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Asymmetric cryo-EM Reconstruction of Phage MS2 Reveals Genome Structure in Situ


Asymmetric cryo-EM Reconstruction of Phage MS2 Reveals Genome Structure in Situ

Roman I Koning et al. Nat Commun.


In single-stranded ribonucleic acid (RNA) viruses, virus capsid assembly and genome packaging are intertwined processes. Using cryo-electron microscopy and single particle analysis we determined the asymmetric virion structure of bacteriophage MS2, which includes 178 copies of the coat protein, a single copy of the A-protein and the RNA genome. This reveals that in situ, the viral RNA genome can adopt a defined conformation. The RNA forms a branched network of stem-loops that almost all allocate near the capsid inner surface, while predominantly binding to coat protein dimers that are located in one-half of the capsid. This suggests that genomic RNA is highly involved in genome packaging and virion assembly.


Figure 1
Figure 1. The asymmetric reconstruction of bacteriophage MS2.
Asymmetric structure of bacteriophage MS2 (green–blue radially coloured) shows the AP (yellow) (a), which replaces one CP dimer (b). Inside the protein capsid a structured genome (grey) is present (c) that is connected to the AP (d). The reconstruction shows the double-stranded helices in the stem loop structures (e). At some positions individual NA's connecting to the capsid are resolved, as shown by fitting of the X-ray structure of the 19-nucleotide TR (magenta) bound to the capsid (blue) (pdb:1ZDH) in the EM density (grey) (f). Scale bar is 100Å.
Figure 2
Figure 2. Proposed RNA densities of known AP binding sites.
Left: nucleotides 3,510–3,527 (magenta) and the RNA secondary structure prediction (asterisk marks binding site). Right: nucleotides 388–414 (blue) with fit in density (red) and the secondary structure of the SL (a). Asymmetric distribution of SL–CP2 interactions; the 23 capsid dimers (red) to which the bound RNA (grey) SLs fit the TR are predominantly localized at one side of the capsid (b). Proposed model for MS2 virion assembly: AP–RNA–(CP2)n complex forms primer (i) to which CP2 dimers bind and start to form the capsid (ii), CP2 is both being recruited by existing SLs and induce new SLs in the RNA (iii) CP2 induced refolding and binding of the 5′ end AP binding site results in condensation of the RNA (iv) that enables efficient packaging and formation of a stable virion (v) (c).

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