Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 189

The small icosahedral capsid (T = 3) of the Escherichia coli bacteriophage MS2 is built from dimers of a single coat protein and one copy of a maturation protein, which is responsible for delivery of the genome to host cells. The capsid contains a single-stranded (+) RNA genome of 3,569 bases, the first genome to be sequenced completely. Cryo-EM of MS2 particles and averaging of more than 300,000 images without imposing any symmetry allowed visualization of >80% of the viral RNA genome at 6-Å resolution. As shown in panel A of the figure, most of the RNA showed prominent major and minor grooves; i.e., it is double-stranded, and is organized as stem-loops. Some of these structures could be examined at higher resolution (3.6 Å), indicating that they are more ordered, and their sequences determined from features of purines and pyrimidines seen in the EM density (panel B in the figure). This study illustrates the power of asymmetric reconstruction of images collected by cryo-EM, and the high degree of viral genome folding that can be imposed by interactions with capsid proteins.

 Dai X, Li Z, Lai M, Shu S, Du Y, Zhou ZH, Sun R. 2017. In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus. Nature 541:112–116.

Structure of a small viral RNA genome. (A) Cut-open view of the asymmetric reconstruction of MS2 (6 Å) with the capsid shell colored yellow to red (by radial distance), the maturation protein in magenta, and the RNA in blue with major and minor grooves indicated. (B) A segment of an RNA stem-loop observed at 3.6-Å resolution, with RNA backbone and bases imposed on the EM density (mesh). Adapted from Dai X et al. 2017. Nature 541:112–116, with permission. Courtesy of H. Zhou, University of California, Los Angeles. See also https://media.nature.com/original/nature-assets/nature/journal/v541/n7635/extref/nature20589-sv1.mp4.

Figure 4.20 Packing of double-stranded DNA genome. (A) Dense packing in the head of bacteriophage T4 DNA. The central section of a 22-Å cryo-EM reconstruction of the head of bacteriophage T4 viewed perpendicular to the fivefold axis is shown. The concentric layers seen underneath the capsid shell have been attributed to the viral DNA genome. The connector, which is derived from the portal structure by which the DNA genome enters the head during assembly, connects the head to the tail. Adapted from Fokine A et al. 2004. Proc Natl Acad Sci U S A 101:6003–6008, with permission. Courtesy of M. Rossmann, Purdue University. (B) (Left) Cryo-EM reconstruction of the polyomavirus BK virus shown as a 40-Å-thick slab, with fitted VP1 density in gray, density assigned to the minor structural proteins VP2 and VP3 in blue/green, and to packaged double-stranded DNA in yellow to pink. These colored densities were not observed in virus-like particles assembled from only VP1. (Right) An enlarged view of the density below a single VP1 penton, indicating the thickness of the radial layers of DNA and the spacing between them. A model of double-stranded DNA as it appears when wrapped on a human histone (blue) is superimposed. Adapted from Hurdiss DL et al. 2016. Structure 24:528–536, licensed under CC BY 4.0. Courtesy of N.A. Ranson, University of Leeds, United Kingdom.

Figure 4.21 Conserved organization of the RNA-packaging proteins of nonsegmented (−) strand RNA viruses. Ribbon diagrams of the N proteins indicated are shown at the top, colored from purple at the N terminus to red at the C terminus. Their electrostatic surfaces from negative (red) to positive (blue) are shown in the space-filling models below, with the molecules rotated as indicated to show the RNA-binding cleft (blue) most clearly. Although differing in structural details, these N proteins share a two-lobed structure (top) and an RNA-binding cleft between the two lobes. Adapted from Ruigrok RW et al. 2011. Curr Opin Microbiol 14:504–510, with permission. Courtesy of D. Kolakofsky, University of Geneva.