RNA Structure Protects the 5’-end of an Uncapped Tombusvirus RNA Genome from Xrn Digestion
One of the many challenges faced by RNA viruses is the maintenance of their genomes during infections of host cells. Members of the family Tombusviridae are plus-strand RNA viruses with unmodified triphosphorylated genomic 5?-termini. The tombusvirus Carnation Italian ringspot virus was used to investigate how it protects its RNA genome from attack by 5?-end-targeting degradation enzymes. In vivo and in vitro assays were employed to determine the role of genomic RNA structure in conferring protection from the 5?-to-3? exoribonuclease Xrn. The results revealed that (i) the CIRV RNA genome is more resistant to Xrn than its sg mRNAs, (ii) the genomic 5?UTR folds into a compact RNA structure that effectively and independently prevents Xrn access, (iii) the RNA structure limiting 5?-access is formed by secondary and tertiary interactions that function cooperatively, (iv) the structure is also able to block access of RNA pyrophosphohydrolase to the genomic 5?-terminus, and (v) the RNA structure does not stall an actively digesting Xrn. Based on its proficiency at impeding Xrn 5?-access, we have termed this 5?-terminal structure an X rn- e vading RNA or xeRNA. These and other findings demonstrate that the 5?UTR of the CIRV RNA genome folds into a complex structural conformation that helps to protect its unmodified 5?-terminus from enzymatic decay during infections. IMPORTANCE The plus-strand RNA genomes of plant viruses in the large family Tombusviridae are not 5?-capped. Here we explored how a species in the type genus Tombusvirus protects its genomic 5?-end from cellular nuclease attack. Our results revealed that the 5?-terminal sequence of the CIRV genome folds into a complex RNA structure that limits access of the 5?-to-3? exoribonuclease Xrn, thereby protecting it from processive degradation. The RNA conformation also impeded access of RNA pyrophosphohydrolase, which converts 5?-triphosphorylated RNA termini into 5?-monophosphorylated forms, the preferred substrate for Xrn. This study represents the first report of a genome-encoded higher-order RNA structure independently conferring resistance to cellular 5?-end-attacking enzymes in an RNA plant virus.