Plus-strand RNA viruses can accumulate viral RNA degradation products during infections. Some of these decay intermediates are generated by the cytosolic 5′-to-3′ exoribonuclease Xrn1 (mammals and yeast) or Xrn4 (plants) and are formed when the enzyme stalls on substrate RNAs upon encountering inhibitory RNA structures. Many Xrn-generated RNAs correspond to 3′-terminal segments within the 3′-UTR of viral genomes and perform important functions during infections. Here we have investigated a 3′-terminal small viral RNA (svRNA) generated by Xrn during infections with Tobacco necrosis virus-D (family Tombusviridae). Our results indicate that (i) unlike known stalling RNA structures that are compact and modular, the TNV-D structure encompasses the entire 212 nt of the svRNA and is not functionally transposable, (ii) at least two tertiary interactions within the RNA structure are required for effective Xrn blocking and (iii) most of the svRNA generated in infections is derived from viral polymerase-generated subgenomic mRNA1. In vitro and in vivo analyses allowed for inferences on roles for the svRNA. Our findings provide a new and distinct addition to the growing list of Xrn-resistant viral RNAs and stalling structures found associated with different plant and animal RNA viruses.

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The replication of plus-strand RNA virus genomes is mediated by virally encoded RNA-dependent RNA polymerases (RdRps). We have investigated the role of the C-proximal region in the RdRp of tomato bushy stunt virus (TBSV) in mediating viral RNA synthesis. TBSV is the prototype species in the genus Tombusvirus, family Tombusviridae, and its RdRp is responsible for replicating the viral genome, transcribing two subgenomic mRNAs, and supporting replication of defective interfering RNAs. Comparative sequence analysis of the RdRps of tombusvirids identified three highly conserved motifs in their C-proximal regions, and these sequences were subsequently targeted for mutational analysis in TBSV. The results revealed that these motifs are important for (i) synthesizing viral genomic RNA and subgenomic mRNAs, (ii) facilitating plus-and/or minus-strand synthesis, and (iii) modulating trans-replication of a defective interfering RNA. These motifs were also found to be conserved in other plant viruses as well as in a fungal and insect virus. The collective findings are discussed in relation to viral RNA synthesis and taxonomy. IMPORTANCELittle is currently known about the structure and function of the viral polymerases that replicate the genomes of RNA plant viruses. Tombusviruses, the prototype of the tombusvirids, have been used as model plus-strand RNA plant viruses for understanding many of the steps in the infectious process; however, their polymerases remain poorly characterized. To help address this issue, the function of the C-terminal region of the polymerase of a tombusvirus was investigated. Three conserved motifs were identified and targeted for mutational analysis. The results revealed that these polymerase motifs are important for determining what type of viral RNA is produced, facilitating different steps in viral RNA production, and amplifying subgenomic RNA replicons. Accordingly, the C-terminal region of the tombusvirus polymerase is needed for a variety of fundamental activities. Furthermore, as these motifs are also present in distantly related viruses, the significance of these results extends beyond tombusvirids. Replication of the genomes of plus-strand RNA viruses is mediated by virally encoded RNA-dependent RNA polymerases (RdRps) (1). RdRps are translated shortly after infection and associate with other viral or host proteins to assemble into RNA replication complexes (RCs) (2). These RCs are responsible for synthesizing complementary minus strands of the genomes that are then used as the templates for production of plus-strand progeny genomes (2). In some viruses, these RCs are also responsible for synthesizing viral subgenomic (sg) mRNAs that are required for expression of other viral proteins (3). Viral RdRps of animal viruses have been the focus of many functional studies, and there are atomic structures available for several viruses (4). In contrast, much less is known about the RdRps of plant viruses (5).The genus Tombusvirus (6) is the prototype member of the family Tombusviridae, whi...

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.