Abstract
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 pre-formed 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 co-self-assembly process. These RNA-containing viral capsids are relatively stable, inert particles while on their journey to the next host cell, but must eventually release their RNA to its host to make it available for further viral life cycle processes. 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 into the viral replication machinery 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 capsid. The probing techniques offer keen insights regarding the ability for RNA to fluctuate outside of its intact capsid in order to engage the host ribosomal machinery. These results are used to interpret corresponding relative translation efficiencies of naked and packaged forms of RNA genes in cell-free extracts, in an effort to determine how ribosomes and their associated factors are involved in this viral RNA uncoating process. Support or Funding Information Research was supported by the NSF, and for R.W.S. by USPHS of the National Institute of Heath, National Research Service Award 5T32GM008496-20 and the UCLA Graduate Office. An in vitro self-assembled virus-like particle, the cherry bomb, a construct used for generating a handle on an encapsidated viral RNA genome. We are using this construct such that we have access to the viral RNA, can pull it out of its capsid and to measure the force necessary to do so.
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