Modular arrangement of viral cis-acting RNA domains in a tombusvirus satellite RNA

Satellite (sat) RNAs are parasitic sub-viral RNA replicons found associated with certain positive-strand RNA viruses. Typical sat RNAs, such as those associated with members of the genus Tombusvirus, share little or no sequence identity with their helper virus genomes. Here, we have investigated a tombusvirus sat RNA and determined that it contains two functionally-relevant higher-order RNA domains, a T-shaped domain and a downstream domain, that are similar to elements shown previously to be present in the 5′ untranslated regions (UTRs) of tombusvirus genomes. Although the two sat RNA domains showed only limited sequence identity with their viral counterparts, they were able to adopt comparably-folded RNA secondary structures. Interestingly, the relative spacing between the domains in the viral and satellite contexts was notably different. In the viral 5′ UTR, the two domains are adjacent and separated by a small hairpin, however, in the sat RNA they are separated by a 137-nt long segment. Despite this distal modular arrangement, the two domains were found to be united spatially in the sat RNA through the formation of an RNA-RNA bridge. This co-localization facilitated an important inter-domain interaction and was essential for efficient helper-mediated sat RNA accumulation in protoplasts. These results indicate that the tombusvirus sat RNA and helper genome contain structurally and functionally equivalent RNA domains. It is proposed that the limited sequence identity observed between these corresponding higher-order RNA structures is related to a strategy that reduces the induction of gene silencing, which presumably would be detrimental to both viral and sat RNA replicons.