RNAs extracted from herpes simplex virus 1 virions: Apparent selectivity of viral but not cellular RNAs packaged in virions
RNAs extracted from herpes simplex virus 1 virions: Apparent selectivity of viral but not cellular RNAs packaged in virions
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DOI：
10.1128/jvi.75.17.8105-8116.2001
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发表时间：
2001-09-01
影响因子：
5.4
通讯作者：
Roizman, B
中科院分区：
文献类型：
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作者：
Sciortino, MT;Suzuki, M;Roizman, B
在最近关于人巨细胞病毒病毒粒子中存在RNA的研究的基础上，我们调查了从纯化的单纯疱疹病毒1型病毒粒子中提取的RNA的存在和鉴定。为了便于这些研究，我们为所有已知的开放阅读框架(ORF)设计了引物，并构建了包含用于检测所有已知转录本的探针的cDNA阵列。在第一系列实验中，从受感染的Hep-2或兔皮肤细胞中提取的聚(A)(+)RNA反转录制成的标记DNA与cDNA阵列中除两个外的所有探针杂交。对从受感染的Hep-2细胞中提取的纯化的细胞外病毒粒子提取的RNA进行了类似的分析，这些病毒粒子与代表24个ORF的探针杂交。在第二系列分析中，我们从受感染的Hep-2或Vero细胞系分离的纯化的细胞内或细胞外病毒粒子中反转录并通过聚合酶链式反应扩增RNA。对阳性的RNA进行了带有和不带有事先反转录的聚合酶链式反应的重新检测，以确保检测的样品没有受到DNA污染。研究结果如下：只有9个RNA(L)10、L 34/U(L)35、L 36、L 42、L 48、L 51、S 1/U(S)1.5、S 8.5、S 10/U(S)11呈阳性。其中，有7个通过与cDNA阵列杂交而呈阳性。(2)从缺乏U(S)8~U(S)12基因的突变病毒感染的细胞中提取的RNA得到与上述结果相似的结果，表明已知的RNA结合蛋白U(S)11不起包装病毒粒子RNA的作用。(3)在病毒粒子中检测到的细胞RNA是丰富的细胞RNA的代表。最后，从病毒粒子中提取的RNA进行体外翻译，并将翻译产物与抗αTIF抗体(VIP16)反应。免疫沉淀物含有一个标记的蛋白质，其表观摩尔重量为αTIF，这表明至少有一个包装在病毒粒子中的mRNA是完整的，能够被翻译。包装在病毒粒子中的病毒RNA的包装具有明显的选择性，其基础尚不清楚。
Following the lead of recent studies on the presence of RNA in virions of human cytomegalovirus, we investigated the presence and identity of RNAs from purified virions of herpes simple virus 1. To facilitate these studies, we designed primers for all known open reading frames (ORFs) and also constructed cDNA arrays containing probes designed to detect all known transcripts. In the first series of experiments, labeled DNA made by reverse transcription of poly(A)(+) RNA extracted from infected HEp-2 or rabbit skin cells hybridized to all but two of the probes in the cDNA array. A similar analysis of the RNA extracted from purified extracellular virions derived from infected HEp-2 cells hybridized to probes representing 24 of the ORFs. In the second series of analyses, we reverse transcribed and amplified by PCR RNAs from purified intracellular or extracellular virions derived from infected HEp-2 or Vero cell lines. The positive RNAs were retested by PCR with and without prior reverse transcription to ensure that the samples tested were free of contaminating DNA. The results were as follows. (i) Only a fraction of viral ORF transcripts were represented in virion RNA, and only nine RNAs (U(L)10, U(L)34/U(L)35, U(L)36, U(L)42, U(L)48, U(L)51, U(S)1/U(S)1.5, U(S)8.5, and U(S)10/U(S)11) were positive in all RT PCR assays. Of these, seven were positive by hybridization to cDNA arrays. (ii) RNA extracted from cells infected with a mutant virus lacking the U(S)8 to U(S)12 genes yielded results similar to those described above, indicating that U(S)11, a known RNA binding protein, does not play a role in packaging RNA in virions. (iii) Cellular RNAs detected in virions were representative of the abundant cellular RNAs. Last, RNA extracted from virions was translated in vitro and the translation products were reacted with antibody to alpha TIF (VIP16). The immune precipitate contained a labeled protein with the apparent molcular weight of alpha TIF, indicating that at least one mRNA packaged in virions was intact and capable of being translated. The basis for the apparent selectivity in the packaging of the viral RNAs packaged in virions is unknown.