the virus-induced proteins varied, in a different manner, with time postinfection and CH pretreatment. Since CH pretreatment appears to increase the levels of early viral proteins, it may be a useful procedure to assist their isolation and functional characterization.