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Chapter 6 : Cell Entry: a Biochemical and Structural Perspective
Category: Viruses and Viral Pathogenesis
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This chapter begins with a discussion of some of the obstacles that have hampered progress in studying cell entry pathways for picornaviruses and other non-enveloped viruses. The chapter reviews what is known about the early steps leading to internalization of the viruses into intracellular vesicles, focusing on examples (for key members of the family) that point out the diversity in the cell entry pathways used as well as the common themes. It finishes with an exploration (admittedly poliovirus centered) of what we know about the machinery that facilitates translocation of the genome across the membrane once the virus has been internalized. The virion- to-135S (or A particle) transition has not been observed in the aphthoviruses (foot-and-mouth disease viruses [FMDVs] and equine rhinitis virus) or cardioviruses (encephalomyocarditis virus, mengovirus). Although clathrin-mediated endocytosis may serve as the predominant entry pathway for some picornaviruses (including FMDV, minor group rhinoviruses, and probably major group rhinoviruses), it is clear that other picornaviruses use a variety of other endocytic pathways. In a recent study it was shown that the coxsackievirus B3 first binds to DAF (CD55) on the apical surfaces of the cells. This study clearly demonstrates the importance of virus-induced signaling in cell entry. Advances in detectors and optics that should become available in the future will improve the resolution achievable in both cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) approaches, making it possible to approach near-atomic resolution from cryo-EM studies of intermediates in vitro and unprecedented resolution for structures determined in situ.
Summary of currently known endocytic pathways and their properties. (Figure obtained from Mercer and Helenius [ 62 ] with permission from the publisher.)
Structure of the virus-receptor-liposome complex as determined by cryo-EM (left) and cryo-ET (right). Both reconstructions are at approximately 30-Å resolution. The reconstructions are consistent with earlier models that suggested that the initial complex of the virus with the receptor on the cell surface would involve five copies of the receptor and bring a particle five-fold axis close to the membrane. The membrane in the reconstructions appears as an isolated patch due to masking and to variability in the membrane structure (both of which result from variabilities in size and deformations of the liposome) outside the immediate footprint of the five bound receptors. The reconstructions clearly show the densities for the three-domain receptor, including bulges that correspond to glycosylation sites in the second domain of the receptor. Both reconstructions also show a prominent crown-shaped bump in the membrane immediately below, where the virus five-fold axis makes its closest approach to the membrane. The appearance of this feature is consistent with an outward deformation of the outer leaflet of the membrane.
Structures of 80S particles. (A) Cryo-EM of preparations of 80S particles, showing that the preparations are heterogeneous with variable levels of density (presumably RNA) inside the particles. (B) A small percentage of these particles (5 to 10%) have density (again, presumably RNA) both inside and outside the particle. The density outside the particle is highly branched, suggesting that the RNA has refolded after exiting the particle, and in many cases, the externalized densities from neighboring particles intermingle. The particles can be classified into two clusters (80Se and 80Sl) based on the capsid structure. Cryo-EM reconstructions of the 80Se particles (C, D, and E) and 80Sl particles (F, G, and H) show the whole particle (C and F), a close-up of the outer surface (D and G), and the inner surface (E and H), including the prominent ridge crossing the two-fold axis that appears to “staple together” two neighboring pentamers.
The structure of 80S particles caught in the act of releasing their RNA. The figure shows a cryo-ET reconstruction of a single virus particle. The density for the capsid (dark grey) is the average of over 20 icosahedrally averaged subtomograms. The density for the RNA comes from a single unaveraged tomogram. Cryo-ET reconstructions of other particles are similar, differing only in the appearance of the RNA, which is unique to each reconstruction. The reconstructions clearly show that the RNA is released from the base of the canyon near a quasi-three-fold axis (see Color Plate 8). This site corresponds to sites of noticeable thinning of the shell in the icosahedral cryo-EM reconstructions of the 80Se and 80Sl particles.
The key cell entry intermediates and their sources in vitro and in vivo