Structural Properties of a Multifunctional T-Shaped RNA Domain That Mediate Efficient Tomato Bushy Stunt Virus RNA Replication

Abstract

In positive-strand RNA viruses, 5 untranslated regions (5 UTRs) mediate many essential viral processes, including genome replication. Previously, we proposed that the 5-terminal portion of the genomic leader sequence of Tomato bushy stunt virus (TBSV) forms an RNA structure containing a 3-helix junction, termed the T-shaped domain (TSD). In the present study, we have carried out structure-function analysis of the proposed TSD and have confirmed an important role for this domain in mediating efficient viral RNA amplification. Using a model TBSV defective interfering RNA replicon and a protoplast system, we demonstrated that various TSD subelements contribute to the efficiency of viral RNA replication. In particular, the stabilities of all three stems (S1, S2, and S4) forming the 3-helix junction are important, while stem-loop 3-a terminal extension of S2-is largely dispensable. Additionally, some of the sequences forming the 3-helix junction are required in an identity-dependent manner. Thus, both secondary structure and nucleotide identity are important for TSDmediated viral RNA replication. Importantly, these results are fully consistent with the dual functions we defined previously for the sequences corresponding to loops 3 and 4, respectively, in facilitating 5 cap-and 3 poly(A) tail-independent translation of the genome by forming a loop-loop interaction with the 3-proximal translational enhancer and in mediating viral RNA replication through formation of a pseudoknot with the adjacent downstream RNA domain. Also, since comparable TSDs and associated interactions are predicted in the 5 UTRs of all sequenced Aureusvirus genomes, members of at least one other genus in the family Tombusviridae appear to utilize this type of multifunctional RNA domain.Genome replication is an essential feature of virus reproduction. For positive-strand RNA viruses, efficient genome replication proceeds through a negative-strand RNA intermediate and is regulated by viral RNA elements, viral proteins, and host factors (2). The 5Ј untranslated regions (5Ј UTRs) in viral genomes contain primary, secondary, and tertiary RNA structures that are involved in genome replication. Accordingly, structural and functional assessments of these 5Ј UTRs have led to important advances in our understanding of viral genome replication and related regulatory mechanisms (2).Tomato bushy stunt virus (TBSV), the prototype member of the genus Tombusvirus in the family Tombusviridae, is a monopartite positive-strand RNA virus (25). Its 4.8-kb genome encodes five viral proteins with known functions (10) (Fig. 1A). Viral RNA replication requires only p33 and its translational readthrough product p92 (the RNA-dependent RNA polymerase, RdRp) (17). These two proteins are also able to replicate defective interfering (DI) RNAs, which are small deleted forms of the genome that maintain cis-acting viral RNA replication elements (e.g., promoters and enhancers) (28). As such, tombusvirus DI RNAs have been very useful for defining RNA sequences and structures involv...

Analysis of a 3′-translation enhancer in a tombusvirus: A dynamic model for RNA–RNA interactions of mRNA termini

“…That is, it can be re-located to different sites relative to either the 59 terminus or the initiation codon in our experimental mRNA and still function efficiently in translation. However, within the viral genomic context, the normal location of SL3 is universally conserved among tombusviruses and is highly integrated with replication elements (Wu et al 2001;Ray et al 2003Ray et al , 2004. Thus, although flexible in terms of a translation-only context, the repositioning of SL3 in the wild-type genome would likely negatively affect the activity of other RNA elements involved in RNA replication and thus reduce the competitiveness of the virus.…”

“…1C). The TSD also plays a critical role in viral RNA replication as mutations in this domain severely compromise viral replicon accumulation in vivo (Wu et al 2001;Ray et al 2003Ray et al , 2004. Translation and replication functions are thus highly integrated within this 59-proximal RNA domain.…”

“…1B,C). All three of these structures are critical for efficient viral RNA replication (Wu et al 2001;Ray et al 2003Ray et al , 2004, whereas SL3 in the TSD represents the key element necessary for efficient TBSV mRNA translation ( Fig. 1C; Fabian and White 2004).…”

Oscillating kissing stem–loop interactions mediate 5′ scanning-dependent translation by a viral 3′-cap-independent translation element

“…6B). The 59-proximal stem-loop plays a key role in replication of many positive-strand viral RNAs (Barton et al 2001;Vlot and Bol 2003), including members of the Tombusviridae (Ray et al 2004), which resemble BYDV in many aspects . Either BYDV is exceptional in that its 59-proximal stem-loop is not involved in replication, or (more likely) it tolerates the single base change that converted SL-A to a BCL and kissing by the BTE, and presence of associated translational machinery does not inhibit the role of SL-A in replication.…”

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

“…1B). RI is important but not essential for efficient DI RNA replication (21,27,30,38,39), while RIV is critical (8,17,24). In addition to these terminal regions, two noncontiguous internal segments, RII and RIII, are present in DI RNAs (Fig.…”