RNA secondary structures play diverse roles in positive-sense (+) RNA virus infections, but those located with the replication protein coding sequence can be difficult to investigate. Structures that regulate the translation of replication proteins pose particular challenges, as their potential involvement in post-translational steps cannot be easily discerned independent of their roles in regulating translation. In the current study, we attempted to overcome these difficulties by providing viral replication proteins in trans. Specifically, we modified the plant-infecting turnip crinkle virus (TCV) into variants that are unable to translate one (p88) or both (p28 and p88) replication proteins, and complemented their replication with the corresponding replication protein(s) produced from separate, non-replicating constructs. This approach permitted us to re-examine the p28/p88 coding region for potential RNA elements needed for TCV replication. We found that, while more than a third of the p88 coding sequence could be deleted without substantially affecting viral RNA levels, two relatively small regions, known as RSE and IRE, were essential for robust accumulation of TCV genomic RNA, but not subgenomic RNAs. In particular, the RSE element, found previously to be required for regulating the translational read-through of p28 stop codon to produce p88, contained sub-elements needed for efficient replication of the TCV genome. Application of this new approach in other viruses could reveal novel RNA secondary structures vital for viral multiplication.
Many positive sense RNA viruses, especially those infecting plants, are known to experience stringent, stochastic population bottlenecks inside the cells they invade, but exactly how and why these populations become bottlenecked are unclear. A model proposed ten years ago advocates that such bottlenecks are evolutionarily favored because they cause the isolation of individual viral variants in separate cells. Such isolation in turn allows the viral variants to manifest the phenotypic differences they encode. Recently published observations lend mechanistic support to this model, and prompt us to refine the model with novel molecular details. The refined model, designated Bottleneck, Isolate, Amplify, Select (BIAS), postulates that these viruses impose population bottlenecks on themselves by encoding bottleneck-enforcing proteins (BNEPs) that function in a concentration-dependent manner. In cells simultaneously invaded by numerous virions of the same virus, BNEPs reach the bottleneck-ready concentration sufficiently early to arrest nearly all internalized viral genomes. As a result, very few (as few as one) viral genomes stochastically escape to initiate reproduction. Repetition of this process in successively infected cells isolate viral genomes with different mutations in separate cells. This isolation prevents mutant viruses encoding defective viral proteins from hitchhiking on sister genome-encoded products, leading to the swift purging of such mutants. Importantly, genome isolation also ensures viral genomes harboring beneficial mutations accrue the cognate benefit exclusively to themselves, leading to the fixation of such beneficial mutations. Further interrogation of the BIAS hypothesis promises to deepen our understanding of virus evolution, and inspire new solutions to virus disease mitigation.
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