Expression of a foreign epitope by porcine reproductive and respiratory syndrome virusM.H. Groot Bramel-Verheije, P.J.M. Rottier and J.J.M. Meulenberg
Institute for Animal Science and Health (ID-DLO), P.O. Box 65, 8200 AB Lelystad, The Netherlands
Abstract - Recently, an infectious cDNA clone of the Lelystad Virus (LV) isolate of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) was constructed. In this study, we used the infectious clone to explore its potential for the development of a viral vector, by the insertion of a foreign sequence into the viral genome. The aim of this study was to identify regions in the PRRSV genome that allow the introduction of a sequence encoding a foreign antigen. For this purpose, we selected an epitope of 9 amino acids from the hemagglutinin of the human influenza A virus (HA-tag), because of its limited size. This reduces the chance to disturb the replication of the virus or the expression or function of the protein to which it is fused. ORF7 was selected as the site for insertion of the HA-tag, because its subgenomic RNA is the most abundant mRNA produced in PRRSV-infected cells and because ORF7 has little overlap with ORF6 at its 5' end and no overlap at its 3' end. The sequence encoding the HA-tag was introduced either immediately downstream of the start codon or directly upstream of the stop codon of ORF7 into the genome-length cDNA clone of LV. Since we could not predict the influence of the terminal extensions on the structure and function of the N protein, we inserted sequences of the self-cleaving protease 2A of the foot-and-mouth disease virus (FMDV) in frame between the HA-tag and ORF7, at the 5' end. We expected this to result in the expression of a polyprotein, which would be proteolytically cleaved, in order to release the HA-tag from the N protein. The RNA-transcripts from HA-encoding full length constructs all properly expressed the PRRSV proteins and the HA epitope when transfected in BHK-21 cells. This indicated that the HA-expressing transcripts replicated in BHK-21 cells, that the subgenomic ORF7 mRNA was produced, and that the N protein to which the HA-tag was fused, was expressed. The transcripts produced the infectious virus, as shown by the expression of PRRSV proteins after infection of porcine alveolar lung macrophages (PAMs) with the culture supernatant from BHK-21 cells. The PRRSV-recombinants containing the HA-tag at the 5' or 3' end of ORF7 expressed the HA-N fusion protein. The full-length mutants that contained an additional protease 2A sequence, produced both the polyprotein as well as the wild type N protein, from which the HA-tag was cleaved off. In addition, we showed that the protease cleaved its polyprotein cotranslationally. The stability of the recombinant PRRSV viruses was studied after serial passage of the viruses on PAMs. Genetic analysis showed that the recombinants that contained the fusion between the HA-tag and the N protein lost (most of) their foreign sequence within several passages. In contrast, we showed that the HA-expressing PRRSV mutants that contained the protease 2A sequences were maintained stable during the 4 passages analysed. This was confirmed by the expression of the HA-tag in immunoperoxidase monolayer assay (IPMA). These results show that PRRSV has a potential as a vector for the expression of foreign antigens, as long as the native structure of the protein, to which the foreign sequence in the genome is attached, is guaranteed.
Corresponding author: M.H. Groot Bramel-Verheije Tel.: (31) 320 238805; fax: (31) 320 238668;
© INRA, EDP Sciences 2000