An infectious RNA with a hepta-adenosine stretch responsible for programmed −1 ribosomal frameshift derived from a full-length cDNA clone of Hibiscus latent Singapore virus

Hibiscus latent Singapore virus (HLSV) is a member of Tobamovirus and its full-length cDNA clones were constructed. The in vitro transcripts from two HLSV full-length cDNA clones, which contain a hepta-adenosine stretch (pHLSV-7A) and an octo-adenosine stretch (pHLSV-8A), are both infectious. The replication level of HLSV-7A in Nicotiana benthamiana protoplasts was 5-fold lower, as compared to that of HLSV-8A. The replicase proteins of HLSV-7A were produced through programmed −1 ribosomal frameshift (−1 PRF) and the 7A stretch was a slippery sequence for −1 PRF. Mutations to the downstream pseudoknot of 7A stretch showed that the pseudoknot was not required for the frameshift in vitro. The stretch was found to be extended to 8A after subsequent replication cycles in vivo. It is envisaged that HLSV employs the monotonous runs of A and −1 PRF to convert its 7A to 8A to reach higher replication for its survival in plants.