Bipartite viral RNA genome heterodimerization influences genome packaging and virion thermostability

Diverse RNA-RNA interactions occur throughout the lifecycle of RNA viruses, including genome dimerization or multimerization through non-covalent association of two or more genomic segments. Both homo-dimerization of retrovirus genomic RNA and hetero-multimerization of genomic segments of multipartite viruses are recognized as important factors that govern genome packaging. The heterodimer of the bipartite alphanodaviruses represents a unique case as its formation is conserved among different virus species but is only observed within the context of fully assembled virus particles. In spite of this, the RNA sequences involved in heterodimerization are unknown. It also remains unclear whether the formation of this heterodimer might impact any stage of the virus lifecycle. In this study, we used Flock House virus (FHV) as a model system to uncover the nucleotide composition of the heterodimer and dissected the impact of heterodimer formation upon numerous stages of the virus lifecycle. We developed a novel Next-Generation Sequencing (NGS) approach called "XL-ClickSeq" to probe candidate heterodimer sequences. We found that the heterodimer is formed via intermolecular base-pairing and its formation is retained in defective virus particles. One heterodimer site was identified by thermodynamic prediction that, upon mutagenic disruption, exhibited significant deficiencies in genome packaging and encapsidation specificity to viral genomic RNA. Furthermore, we demonstrate that disruption of this RNA secondary structure directly impacts the thermostability of mature virus particles. These results demonstrated that the intermolecular RNA-RNA interactions within the encapsidated genome of an RNA virus can have an important impact on virus particle integrity and stability and thus its transmission to a new host.