Structural Plasticity and Rapid Evolution in a Viral RNA Revealed by In Vivo Genetic Selection

Abstract

Satellite RNAs usually lack substantial homology with their helper viruses. The 356-nucleotide satC of Turnip crinkle virus (TCV) is unusual in that its 3-half shares high sequence similarity with the TCV 3 end. Computer modeling, structure probing, and/or compensatory mutagenesis identified four hairpins and three pseudoknots in this TCV region that participate in replication and/or translation. Two hairpins and two pseudoknots have been confirmed as important for satC replication. One portion of the related 3 end of satC that remains poorly characterized corresponds to juxtaposed TCV hairpins H4a and H4b and pseudoknot 3 , which are required for the TCV-specific requirement of translation (V. A. Stupina et al., RNA 14:2379-2393, 2008). Replacement of satC H4a with randomized sequence and scoring for fitness in plants by in vivo genetic selection (SELEX) resulted in winning sequences that contain an H4a-like stem-loop, which can have additional upstream sequence composing a portion of the stem. SELEX of the combined H4a and H4b region in satC generated three distinct groups of winning sequences. One group models into two stem-loops similar to H4a and H4b of TCV. However, the selected sequences in the other two groups model into single hairpins. Evolution of these single-hairpin SELEX winners in plants resulted in satC that can accumulate to wild-type (wt) levels in protoplasts but remain less fit in planta when competed against wt satC. These data indicate that two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts.Defective interfering (DI) and satellite (sat) RNAs are subviral RNAs that associate with viruses, require virus-encoded proteins for replication and other activities, and are capable of modifying viral infections. DI RNAs, found primarily in infections of animal hosts, are generated from viral genomic sequence, while satRNAs, more common to plant viruses, feature sequence that usually is unrelated to the helper virus. Most of the subviral RNAs with limited genome sizes are not translated, and thus their effect on virus infection must be mediated by the RNA primary or higher-ordered structure. DI and satRNAs that associate with different positive-strand RNA viruses can either intensify or attenuate viral symptoms (3, 10), which can involve inhibition of virus-encoded posttranscriptional gene silencing suppressors (9) or activation of posttranscriptional gene silencing (23).satRNAs and DI RNAs that share partial or near-complete sequence similarity with their helper virus contain related regions that allow for recognition by the helper virus RNAdependent RNA polymerase (RdRp). While this had led to the supposition that subviral RNAs would be useful models for examining replication elements that also exist in the much larger helper virus genome, more recent findings that RNA viruses contain elements in both their 5Ј and 3Ј untranslated regions that participate in translation (11,15,16), a function not required by small subviral RNAs, suggest that subvira...

Dynamic RNA structures in the dengue virus genome

“…23 Studies from many different laboratories using infectious clones and replicon systems provided compelling evidence for the essential role of genome cyclization during flavivirus replication. 23,76,86,[90][91][92][93] Mismatches within complementary regions did not alter translation of the viral RNA but greatly decreased RNA synthesis, leading in some cases to undetectable levels of viral replication. Compensatory mutations that restored 5'-3' base pairing rescued RNA synthesis indicating a role of RNA-RNA complementarity rather than the nucleotide sequence per se for viral replication.…”

RNA-RNA Recombination in Plant Virus Replication and Evolution

“…The phylogenetic-based sequence comparison and experimental approaches led to the suggestion that viral genomes comprise a series of modules that can be exchanged via recombination during mixed infection. This process, referred to as the modular evolution, gives plant viruses an enormous level of flexibility and a capacity for very rapid evolutionary changes (23,56,79).…”

Requirement for Host RNA-Silencing Components and the Virus-Silencing Suppressor when Second-Site Mutations Compensate for Structural Defects in the 3′ Untranslated Region

“…The Pr terminal loop additionally engages in a long-distance RNA: RNA interaction with a bulge loop in a hairpin just downstream from the ribosome readthrough site at the termination of the p28 ORF (7). 3= UTR hairpins H4a, H4b, and H5 are important for replication of satC (28,29) and, along with pseudoknots ⌿ 2 and ⌿ 3 , form a T-shaped structure (TSS) in the gRNA that functions as a translational enhancer through interaction with the 60S ribosomal subunit and/or 80S ribosome (30,31). Upstream of the TSS is H4, which is critical for both replication and translation (31)(32)(33).…”