Replication of cucumber mosaic virus RNA 1 in cis requires functional helicase-like motifs of the 1a protein.

The cucumber mosaic virus (CMV) encoded 1a protein contains an NTPase/helicase-like domain. To investigate whether various helicase motifs were required for efficient replication and to establish whether CMV RNA 1 could be replicated efficiently in cis, we constructed deletion mutations in helicase motifs I, III and VI and analyzed their effects on CMV RNA replication in tobacco. CMV replication was not detectable for any of the three helicase mutants, indicating that the helicase domain is crucial for efficient CMV replication. Both the wild-type and mutant 1a proteins could be detected at similar levels after transient expression in infiltrated tissues, indicating that the helicase-motif mutations did not affect the stability of the proteins. Co-inoculation tests with various mutant combinations did not result in complementation. In protoplasts derived from CMV RNA 1-transgenic tobacco, which supported replication of CMV RNAs 2 and 3, the RNA 1 helicase mutants were not replicated detectably in trans, but also did not interfere with the replication of the genomic RNAs, indicating that the conserved helicase motifs of the 1a protein are required in cis for the effective accumulation of RNA 1.