Long-Distance Base Pairing in Flock House Virus RNA1 Regulates Subgenomic RNA3 Synthesis and RNA2 Replication

Abstract

Replication of flock house virus (FHV) RNA1 and production of subgenomic RNA3 in the yeast Saccharomyces cerevisiae provide a useful tool for the dissection of FHV molecular biology and host-encoded functions involved in RNA replication. The replication template activity of RNA1 can be separated from its coding potential by supplying the RNA1-encoded replication factor protein A in trans. We constructed a transreplication system in yeast to examine cis-acting elements in RNA1 that control RNA3 production, as well as RNA1 and RNA2 replication. Two cis elements controlling RNA3 production were found. A proximal subgenomic control element was located just upstream of the RNA3 start site (nucleotides [nt] 2282 to 2777). A short distal element also controlling RNA3 production (distal subgenomic control element) was identified 1.5 kb upstream, at nt 1229 to 1239. Base pairing between these distal and proximal elements was shown to be essential for RNA3 production by covariation analysis and in vivo selection of RNA3-expressing replicons from plasmid libraries containing random sequences in the distal element. Two distinct RNA1 replication elements (RE) were mapped within the 3 quarter of RNA1: the intRE (nt 2322 to 2501) and the 3RE (nt 2735 to 3011). The 3RE significantly overlaps the RNA3 region in RNA1, and this information was applied to produce improved RNA3-based vectors for foreign-gene expression. In addition, replication of an RNA2 derivative was dependent on RNA1 templates capable of forming the long-distance interaction that controls RNA3 production.Viruses with positive-strand RNA genomes include numerous human, animal, and plant pathogens of medical and economic importance. Following binding and entry into a permissive cell, the viral genome is translated to produce nonstructural proteins that assemble with genomic RNA and presumably some host factors to form RNA replication complexes. Replication proceeds through a negative-strand RNA intermediate, which serves as a template for multiple rounds of positive-strand synthesis (reviewed in reference 11). Later in infection, nascent genomic positive strands interact with viral structural components to form new virus particles. The competing processes of genome translation, replication, and packaging are coordinated in part via differential viral gene expression. Mechanisms regulating viral gene expression include translational control such as readthrough at frameshifts or nonsense codons, postranslational control such as proteolytic cleavage of polyprotein precursors, and transcriptional control via production of subgenomic RNA (sgRNA). sgRNAs are colinear with the 3Ј region of genomic RNA and are produced during replication via one of several possible mechanisms (reviewed in reference 38). Since ribosomal scanning takes place in a 5Ј-to-3Ј direction, sgRNAs permit gene expression from downstream open reading frames (ORFs) that are not translatable from genomic RNA. Understanding the mechanisms that control sgRNA production should reveal novel aspects of vira...

The Cellular Decapping Activators LSm1, Pat1, and Dhh1 Control the Ratio of Subgenomic to Genomic Flock House Virus RNAs

“…Plasmid pCupF1 fs is a pF1 fs derivative (34,42) in which the GAL1 promoter was replaced by the CUP1 promoter. Plasmids pF1 fs mut1, pFA, pFA D692E (35,45), and pFA-HCV (46) were described previously. Plasmid PV167 expresses RNA3 under the GAL1 promoter and a 3=-flanking antigenomic hepatitis delta virus ribozyme (␦Rz).…”

“…In addition, RNA3 fulfills an important role in the regulation of FHV gene expression by coordinating the production of proper RNA1 and RNA2 levels. RNA3 transactivates RNA2 replication, while high levels of RNA2 inhibit RNA3 synthesis (35,37,38). The downregulation of RNA3 most probably disallows disproportionately high RNA2/RNA1 ratios within FHV-infected cells.…”

“…During replication, RNA1 also produces subgenomic RNA3 (387 nucleotides). This RNA3 corresponds to the 3= end of RNA1, and its synthesis requires a long-distance base pairing between two cisacting elements in RNA1 (35). Thus, different RNP rearrangements within genomic RNA1 are predicted to lead to the synthesis of either negative-stranded RNA1 [(Ϫ)RNA1), when the polymerase synthesizes a complete cRNA, or (Ϫ)RNA3, when the polymerase prematurely stops due to the secondary or tertiary structures formed by the long-distance base pairing.…”

Subgenomic mRNA transcription in Tombusviridae

“…Outside of Tombusviridae, there is some evidence that a PT mechanism may be utilized by unrelated nodaviruses, 39 closteroviruses 40 and/or toroviruses. 41 Additionally, umbraviruses and luteoviruses are closely related to Tombusviridae in terms of both RNA replication proteins and cis-acting RNA replication signals, 42,43 thus these genera also represent possible candidates for using a PT mechanism.…”

Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy

“…In addition to the primary sequence that directly codes for the viral proteins, single-stranded (ss)-RNA viruses exploit their secondary and tertiary structures to direct various events during infection. For example, specific secondary structure motifs within the viral genome have been implicated in packaging (Valegård et al 1994;Qu and Morris 1997;Choi et al 2002;Bunka et al 2011), genome replication (Frolov et al 2001;Lindenbach et al 2002;Damgaard et al 2004), the regulation of protein expression (Barends et al 2003), protein folding (Watts et al 2009), conformational switching (Stockley et al 2007), and evading degradation by the host cell (Athavale et al 2013).…”