Mo Qingrong, Wang Hao, He Wei, Lin Siyuan, Xie Xin, Wang Yuxu, Wang Xindong, Ouyang Kang, Chen Ying, Huang Weijian, Wei Zuzhang
Laboratory of Animal infectious Diseases and molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning 530005, China; Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning 530005, China; Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, Nanning 530004, PR China; Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease, Nanning 530005, China.
Laboratory of Animal infectious Diseases and molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning 530005, China; Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning 530005, China; Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, Nanning 530004, PR China; Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease, Nanning 530005, China.
J Virol Methods. 2023 Jun;316:114711. doi: 10.1016/j.jviromet.2023.114711. Epub 2023 Mar 13.
The mechanism of discontinuous transcription for the synthesis of a series of sub-genomic mRNAs to express the structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) potentially allows for the simultaneous expression of multiple foreign genes. This can occur by insertion of multiple novel independent transcription units between the ORF sequences of the PRRSV genome. Here, an expression cassette consisting of a red fluorescent protein (RFP) gene flanked at its 3' end by transcription-regulating sequences (TRS) and an expression cassette consisting of an iLOV gene flanked at its 5' end by TRS, was constructed. The resulting expression cassette containing a RFP and an iLOV gene were introduced between ORF1b and 2 as well as ORF7 and 3'UTR, respectively, in an infectious PRRSV cDNA clone. Transfection of the resulting clone (pGX-12RFP-73iLOV) into cells resulted in the recovery of a recombinant virus (rGX-12RFP-73iLOV). Simultaneous expression of RFP and iLOV was observed in MARC-145 cells infected with rGX-RFP-iLOV. To test the ability of the PRRSV genome to express all three reporter genes simultaneously, an expression cassette containing the Gluc gene and one containing the iLOV gene were also inserted in between ORF1b and 2 as well as ORF7 and 3'UTR, respectively. This was performed in a recently obtained infectious PRRSV cDNA clone carrying a RFP gene in nsp2. Transfection of the construct (pGX-R-Gluc-iLOV) carrying the three reporter genes into cells allowed the rescue of the recombinant reporter virus (rGX-R-Gluc-iLOV) which showed similar growth characteristics to the parental virus but yielded 100-fold less infectious viruses. Fluorescence microscopy of cells infected with rGX-R-Gluc-iLOV demonstrated the presence of both RFP and iLOV genes. Gluc activities in supernatants harvested at different time points from cells infected with recombinant viruses carrying Gluc showed increased levels of Gluc activity as the infection progressed. This indicated that Gluc gene as well as its activity were acceptable parameters to monitor viral propagation. Our results indicate that it is possible to introduce at least three foreign proteins simultaneously in a PRRSV-based vector and such studies will prove invaluable in our future understanding of these viruses.
猪繁殖与呼吸综合征病毒（PRRSV）合成一系列亚基因组mRNA以表达结构蛋白的不连续转录机制，可能允许同时表达多个外源基因。这可以通过在PRRSV基因组的开放阅读框（ORF）序列之间插入多个新的独立转录单元来实现。在此，构建了一个表达盒，其由在3'端侧翼有转录调控序列（TRS）的红色荧光蛋白（RFP）基因组成，以及一个表达盒，其由在5'端侧翼有TRS的iLOV基因组成。将所得的包含RFP和iLOV基因的表达盒分别引入感染性PRRSV cDNA克隆的ORF1b和2之间以及ORF7和3'非翻译区（UTR）之间。将所得克隆（pGX - 12RFP - 73iLOV）转染到细胞中，获得了重组病毒（rGX - 12RFP - 73iLOV）。在感染rGX - RFP - iLOV的MARC - 145细胞中观察到RFP和iLOV的同时表达。为了测试PRRSV基因组同时表达所有三个报告基因的能力，还分别在ORF1b和2之间以及ORF7和3'UTR之间插入了一个包含Gluc基因的表达盒和一个包含iLOV基因的表达盒。这是在最近获得的在nsp2中携带RFP基因的感染性PRRSV cDNA克隆中进行的。将携带三个报告基因的构建体（pGX - R - Gluc - iLOV）转染到细胞中，获得了重组报告病毒（rGX - R - Gluc - iLOV），其生长特性与亲本病毒相似，但产生的感染性病毒少100倍。对感染rGX - R - Gluc - iLOV的细胞进行荧光显微镜检查，证明了RFP和iLOV基因的存在。从感染携带Gluc的重组病毒的细胞在不同时间点收获的上清液中的Gluc活性，随着感染的进展显示出Gluc活性水平增加。这表明Gluc基因及其活性是监测病毒增殖的可接受参数。我们的结果表明，在基于PRRSV的载体中同时引入至少三种外源蛋白是可能的，并且此类研究将对我们未来对这些病毒的理解具有不可估量的价值。
