Visualizing viral RNA packaging signals in action

Here we confirm, using genome-scale RNA fragments in assembly competition assays, that multiple sub-sites (Packaging Signals, PSs) across the 5́ two-thirds of the gRNA of Satellite Tobacco Necrosis Virus-1 make sequence-specific contacts to the viral CPs helping to nucleate formation of its T=1 virus-like particle (VLP). These contacts explain why natural virions only package their positive-sense genomes. Asymmetric cryo-EM reconstructions of these VLPs suggest that these interactions occur between amino acid residues in the N-terminal ends of the CP subunits and the gRNA PS loop sequences. The base-paired stems of PSs also act non-sequence-specifically by electrostatically promoting the assembly of CP trimers. Importantly, alterations in PS-CP affinity result in an asymmetric distribution of bound PSs inside VLPs, with fuller occupation of the higher affinity 5́ PS RNAs around one vertex, decreasing to an RNA-free opposite vertex within the VLP shell. This distribution suggests that gRNA folding regulates cytoplasmic genome extrusion so that the weakly bound 3́ end of the gRNA, containing the RNA polymerase binding site, extrudes first. This probably occurs after cation-loss induced swelling of the CP-shell, weakening contacts between CP subunits. These data reveal for the first time in any virus how differential PS folding propensity and CP affinities support the multiple roles genomes play in virion assembly and infection. The high degree of conservation between the CP fold of STNV-1 and those of the CPs of many other viruses suggests that these aspects of genome function will be widely shared.