Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 247

Most virus particles that enter cells by receptor-mediated endocytosis leave the pathway before the vesicles reach the lysosomal compartment. This departure is not surprising, for lysosomes contain proteases and nucleases that would degrade virus particles. However, these enzymes play an important role during the uncoating of members of the Reoviridae.

Orthoreoviruses are naked icosahedral viruses containing a double-stranded RNA genome of 10 segments. The viral capsid is a double-shelled structure assembled from eight different proteins. These virus particles bind to cell receptors via protein σ1 and are internalized into cells by endocytosis (Fig. 5.23). The intact virus particle comprises two concentric, icosahedrally organized protein capsids. The outer capsid is made up largely of σ3 and μ1. The dense core shell is formed mainly by λ1 and σ2.

Infection of cells by reoviruses is sensitive to bafilomycin A1, an inhibitor of the endosomal proton pump, indicating that acidification is required for entry. Disassembly occurs in multiple steps while the virus particle travels within endosomes to the lysosome (Fig. 5.23A). The process is initiated with the acid-induced proteolysis that releases the 600 σ3 subunits of the capsid. The μ1 protein changes from a compact form to an extended flexible fiber, producing an infectious subviral particle (ISVP). The μ1 protein undergoes significant conformational changes and is cleaved at three sites, one of which releases the myristoylated N terminus, μ1N, which can insert into membranes (Fig. 5.23B). Both μ1N and μ1C are required for membrane penetration. Isolated ISVPs cause cell membranes to become permeable to toxins and produce pores in artificial membranes. These can also initiate an infection by penetrating the plasma membrane, entering the cytoplasm directly. Their infectivity is not sensitive to bafilomycin A1, further supporting the idea that these particles are primed for membrane entry and do not require further acidification for this process.

The core particles generated from infectious subviral particles after penetration into the cytoplasm adopt a third morphology and carry out viral mRNA synthesis. The core is produced by the release of 12 σ1 fibers and 600 μ1 subunits. In the transition from ISVP to core, domains of λ2 rotate upward and outward to form a turret-like structure (Fig. 5.23A).

Figure 5.23 Entry of reovirus into cells. (A) The different stages in cell entry of reovirus. After the attachment of σ1 protein to the cell receptor, the virus particle enters the cell by clathrin-mediated endocytosis. Proteolysis in the late endosome produces the infectious subviral particle (ISVP). The viral μ1, a myristoylated protein, is located at the surface of these particles and interacts with membranes. Consequently, subviral particles penetrate the lysosomal membrane and escape into the cytosol. (Insets) Close-up views of the emerging turret-like structure as the virus progresses through the ISVP and core stages. This structure may facilitate the entry of nucleotides into the core and the exit of newly synthesized viral mRNAs. (B) Schematic of the μ1 protein, showing locations of myristate and the protease cleavage sites flanked by the amphipathic α-helices. Virus images based on studies performed with mammalian reovirus type I Lang, reprinted from Dryden KA et al. 1993. J Cell Biol 122:1023–1041, with permission. Courtesy of Norm Olson and Tim Baker, Purdue University.