Dynamic network approach for the modelling of genomic sub-complexes in multi-segmented viruses

Abstract

Viruses with segmented genomes, including pathogens such as influenza virus, Rotavirus and Bluetongue virus (BTV), face the collective challenge of packaging their genetic material in terms of the correct number and types of segments. Here we develop a novel network approach to predict RNA–RNA interactions between different genomic segments. Experimental data on RNA complex formation in the multi-segmented BTV genome are used to establish proof-of-concept of this technique. In particular, we show that trans interactions between segments occur at multiple specific sites, termed segment assortment signals (SASs) that are dispersed across each segment. In order to validate the putative trans acting networks, we used various biochemical and molecular techniques which confirmed predictions of the RNA network approach. A combination of mutagenesis and reverse genetics systems revealed that the RNA–RNA interacting sites identified are indeed responsible for segment assortment and complex formation, which are essential criteria for genome packaging. This paves the way for their exploitation as novel types of drug target, either to inhibit assembly, or for designing defective interfering particles containing an incomplete set of genomic segments.

The Interaction of Bluetongue Virus VP6 and Genomic RNA Is Essential for Genome Packaging

“…It is highly likely that RNA segments interact with each other in a sequential manner prior to the packaging of the RNA complex into the capsid (1, 5, 15). In a recent report, we showed that RNA segment assortment is based mainly on RNA-RNA interactions via specific sequences (32). It is possible that VP6 is also actively involved in assortment, but our current data do not support this notion.…”

Trans-Acting RNA–RNA Interactions in Segmented RNA Viruses

“…Interestingly, while the loss of function of one of the mRNA S10-S6 interactions caused by substitutions could be recovered by compensatory substitutions, the same was not true for a second interaction that was tested. The latter result suggests that localized changes can have global effects in a segment that lead to interference with other parts of the interactive network [37]. Additional functionally relevant BTV segment interactions have also been reported.…”

“…Recently, a computational approach was used to predict a sequential assembly pathway to describe mRNA complex formation for segments S6 through S10 (Figure 3B) [37]. Briefly, the approach involved first identifying predicted ssRNA regions in each mRNA and then assessing potential tRRIs between these regions.…”

“…The results yielded a pathway for segment assembly that involved a dynamic network of interactions (Figure 3B), with the lengths of interacting regions ranging from 9 to 18 base pairs and free energies spanning −16 to 27 kcal/mol. Interestingly, transitions from one stage to the next were predicted to involve both the addition of new interactions, as well as the loss of existing interactions [37]. The latter observation highlights the possible involvement of transient interactions during complex assembly.…”

Reovirus RNA recombination is sequence directed and generates internally deleted defective genome segments during passage

“…Reoviridae +RNA sequences required for packaging, assortment, transcription, or translation encompass the UTR and extend into the ORF (17)(18)(19)(20)(21). Experimental and computational evidence suggests inter-segment complementarity drives assortment of Reoviridae +RNAs, with shorter gene segments nucleating +RNA complexes that are packaged into assembling viral capsids (22)(23)(24)(25)(26).…”