Messenger RNA synthesis by a "coated" viral genome.
Messenger RNA synthesis by a "coated" viral genome.
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由“包被”病毒基因组合成信使 RNA。
DOI：
10.1073/pnas.57.2.314
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发表时间：
1967
影响因子：
11.1
通讯作者：
B. R. TAIcAusLAN
中科院分区：
文献类型：
--
作者：
BY J. R. Kates;B. R. TAIcAusLAN
痘病毒的脱壳是一个相当令人感兴趣的过程，因为它需要蛋白质合成，但它发生在病毒基因组被认为是无活性的时候。据推测，脱壳是由宿主细胞基因组编码的蛋白质介导的，并由进入的病毒颗粒诱导。2 ' 3放线菌素D抑制脱壳的观察结果与基因被去抑制的假设一致。2' 3我们对痘病毒感染早期事件的了解，基于电子显微镜研究2 ' 4，5或基于放射性标记病毒粒子命运的研究，3 6可以概括如下：（1）病毒在吞噬空泡或溶酶体内转运到细胞内。(2)在溶酶体内，病毒外壳的蛋白质外层通过一种不依赖于新蛋白质合成的组成性过程被除去。病毒粒子的这种初步降解导致病毒核心的产生，病毒核心由包封在蛋白质外壳内的核酸部分组成。(3)然后病毒核心穿过溶酶体膜并进入细胞的细胞质基质。(4)一旦进入细胞质，病毒核心就会释放出它们的核酸成分。最后一步是脱膜。在这篇文章中，我们将表明，必须从新的角度重新审视脱壳的问题，因为病毒基因在核心中不表达的先验假设似乎是没有根据的。目前的证据表明，痘病毒核心，事实上，表达了相当大的比例，他们的gertome。这些发现也与病毒基因表达调控的一般问题有一定关系。材料与方法病毒感染程序：仅使用在Spinner培养物中生长的HeLa S3细胞和兔痘病毒（RP）的Utrecht株。病毒学程序已在前面描述。8 ' 9放射性病毒的制备：用RP（2.5PFU/细胞）感染HeLa细胞（2 × 108），并在0.5ml H ′-胸苷（sp. act. 10 c/mmole）。用Joklik法从这些细胞中纯化病毒。在蔗糖梯度离心之前，用胰DNA酶（200 μ g/ml）在50 ℃处理病毒制备物12小时，在37 ℃处理30分钟。通过该方法获得的病毒制剂每107个麻点形成单位（PFU）产生1.5 × 104 cpm。细胞内去包被试验：去包被的测定方法基本上与Joklik 6所述的方法相同。测定方法中的一些差异如下：（1）用H3胸苷代替p32标记病毒，因此用Bray溶液中的液体闪烁法估计放射性，效率约为10%;和（2）通过测量总细胞相关放射性的比例来计算未包被的百分比，所述总细胞相关放射性被赋予酸性-如在Joklik的研究中，通过用DNA酶处理细胞超声处理可溶，但是在DNA酶处理之前对酸溶性背景进行校正（通常为零）和病毒吸附后立即发现的DNA酶敏感背景放射性（小于总量的5%）。标记的RP-RNA的制备：将用痘病毒感染并用各种抑制剂处理的HeLa细胞（见下文）暴露于H3-尿苷（sp. act. 7 c/mmole;如前所述1制备细胞质部分，并通过离心（104，000 X g，60分钟）获得核糖体沉淀。将核糖体沉淀重悬于小体积的10-2 M Tris-HCl，pH 7.8，+ 1.5 X 10-3 M MgCl 2 + 10-2 M
The uncoating of the poxvirus is a process of considerable interest since it requires protein synthesis,1' 2 yet it takes place when the viral genome is believed to be inactive. It has been postulated that uncoating is mediated by proteins coded by the host cell genome, and induced by incoming virus particles.2' 3 The observation that actinomycin D inhibits uncoating is consistent with the hypothesis that a gene is derepressed.2' 3 Our knowledge of the early events in poxvirus infection, based either on electron microscope studies2' 4, 5or on studies of the fate of radioactively labeled virions,3 6 can be summarized as follows: (1) Viruses are transported into the cell within phagocytic vacuoles or lysosomes. (2) Within the lysosomes the outer protein layers of the virus coat are removed by a constitutive process not dependent upon the synthesis of new proteins. This preliminary degradation of the virions results in the production of viral cores which consist of the nucleic-acid moiety encapsulated within a protein coat. (3) The viral cores then traverse the lysosomal membrane and enter the cytoplasmic matrix of the cell. (4) Once in the cytoplasm, the viral cores release their nucleic acid component. This last step is uncoating. It will be shown in this communication that the problem of uncoating must be re-examined in a new light, since the a priori assumption that viral genes are not expressed in the cores'-3' I does not seem warranted. The present evidence indicates that poxvirus cores do, in fact, express a sizable proportion of their gertome. The findings have also some bearing on the general problem of regulation of viral gene expression. Materials and Methods.-Virus infection procedure: HeLa S3 cells grown in Spinner cultures and the Utrecht strain of rabbit poxvirus (RP) were used exclusively. The virological procedures have been described previously.8' 9 Preparation of radioactive virus: HeLa cells (2 X 108) were infected with RP (2.5 PFU/cell) and incubated for 24 hr in the presence of 0.5 me of H'-thymidine (sp. act. 10 c/mmole). Thevirus was purified from such cells by the method of Joklik.'0 The virus preparation was treated with pancreatic DNase (200 ,ug/ml) for 12 hr at 50C and 30 min at 370C prior to sucrose gradient centrifugation. The virus preparation obtained by this procedure gave 1.5 X 104 cpm per 107 pock-forming units (PFU). Assay jor intracellular uncoating: The assay procedure for uncoating was essentially the same as that described by Joklik.6 Some differences in the assay procedure are as follows: (1) H3thymidine instead of p32 was employed for labeling virus, and consequently radioactivity was estimated by liquid scintillation in Bray's solution at an efficiency of about 10%; and (2) the percentage of uncoating was calculated by a measurement of the proportion of the total cellassociated radioactivity which was rendered acid-soluble by treatment of cell sonicates with DNase as in the study by Joklik' but a correction was applied for the acid-soluble background prior to DNase treatment (usually nil) and for the DNase-sensitive background radioactivity found immediately following viral adsorption (less than 5%0 of the total). Preparation of labeled RP-RNA: HeLa cells infected with poxvirus and treated with various inhibitors (see below) were exposed to H3-uridine (sp. act. 7 c/mmole; 0.3 me per 108 cells) for a period of 60 min. The cytoplasmic fraction was prepared as previously described1 and the ribosomal pellet was obtained by centrifugation (104,000 X g for 60 min). The ribosomal pellet was resuspended in a small volume of 10-2 M Tris-HCl, pH 7.8, + 1.5 X 10-3 M MgCl2 + 10-2 M