Evans Sharon, Cavanagh David, Britton Paul
Division of Molecular Biology, Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, UK1.
J Gen Virol. 2000 Dec;81(Pt 12):2855-2865. doi: 10.1099/0022-1317-81-12-2855.
Coronavirus defective RNAs (D-RNAs) have been used as RNA vectors for the expression of heterologous genes and as vehicles for reverse genetics by modifying coronavirus genomes by targetted recombination. D-RNAs based on the avian coronavirus infectious bronchitis virus (IBV) D-RNA CD-61 have been rescued (replicated and packaged into virions) in a helper virus-dependent manner following electroporation of in vitro-generated T7 transcripts into IBV-infected cells. In order to increase the efficiency of rescue of IBV D-RNAs, cDNAs based on CD-61, under the control of a T7 promoter, were integrated into the fowlpox virus (FPV) genome. The 3'-UTR of the D-RNAs was flanked by a hepatitis delta antigenomic ribozyme and T7 terminator sequence to generate suitable 3' ends for rescue by helper IBV. Cells were co-infected simultaneously with IBV, the recombinant FPV (rFPV) containing the D-RNA sequence and a second rFPV expressing T7 RNA polymerase for the initial expression of the D-RNA transcript, subsequently rescued by helper IBV. Rescue of rFPV-derived CD-61 occurred earlier and with higher efficiency than demonstrated previously for electroporation of in vitro T7-generated RNA transcripts in avian cells. Rescue of CD-61 was also demonstrated for the first time in mammalian cells. The rescue of rFPV-derived CD-61 by M41 helper IBV resulted in leader switching, in which the Beaudette-type leader sequence on CD-61 was replaced with the M41 leader sequence, confirming that helper IBV virus replicated the rFPV-derived D-RNA. An rFPV-derived D-RNA containing the luciferase gene under the control of an IBV transcription-associated sequence was also rescued and expressed luciferase on serial passage.
冠状病毒缺陷RNA（D-RNAs）已被用作表达异源基因的RNA载体，并通过靶向重组修饰冠状病毒基因组作为反向遗传学的工具。基于禽冠状病毒传染性支气管炎病毒（IBV）D-RNA CD-61的D-RNAs在体外转录的T7转录本电穿孔导入IBV感染细胞后，已以依赖辅助病毒的方式被拯救（复制并包装到病毒粒子中）。为了提高IBV D-RNAs的拯救效率，基于CD-61且在T7启动子控制下的cDNA被整合到禽痘病毒（FPV）基因组中。D-RNAs的3'-UTR侧翼有丁型肝炎抗原基因组核酶和T7终止子序列，以产生适合由辅助IBV拯救的3'末端。细胞同时被IBV、含有D-RNA序列的重组FPV（rFPV）和表达T7 RNA聚合酶以初始表达D-RNA转录本的第二个rFPV共感染，随后由辅助IBV拯救。rFPV衍生的CD-61的拯救比之前在禽细胞中对体外T7生成的RNA转录本进行电穿孔所证明的时间更早且效率更高。CD-61的拯救也首次在哺乳动物细胞中得到证明。M41辅助IBV对rFPV衍生的CD-61的拯救导致了前导序列切换，其中CD-61上的Beaudette型前导序列被M41前导序列取代，证实辅助IBV病毒复制了rFPV衍生的D-RNA。一个含有在IBV转录相关序列控制下的荧光素酶基因的rFPV衍生的D-RNA也被拯救，并在连续传代时表达了荧光素酶。
