Abstract
Dengue virus (DENV) is a single-stranded (+)-sense RNA virus that infects humans and mosquitoes, posing a significant health risk in tropical and subtropical regions. Mature virions are composed of an icosahedral shell of envelope (E) and membrane (M) proteins circumscribing a lipid bilayer, which in turn contains a complex of the approximately 11 kb genomic RNA with capsid (C) proteins. Whereas the structure of the envelope is clearly defined, the structure of the packaged genome in complex with C proteins remains elusive. Here, we investigated the interactions of C proteins with viral RNA, in solution and inside mature virions, via footprinting and cross-linking experiments. We demonstrated that C protein interaction with DENV genomes saturates at an RNA:C protein ratio below 1:250. Moreover, we also showed that the length of the RNA genome interaction sites varies, in a multimodal distribution, consistent with the C protein binding to each RNA site mostly in singlets or pairs (and, in some instances, higher numbers). We showed that interaction sites are preferentially sites with low base pairing, as previously measured by 2'-acetylation analyzed by primer extension (SHAPE) reactivity indicating structuredness. We found a clear association pattern emerged: RNA-C protein binding sites are strongly associated with long-range RNA-RNA interaction sites, particularly inside virions. This, in turn, explains the need for C protein in viral genome packaging: the protein has a chief role in coordinating these key interactions, promoting proper packaging of viral RNA. Such sites are, thus, highly consequential for viral assembly, and, as such, may be targeted in future drug development strategies against these and related viruses.