Recovery of infectious respiratory syncytial virus expressing an additional, foreign gene
Recovery of infectious respiratory syncytial virus expressing an additional, foreign gene
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DOI：
10.1128/jvi.70.10.6634-6641.1996
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发表时间：
1996-10-01
影响因子：
5.4
通讯作者：
Collins, PL
中科院分区：
文献类型：
--
作者：
Bukreyev, A;Camargo, E;Collins, PL
以前的报告描述了从感染性重组呼吸道合胞病毒（RSV）A2株（P.L.）的cDNA中回收病毒。柯林斯，M. G.希尔，E.卡马戈湾格罗斯费尔德河M.查诺克和B. R. Murphy，Proc. Natl. Acad. Sci，USA，92：11563-11567，1995）。在这里，该系统用于构建含有编码氯霉素乙酰转移酶（CAT）的额外基因的重组RSV。CAT编码序列两侧是RSV特异性基因起始和基因末端基序;病毒RNA依赖性RNA聚合酶的转录信号。将RSV-CAT嵌合转录盒插入完整cDNA编码的正义RSV反基因组的G和F基因之间的区域，并回收感染性CAT表达重组RSV。通过北方（RNA）印迹杂交分析证明插入基因转录到预测的亚基因组聚腺苷酸化mRNA中，并通过酶测定和放射免疫沉淀检测编码的蛋白。细胞内CAT、SH、G和F mRNA的定量显示CAT mRNA被有效表达，并且G和F mRNA（其代表插入的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。与这一发现相一致的是，含CAT的酸性温和型病毒在主要病毒蛋白的合成水平方面非常相似。通过噬斑纯化获得的25个RSV分离株中的每一个在8次连续传代后表达CAT，表明外源基因忠实地保持在功能形式中。通过逆转录和PCR分析没有发现外源序列缺失的证据。这一发现表明，RSV基因组可以接受并保持外源序列长度增加762个核苷酸，并且可以被工程化以编码另外的第11种mRNA。额外基因的存在导致噬菌斑直径减少10%，并与病毒生长延迟和体外病毒产量减少20倍有关。因此，将另外的基因引入RSV基因组中可能代表减毒的方法。通过重组RSV表达外源基因的能力对于基础研究以及活重组疫苗的开发具有意义。
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,vas 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 acid mild-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, shoeing 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.