Principles of Virology. Jane Flint

Principles of Virology - Jane Flint


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charges of the sugar-phosphate backbone. Neutralization might be accomplished by proteins that form the inner surface of the capsid, or by the incorporation of small, basic peptides made by the host cell, such as spermine and spermidine.

      Electron microscopy of cores released from adenovirus particles and cryo-EM of virus particles have suggested that the internal nucleoprotein is also arranged in some regular fashion. However, how the viral DNA genome is organized and condensed by the core proteins is not known: the nucleoprotein was not observed in the high-resolution structures of adenovirus particles described previously, and the structures of core proteins have not been determined. The fundamental DNA packaging unit is a multimer of protein VII, which appears as beads on a string of adenoviral DNA when other core proteins are removed. Protein VII binds tightly to and condenses double-stranded DNA in vitro, consistent with a packaging function, but has been reported to be dispensable for assembly of virus particles (Chapter 13). Protein VII and the other core proteins are basic, as would be expected for proteins that bind to a negatively charged DNA molecule without sequence specificity.

      The final mechanism for condensing the viral genome, by cellular proteins, is unique to polyomaviruses, such as simian virus 40, and papillomaviruses. The circular, double-stranded DNA genomes released from these virus particles are organized into nucleosomes that contain the four cellular core histones, H2A, H2B, H3, and H4, to form a minichromosome. Comparison of cryo-EM structures of purified particles of the human polyomavirus BK virus and virus-like particles formed only from VP1 (the major structural protein) has revealed two radial shells of the DNA genome within virus particles (Fig. 4.20B). The thickness of these shells (24 Å) and the distance between them match those of double-stranded DNA within a human nucleosome. The 20 or so nucleosomes that are associated with polyomaviral genomes condense the DNA by a factor of ~7. This packaging mechanism is elegant, with two major advantages: none of the limited viral genetic information needs to be devoted to DNA-binding proteins, and the viral genome, which is transcribed by cellular RNA polymerase II, enters the infected cell nucleus as a nucleoprotein closely resembling the cellular templates for this enzyme.

      EXPERIMENTS

       A high-resolution view of an encapsidated viral genome

      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.

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      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.

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