Bukreyev A, Camargo E, Collins P L
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0720, USA.
J Virol. 1996 Oct;70(10):6634-41. doi: 10.1128/JVI.70.10.6634-6641.1996.
A previous report described the recovery from cDNA of infectious recombinant respiratory syncytial virus (RSV) strain A2 (P. L. Collins, M. G. Hill, E. Camargo, H. Grosfeld, R. M. Chanock, and B. R. Murphy, Proc. Natl. Acad. Sci. USA, 92:11563-11567, 1995). Here, the system was used to construct recombinant RSV containing an additional gene encoding chloramphenicol acetyltransferase (CAT). The CAT coding sequence was flanked by RSV-specific gene-start and gene-end motifs, the transcription signals for the viral RNA-dependent RNA polymerase. The RSV-CAT chimeric transcription cassette was inserted into the region between the G and F genes of the complete cDNA-encoded positive-sense RSV antigenome, and infectious CAT-expressing recombinant RSV was recovered. Transcription of the inserted gene into the predicted subgenomic polyadenylated mRNA was demonstrated by Northern (RNA) blot hybridization analysis, and the encoded protein was detected by enzyme assay and by radioimmunoprecipitation. Quantitation of intracellular CAT, SH, G, and F mRNAs showed that the CAT mRNA was efficiently expressed and that the levels of the G and F mRNAs (which represent the genes on either side of the inserted CAT gene) were comparable to those expressed by a wild-type recombinant RSV. Consistent with this finding, the CAT-containing and wild-type viruses were very similar with regard to the levels of synthesis of the major viral proteins. Each of 25 RSV isolates obtained by plaque purification following eight serial passages expressed CAT, showing that the foreign gene was faithfully maintained in functional form. Analysis by reverse transcription and PCR did not reveal evidence of deletion of the foreign sequence. This finding demonstrated that the RSV genome can accept and maintain an increase in length of 762 nucleotides of foreign sequence and can be engineered to encode an additional, 11th mRNA. The presence of the additional gene resulted in a 10% decrease in plaque diameter and was associated with delay in virus growth and 20-fold decrease in virus yield in vitro. Thus, introduction of an additional gene into the RSV genome might represent a method of attenuation. The ability to express foreign genes by recombinant RSV has implications for basic studies as well as for the development of live recombinant vaccines.
先前的一份报告描述了从感染性重组呼吸道合胞病毒（RSV）A2株的cDNA中恢复病毒（P.L.柯林斯、M.G.希尔、E.卡马戈、H.格罗斯费尔德、R.M.查诺克和B.R.墨菲，《美国国家科学院院刊》，92:11563 - 11567，1995年）。在此，该系统被用于构建含有一个额外编码氯霉素乙酰转移酶（CAT）基因的重组RSV。CAT编码序列两侧是RSV特异性的基因起始和基因终止基序，即病毒RNA依赖的RNA聚合酶的转录信号。将RSV - CAT嵌合转录盒插入完整cDNA编码的正链RSV抗原基因组的G基因和F基因之间的区域，并回收了表达CAT的感染性重组RSV。通过Northern（RNA）印迹杂交分析证实了插入基因转录成了预测的亚基因组多聚腺苷酸化mRNA，并且通过酶测定和放射免疫沉淀检测到了编码的蛋白质。对细胞内CAT、SH、G和F mRNA的定量分析表明，CAT mRNA得到了有效表达，并且G和F mRNA（代表插入的CAT基因两侧的基因）的水平与野生型重组RSV表达的水平相当。与这一发现一致，含CAT的病毒和野生型病毒在主要病毒蛋白的合成水平方面非常相似。经过八次连续传代后通过噬斑纯化获得的25株RSV分离株均表达CAT，表明外源基因以功能形式被忠实地保留。通过逆转录和PCR分析未发现外源序列缺失的证据。这一发现表明，RSV基因组能够接受并保留762个核苷酸的外源序列长度增加，并且可以进行改造以编码额外的第11种mRNA。额外基因的存在导致噬斑直径减小10%，并与病毒生长延迟以及体外病毒产量降低20倍相关。因此，将额外基因引入RSV基因组可能代表一种减毒方法。通过重组RSV表达外源基因的能力对基础研究以及活重组疫苗的开发都有意义。
