The p92 Polymerase Coding Region Contains an Internal RNA Element Required at an Early Step in Tombusvirus Genome Replication

Abstract

The replication of positive-strand RNA viral genomes involves various cis-acting RNA sequences. Generally, regulatory RNA sequences are present at or near genomic termini; however, internal replication elements (IREs) also exist. Here we report the structural and functional characterization of an IRE present in the readthrough portion of the p92 polymerase gene of Tomato bushy stunt virus. Analysis of this element in the context of a noncoding defective interfering RNA revealed a functional core structure composed of two noncontiguous segments of sequence that interact with each other to form an extended helical conformation. IRE activity required maintenance of several base-paired sections as well as two distinct structural features: (i) a short, highly conserved segment that can potentially form two different and mutually exclusive structures and (ii) an internal loop that contains a critical CC mismatch. The IRE was also shown to play an essential role within the context of the viral genome. In vivo analysis with novel RNA-based temperature-sensitive genomic mutants and translationally active subgenomic viral replicons revealed the following about the IRE: (i) it is active in the positive strand, (ii) it is dispensable late in the viral RNA replication process, and (iii) it is functionally inhibited by active translation over its sequence. Together, these results suggest that IRE activity is required in the cytosol at an early step in the viral replication process, such as template recruitment and/or replicase complex assembly.Positive-strand RNA viruses replicate their genomes via a negative-strand RNA intermediate (2). This process is catalyzed by a virally encoded RNA-dependent RNA polymerase that is the core subunit of a viral replicase complex. Replicase activity is regulated in part by cis-acting RNA elements in the viral genome (2). The core promoters for the synthesis of positive and negative strands are found at the termini of RNA templates, while other important RNA elements that modulate replicase function are more internally located. These auxiliary RNA elements can function directly by either enhancing or silencing promoter activity (18,20,24,28,29,41) or indirectly by influencing replicase assembly, template recruitment, or primer synthesis functions (4,22,23,26,33).RNA elements involved in genome replication have been studied extensively in the genus Tombusvirus (37). The type species of this genus, Tomato bushy stunt virus (TBSV), is an icosahedral virus that contains a 4.8-kb-long nonsegmented positive-strand RNA genome (11) (Fig. 1A). Replication of the TBSV genome requires two 5Ј-proximally encoded proteins, p33, an auxiliary replication protein, and p92, the RNA-dependent RNA polymerase (19) (Fig. 1A). p92 is produced by translational readthrough of the p33 stop codon and accumulates at about 5% the level of p33 in infected tissue (31).Most studies investigating the role of RNA elements in TBSV replication have been carried out by using TBSV defective interfering (DI) RNAs (34, 37). Typica...

Screening of the Yeast yTHC Collection Identifies Essential Host Factors Affecting Tombusvirus RNA Recombination

“…The two Nterminally overlapping proteins, termed p33 and p92, are essential for replication. The p92 is the viral RNA-dependent RNA polymerases, whereas p33 replication cofactor, an RNAbinding protein (46,49,50), is involved in template selection and recruitment of viral RNA into replication (29,43,49). These proteins interact with each other and the viral RNA in cells (43, 51), which leads to replication complexes formed on peroxisomal membranes (41,43).…”

Conformational organization of the 3′ untranslated region in the tomato bushy stunt virus genome

“…For example, RNA elements involved in genome replication can be located in coding regions, thereby directing both translational and replication functions (McKnight and Lemon 1998;Goodfellow et al 2000;You et al 2004;Monkewich et al 2005;Pogany et al 2005). Even when a distinct RNA element is used for one specific function, it is often highly integrated with other operative RNA elements.…”

Identification of Essential Host Factors Affecting Tombusvirus RNA Replication Based on the Yeast Tet Promoters Hughes Collection

“…p92 pol is the viral RNA-dependent RNA polymerase (RdRp), whereas p33 replication cofactor (which overlaps with the Nterminal pre-readthrough segment of p92 pol ) is an RNA-binding protein (28,32,33). Earlier work defined that p33 is involved in template selection and recruitment of viral RNA into replication (18,25,32). These proteins interact with each other, the viral RNA, and the host proteins in cells (25,34,35,39), which leads to the assembly of RC on peroxisomal membranes (22,25).…”