Global Organization of a Positive-strand RNA Virus Genome

Abstract

The genomes of plus-strand RNA viruses contain many regulatory sequences and structures that direct different viral processes. The traditional view of these RNA elements are as local structures present in non-coding regions. However, this view is changing due to the discovery of regulatory elements in coding regions and functional long-range intra-genomic base pairing interactions. The ∼4.8 kb long RNA genome of the tombusvirus tomato bushy stunt virus (TBSV) contains these types of structural features, including six different functional long-distance interactions. We hypothesized that to achieve these multiple interactions this viral genome must utilize a large-scale organizational strategy and, accordingly, we sought to assess the global conformation of the entire TBSV genome. Atomic force micrographs of the genome indicated a mostly condensed structure composed of interconnected protrusions extending from a central hub. This configuration was consistent with the genomic secondary structure model generated using high-throughput selective 2′-hydroxyl acylation analysed by primer extension (i.e. SHAPE), which predicted different sized RNA domains originating from a central region. Known RNA elements were identified in both domain and inter-domain regions, and novel structural features were predicted and functionally confirmed. Interestingly, only two of the six long-range interactions known to form were present in the structural model. However, for those interactions that did not form, complementary partner sequences were positioned relatively close to each other in the structure, suggesting that the secondary structure level of viral genome structure could provide a basic scaffold for the formation of different long-range interactions. The higher-order structural model for the TBSV RNA genome provides a snapshot of the complex framework that allows multiple functional components to operate in concert within a confined context.

Author Summary

The genomes of many important pathogenic viruses are made of RNA. These genomes encode viral proteins and contain regulatory sequences and structures. In some viruses, distant regions of the RNA genome can interact with each other via base pairing, which suggests that certain genomes may take on well-defined conformations. This concept was investigated using a tombusvirus RNA genome that contains several long-range RNA interactions. The results of microscopic and biochemical analyses indicated a compact genome conformation with structured regions radiating from a central core. The structural model was compatible with some, but not all, long-range interactions, suggesting that the genome is a dynamic molecule that assumes different conformations. The analysis also revealed new structural features of the genome, some of which were shown to be functionally relevant. This study advances our understanding of the role played by global structure in virus genome function and provides a model to further investigate its in role virus reproduction. We anticipate that organizational principles revealed by this investigation will be applicable to other viruses.