Recombinant equine arteritis virus as an expression vector

Equine arteritis virus (EAV) is the prototypic member of the family Arteriv iridae, which together with the Corona- and Toroviridae constitutes the ord er Nidovirales, A common trait of these positive-stranded RNA viruses is th e 3'-coterminal nested set of six to eight leader-containing subgenomic mRN As which are generated by a discontinuous transcription mechanism and from which the viral open reading frames downstream of the polymerase gene are e xpressed. In this study, we investigated whether the unique gene expression strategy of the Nidovirales could be utilized to convert them into viral e xpression vectors by introduction of an additional transcription unit into the EAV genome directing the synthesis of an extra subgenomic mRNA. To this end, an expression cassette consisting of the gene for a green fluorescent protein (GFP) flanked at its 3' end by EAV-specific transcription-regulati ng sequences was constructed. This genetic module was inserted into the rec ently obtained mutant infectious EAV cDNA clone pBRNX1.38-5/6 (A. A. F. de Vries, et al., 2000, Virology 270, 84-97) between the genes for the M and t he G(L) proteins. Confocal fluorescence microscopy of BHK-21 cells electrop orated with capped RNA transcripts derived from the resulting pasmid (pBRNX 1.38-5/6-GFP) demonstrated that the GFP gene was expressed in the transfect ed cells, while the gradual spread of the infection through the cell monola yer showed that the recombinant virus was replication competent. The develo pment of the cytopathic effect was, however, much slower than in cells that had received equivalent amounts of pBRNX1.38-5/6 RNA, indicating that the vector virus had a clear growth disadvantage compared to its direct precurs or. Immunoprecipitation analyses of proteins from metabolically labeled BHK -21 cells infected with supernatant of the transfected cultures confirmed t hat the recombinant virus vector was viable and expressed viral genes as we ll as the GFP gene. Reverse transcription-PCR of the viral mRNAs extracted from cells infected with the vector virus revealed that it directed the syn thesis of nine instead of eight different EAV RNAs. These findings were cor roborated by hybridization analyses. Mapping of the leader-to-body junction s of the ninth mRNA indicated that the 3' part of the GFP gene contains cry ptic transcription signals which gave rise to at least five different RNA s pecies ranging in sire from 1277 to 1439 nt [without oligo(A) tract], Furth ermore, translation of the unintended mRNA resulted in the production of an extended version of the EAV M protein. Serial passage of the recombinant v irus vector led to its gradual replacement by viral mutants carrying deleti ons in the GFP gene. The reduction in viral fitness associated with the ins ertion of the expression cassette into the EAV genome apparently caused gen etic instability of the recombinant virus. (C) 2001 Academic Press.