Efficient homologous RNA recombination and requirement for an open readingframe during replication of equine arteritis virus defective interfering RNAs

Equine arteritis virus (EAV), the prototype arterivirus, is an enveloped pl us-strand RNA virus with a genome of approximately 13 kb. Based on similari ties in genome organization and protein expression, the arteriviruses have recently been grouped together with the coronaviruses and toroviruses in th e newly established order Nidovirales. Previously, we reported the construc tion of pEDI, a full-length cDNA copy of EAV DI-b, a natural defective inte rfering (DI) RNA of 5.6 kb (R. Molenkamp et al., J. Virol. 74:3156-3165, 20 00). EDI RNA consists of three noncontiguous parts of the EAV genome fused in frame with respect to the replicase gene. As a result, EDI RNA contains a truncated replicase open reading frame (EDI-ORF) and encodes a truncated replicase polyprotein. Since some coronavirus DI RNAs require the presence of an ORF for their efficient propagation, we have analyzed the importance of the EDI-ORF in EDI RNA replication. The EDI-ORF was disrupted at differe nt positions by the introduction of frameshift mutations. These were found either to block DI RNA replication completely or to be removed within one v irus passage, probably dug to homologous recombination with the helper viru s genome. Using recombination assays based on EDI RNA and full-length EAV g enomes containing specific mutations, the rates of homologous RNA recombina tion in the 3'- and 5'-proximal regions of the. EAV genome were studied. Re markably, the recombination frequency in the 5'-proximal region was found t o be approximately 100-fold lower than that in the 3'-proximal part of the genome.