Coaxing a Viral RNA Out of its Shell: How does a Viral RNA Genome Initiate Contact with its Host?

A conceptual challenge in understanding the life cycles of all viruses is how a rigid protein shell - the “capsid” - can both protect the viral genome from nucleases and other insults and, at the appropriate downstream stage, make its genetic information available to the host cell machinery. For double-stranded DNA viruses, like bacteriophages or Herpes Simplex Virus, genomes are packaged into preformed empty capsids, and released by pressure-driven ejection, with the capsid remaining intact: work is done to package the genome, but its delivery is spontaneous. In contrast, for single-stranded RNA viruses, like polio or SARS, packaging of their genomes is spontaneous, with capsid protein organizing around the RNA in a self-assembly process. This immediately raises the question of whether work is needed to release the RNA from its protective capsid. In the case of positive-strand RNA viruses, whose genomes are directly translated following their entry into the host cell cytoplasm, ribosomes have been implicated in the delivery/release of the viral RNA. We report here on the synthesis of constructs designed to measure the force needed to pull RNA out of its capsid, using virus-like particles reconstituted in vitro from RNA and purified capsid protein from a particularly well-studied virus, cowpea chlorotic mottle virus (CCMV). We also investigate the extent to which the ends of a packaged RNA are accessible for interaction with probes impermeable to the interior of the capsids. These results are used to interpret relative translation efficiencies of naked and packaged forms of RNA genes in cell-free extracts, in an effort to determine how ribosomes are involved in this RNA uncoating process.