Protein-mediated RNA folding governs sequence-specific interactions between rotavirus genome segments

Abstract

Segmented RNA viruses are ubiquitous pathogens, which include influenza viruses and rotaviruses. A major challenge in understanding their assembly is the combinatorial problem of a non-random selection of a full genomic set of distinct RNAs. This process involves complex RNA-RNA and protein-RNA interactions, which are often obscured by non-specific binding at concentrations approaching in vivo assembly conditions. Here, we present direct experimental evidence of sequence-specific inter-segment interactions between rotavirus RNAs, taking place in a complex RNA- and protein-rich milieu. We show that binding of the rotavirus-encoded non-structural protein NSP2 to viral ssRNAs results in the remodeling of RNA, which is conducive to formation of stable inter-segment contacts. To identify the sites of these interactions, we have developed an RNA-RNA SELEX approach for mapping the sequences involved in inter-segment base-pairing. Our findings elucidate the molecular basis underlying inter-segment interactions in rotaviruses, paving the way for delineating similar RNA-RNA interactions that govern assembly of other segmented RNA viruses.

eLife digest

Rotavirus is a highly infectious virus that affects children worldwide, causing severe diarrhoea. Despite the introduction of several highly effective vaccines, more than 200,000 children still die from rotavirus each year. There are currently no drugs that can combat this disease once a child has been infected.

Viruses carry the instructions that determine their properties and behavior in molecules of DNA or RNA. Unlike many other viruses, which typically have a single molecule of DNA or RNA, rotavirus has 11 distinct “RNA segments”. After invading a cell the virus begins to replicate itself. During replication, the RNA segments (which consist of two strands of RNA paired together) are copied many times. It is not clear how rotaviruses ‘count’ up to 11 so that each new virus acquires a single copy of each segment.

Previous biochemical and structural studies of rotavirus replication suggest that selecting 11 distinct RNA segments must involve the RNAs forming complex interactions with proteins and other RNA molecules. Using a highly sensitive fluorescence-based approach, termed fluorescence cross-correlation spectroscopy, Borodavka et al. now present direct experimental evidence of interactions between the RNA segments that occur via single strands of the rotavirus RNA.

These RNA-RNA interactions require the binding of a rotavirus protein NSP2 to the RNA strands, which results in the remodeling of the RNA; this remodeling is required to form stable contacts between different RNA segments. Furthermore, a new experimental approach (called RNA-RNA SELEX) developed by Borodavka et al. identified the parts of the RNA segments that may take part in these interactions.

The results presented by Borodavka et al. pave the way for identifying the RNA-RNA interactions that govern how other segmented RNA viruses can package their genetic material. Further work to uncover the entire RNA interaction network in rotaviruses would also accelerate the design of new vaccines and may help us to develop antiviral drugs to treat infections.