Collins P L, Huang Y T, Wertz G W
J Virol. 1984 Feb;49(2):572-8. doi: 10.1128/JVI.49.2.572-578.1984.
Nine mRNAs, their cDNA clones, and a genome transcriptional map have been reported previously for respiratory syncytial virus (P. L. Collins and G. W. Wertz, Proc. Natl. Acad. Sci. U.S.A. 80:3208-3212, 1983). We report here the identification of a 10th viral mRNA, designated mRNA 2b (molecular weight [MW] ca. 0.39 X 10(6)), that was detected by RNA (Northern) blot hybridization with cDNA clones. Analysis of a polycistronic readthrough transcript was used to deduce the position in the viral transcriptional map of the gene encoding the newly identified mRNA. The polypeptide coding assignments of 9 of the 10 respiratory syncytial virus mRNAs were determined. Individual viral mRNAs were purified by hybridization selection with nine unique, nonoverlapping cDNA clones and analyzed by translation in vitro. Each of the nine mRNAs encoded a single polypeptide chain. The coding assignments were as follows: RNA 1a (MW ca. 0.24 X 10(6)), a 9,500-dalton (9.5K) protein; RNA 1b (MW 0.26 X 10(6)), an 11K protein; RNA 1c (MW 0.26 X 10(6)), a 14K protein; RNA 2a (MW 0.38 X 10(6)), the 34K phosphorylated (P) protein; RNA 2b (MW 0.39 X 10(6)), a 36K protein; RNA 3a (MW 0.40 X 10(6)), the 26K matrix (M) protein; RNA 3b (MW 0.40 X 10(6)), a 24K protein; RNA 4 (MW 0.47 X 10(6)), the 42K major nucleocapsid (N) protein; and RNA 5 (MW 0.74 X 10(6)), a 59K protein. The cDNA clones used for the hybridization selections were respiratory syncytial virus specific and did not hybridize with uninfected-cell mRNA; therefore the proteins synthesized with the selected mRNAs were virus specific. The 9.5K, 11K, 14K, 24K, M, P, 36K, N, and 59K proteins were encoded by different mRNAs; therefore these nine proteins are all unique. The 9.5K, 11K, 14K, 24K, M, P, and N proteins synthesized in vitro with hybrid-selected mRNAs each had counterparts with the same electrophoretic mobilities in extracts of virus-infected cells. The in vitro polypeptides and their authentic counterparts were shown to be closely related by limited digest peptide mapping. The 36K and 59K polypeptides lacked counterparts with the same electrophoretic mobilities in infected cells and therefore are candidates for the unprocessed precursors of the viral F and G glycoproteins. The 10th viral mRNA, the 2,500K RNA 7, was not tested directly but is the only known mRNA of the appropriate size to encode the 200K large (L) protein of the viral nucleocapsid. These assignments account for all 10 of the reported viral mRNAs and bring to 10 the number of known unique viral proteins.
此前已报道过呼吸道合胞病毒的9种信使核糖核酸（mRNA）、它们的互补脱氧核糖核酸（cDNA）克隆以及基因组转录图谱（P. L. 柯林斯和G. W. 韦茨，《美国国家科学院院刊》80:3208 - 3212, 1983）。我们在此报告鉴定出第10种病毒mRNA，命名为mRNA 2b（分子量[MW]约0.39×10⁶），它是通过与cDNA克隆进行RNA（Northern）印迹杂交检测到的。对多顺反子通读转录本的分析用于推断编码新鉴定mRNA的基因在病毒转录图谱中的位置。确定了10种呼吸道合胞病毒mRNA中9种的多肽编码分配。通过与9个独特的、不重叠的cDNA克隆进行杂交选择来纯化各个病毒mRNA，并通过体外翻译进行分析。9种mRNA中的每一种都编码一条单一的多肽链。编码分配如下：RNA 1a（MW约0.24×10⁶），一种9500道尔顿（9.5K）的蛋白质；RNA 1b（MW 0.26×10⁶），一种11K的蛋白质；RNA 1c（MW 0.26×10⁶），一种14K的蛋白质；RNA 2a（MW 0.38×10⁶），34K的磷酸化（P）蛋白；RNA 2b（MW 0.39×10⁶），一种36K的蛋白质；RNA 3a（MW 0.40×10⁶），26K的基质（M）蛋白；RNA 3b（MW 0.40×10⁶），一种24K的蛋白质；RNA 4（MW 0.47×10⁶），42K的主要核衣壳（N）蛋白；以及RNA 5（MW 0.74×10⁶），一种59K的蛋白质。用于杂交选择的cDNA克隆是呼吸道合胞病毒特异性的，不会与未感染细胞的mRNA杂交；因此，用所选mRNA合成的蛋白质是病毒特异性的。9.5K、11K、14K、24K、M、P、36K、N和59K蛋白由不同mRNA编码；因此这9种蛋白质都是独特的。用杂交选择的mRNA在体外合成的9.5K、11K、14K、24K、M、P和N蛋白在病毒感染细胞提取物中各自都有具有相同电泳迁移率的对应物。通过有限消化肽图谱分析表明，体外多肽及其真实对应物密切相关。36K和59K多肽在感染细胞中缺乏具有相同电泳迁移率对应的物，因此是病毒F和G糖蛋白未加工前体的候选物。第10种病毒mRNA，2500K的RNA 7，未直接进行测试，但它是已知唯一具有合适大小来编码病毒核衣壳200K大（L）蛋白的mRNA。这些分配涵盖了所有报道的10种病毒mRNA，并使已知独特病毒蛋白的数量达到10种。
