Simultaneous expression of three reporter proteins from a porcine reproductive and respiratory syndrome virus-based vector

Abstract

Abstract The mechanism of discontinuous transcription for the synthesis of a series of sub-genomic mRNAs to express the porcine reproductive and respiratory syndrome virus (PRRSV) structural proteins potentially allows for the simultaneous expression of multiple foreign genes. This can occur by insertion of multiple novel independent transcription units between 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 gene and containing iLOV gene was introduced between ORF1b and 2, and between 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 three reporter genes simultaneously, an expression cassette containing the Gluc gene and an expression cassette containing iLOV gene were also inserted in between ORF1b and 2, and between 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 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 and yet yielded 100-fold less infectious viruses. Fluorescence microscopy of cells infected with rGX-R-Gluc-iLOV demonstrated the presence of both GFP and iLOV genes. Gluc activities in supernatants harvested at different time points from cells infected with recombinant viruses carrying Gluc showed the levels of Gluc activity increased as the infection progressed, indicating that the expression of Gluc gene and its activity were parameters for monitoring viral propagation. These results indicate that it is possible to introduce at least three foreign proteins simultaneously in a PRRSV-based vector and this will prove invaluable in our future understanding of these viruses.