Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 246

Following receptor-mediated endocytosis, nonenveloped (+) strand RNA viruses can escape from the endosome by forming pores in the membrane. For example, the interaction of poliovirus with its Ig-like cell receptor, CD155, leads to major conformational rearrangements in the virus particle and the production of an expanded form called an altered (A) particle (Fig. 5.22). VP4 and part of VP1 move from the inner surface of the capsid to the exterior and can associate with membranes. Shortly after internalization, the RNA is released into the cytoplasm. Early hypotheses suggested that VP1, VP4, and RNA were released from a channel at the 5-fold axes. However, structures of particles in the process of uncoating, and empty particles devoid of RNA, indicate that holes in the capsid that form at the 2-fold and quasi-3-fold axes of symmetry are sites of RNA exit. A long, “umbilical” connector appears to connect the virus particles to membranes and protect RNA as it passes into the cell.

The properties of a virus with substitutions in VP4 indicate that this protein is required for an early stage of cell entry. Virus particles with such amino acid alterations can bind to target cells and convert to A particles but are blocked at a subsequent, unidentified step. During poliovirus assembly, VP4 and VP2 are part of the precursor VP0, which remains uncleaved until the viral RNA has been encapsidated. The cleavage of VP0 during poliovirus assembly therefore primes the capsid for uncoating by separating VP4 from VP2.

In cells in culture, release of the poliovirus genome occurs from within early endosomes located close (within 100 to 200 nm) to the plasma membrane (Fig. 5.22). Uncoating is dependent on actin and tyrosine kinases, possibly for movement of the capsid via the network of actin filaments. Movement is not dependent on dynamin, clathrin, caveolin, or flotillin (a marker protein for clathrin- and caveolin-independent endocytosis); endosome acidification; or microtubules. The trigger for RNA release from early endosomes is not known but is clearly dependent on prior interaction with CD155. This conclusion derives from the finding that antibody-poliovirus complexes can bind to cells that produce Fc receptors but cannot infect them. As the Fc receptor is known to be endocytosed, these results suggest that interaction of poliovirus with CD155 is required to induce the conformational changes in the particle that are necessary for uncoating.

Figure 5.21 Stepwise uncoating of adenovirus. (A) Adenovirus fiber proteins bind a primary cell receptor, often CAR (Coxsackievirus and adenovirus receptor). Subsequently, interaction of the penton base with vibronectin-binding integrins α_vβ₃ and α_vβ₅ leads to internalization by endocytosis. Fibers are released from the capsid during uptake. The capsid protein is further destabilized in the endosome, likely triggered by low pH, and releases several viral proteins including protein VI (yellow). The hydrophobic N terminus of protein VI disrupts the endosome membrane, leading to release of the subviral particle into the cytoplasm. This particle is transported in the cytoplasm along microtubules and docks onto the nuclear pore complex, where further disassembly occurs to release the viral DNA into the nucleus. Individual steps in entry have been timed, and the overall process from receptor binding to nuclear entry takes a total 85 to 105 minutes. Data from Greber UF et al. 1993. Cell 75:477–486, 1993; and Trotman LC et al. 2001. Nat Cell Biol 3:1092–1100. (B) Electron micrograph of adenovirus type 2 particles bound to a microtubule (top) and bound to the cytoplasmic face of the nuclear pore complex (bottom). Reprinted from Greber UF et al. 1994. Trends Microbiol 2:52–56, with permission. Courtesy of Ari Helenius, Urs Greber, and Paul Webster, University of Zurich.

A critical regulator of the receptor-induced structural transitions of poliovirus particles appears to be a hydrophobic tunnel located below the surface of each structural unit (Fig. 5.22). The tunnel opens at the base of the canyon and extends toward the 5-fold axis of symmetry. In poliovirus type 1, each tunnel is occupied by a molecule of sphingosine. Similar lipids have been observed in the capsids of other picornaviruses. Because of the symmetry of the capsid, each virus particle may contain up to 60 lipid molecules. These lipids are thought to contribute to the stability of the native virus particle by locking the capsid in a stable conformation. Consequently, removal of the lipid is probably necessary to endow the particle with sufficient flexibility to permit the RNA to leave the protein shell.

The viral genome is released from the endosome, and it is usually assumed that the 5′ end of (+) strand RNAs is the first to leave the capsid, to allow immediate initiation of translation by ribosomes. This assumption is incorrect for rhinovirus type 2: exit of viral RNA starts from the 3′ end. This directionality is a consequence of how the viral RNA is packaged in the virus particle, with the 3′ end near the location of pore formation in the altered particle. Whether such directionality is a general feature of nonenveloped (+) strand RNA viruses is unknown.

Similar to picornaviruses, another family of nonenveloped (+) strand RNA viruses, caliciviruses, also form pores in the endosomal membrane. Binding to the receptor triggers conformational changes in the viral capsid, and following endocytosis, the capsid protein VP2 forms a large portal at the 3-fold axis of symmetry. This portal would allow delivery of the RNA genome to the cytoplasm.

Figure 5.22 Model for poliovirus entry into cells. The native virus particle (160S) binds to its cell receptor, CD155, and undergoes a receptor-mediated conformational transition resulting in the formation of altered (A) particles. Shortly after endocytosis and close to the plasma membrane, the viral RNA leaves the capsid. A long, umbilical connector is formed between the particles and the endosomal membrane that allows the RNA to escape. (Inset) Cross-section of poliovirus particle bound to CD155. Capsid pockets are occupied by lipids that may contribute to capsid stability.