Viral Uncoating Is Directional: Exit of the Genomic RNA in a Common Cold Virus Starts with the Poly-(A) Tail at the 3′-End

Shushan Harutyunyan, Mohit Kumar, Arthur Sedivy, Xavier Subirats, Heinrich Kowalski, Gottfried Köhler and Dieter Blaas

PLOS Pathogens, 2013, vol. 9, issue 4, 1-14

Abstract: Upon infection, many RNA viruses reorganize their capsid for release of the genome into the host cell cytosol for replication. Often, this process is triggered by receptor binding and/or by the acidic environment in endosomes. In the genus Enterovirus, which includes more than 150 human rhinovirus (HRV) serotypes causing the common cold, there is persuasive evidence that the viral RNA exits single-stranded through channels formed in the protein shell. We have determined the time-dependent emergence of the RNA ends from HRV2 on incubation of virions at 56°C using hybridization with specific oligonucleotides and detection by fluorescence correlation spectroscopy. We report that psoralen UV crosslinking prevents complete RNA release, allowing for identification of the sequences remaining inside the capsid. We also present the structure of uncoating intermediates in which parts of the RNA are condensed and take the form of a rod that is directed roughly towards a two-fold icosahedral axis, the presumed RNA exit point. Taken together, in contrast to schemes frequently depicted in textbooks and reviews, our findings demonstrate that exit of the RNA starts from the 3′-end. This suggests that packaging also occurs in an ordered manner resulting in the 3′-poly-(A) tail becoming located close to a position of pore formation during conversion of the virion into a subviral particle. This directional genome release may be common to many icosahedral non-enveloped single-stranded RNA viruses. Author Summary: Viral infection requires safe transfer of the viral genome from within the protective protein shell into the host cell's cytosol. For many viruses this happens after uptake into endosomes, where receptor-binding and/or the acidic pH trigger conformational modifications or disassembly of the shell, allowing the nucleic acids to escape. For example, common cold viruses are converted into subviral particles still containing the single-stranded positive sense RNA genome; subsequently, the RNA escapes into the cytoplasm, leaving behind empty capsids. We triggered this process by heating HRV2 to 56°C and found that 3′- and 5′-end emerged with different kinetics. Crosslinking prevented complete RNA egress and upon nuclease digestion only sequences derived from the 5′-end were protected. Part of the RNA remaining within the viral shell adopted a rod-like shape pointing towards one of the two-fold axes where the RNA is presumed to exit in single-stranded form. Egress thus commences with the poly-(A) tail and not with the genome-linked peptide VPg. This suggests that assembly and uncoating are well-coordinated to avoid tangling, kinetic traps, and/or simultaneous exit of the two RNA ends at different sites.

Date: 2013
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Persistent link: https://EconPapers.repec.org/RePEc:plo:ppat00:1003270

DOI: 10.1371/journal.ppat.1003270

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