Synthesis of infectious transcripts of olive latent virus 1: genes required for RNA replication and virus movement

Genomic RNA of olive latent virus I (OLV-1) contains five open reading fram es (ORFs) encoding proteins of 23, 82, 8, 6 and 30 (CP) kDa. A full-length cDNA copy of OLV-1 RNA was prepared and cloned in a low-copy-number vector (pMUC-19) downstream of or T7 RNA polymerase promoter. Transcripts derived from this template, denoted pMUC-OLV, were highly infectious when inoculate d in local and systemic hosts and infected tissues contained virus-like par ticles. Genes required for replication and virus movement were mapped by si te-directed and deletion mutogenesis of the pMUC-OLV. ORF1 and ORF2 mutants were not viable, suggesting that replication requires the 23 and 82 kDa pr oteins. The 8 and 6 kDa polypeptides were involved in cell-to-cell movement , since their absence did not interfere with RNA replication but prevented systemic infection of inoculated plants. Mutant clones in R and S domains o f the CP gene could replicate, but they did not systemically infect Nicotia na benthamiana, indicating that the CP gene is required for OLV-1 long-dist ance translocation. Mutant clones with large deletions in the CP gene were not viable, probably due to loss of 3'-proximal sequences required for RNA replication.