Principles of Virology. Jane Flint
the cytoplasm by pH-independent mechanisms. The icosahedral nucleocapsid of this virus is built from a single viral protein, the C protein, which encloses the (+) strand viral RNA. This structure is surrounded by an envelope containing viral glycoproteins E1 and E2, which are arranged as heterodimers clustered into groups of three, each cluster forming a spike on the virus particle surface.
Fusion of the viral and endosomal membranes exposes the nucleocapsid to the cytoplasm (Fig. 5.20). To begin translation of (+) strand viral RNA, the nucleocapsid must be disassembled, a process mediated by an abundant cellular component, the ribosome. Each ribosome binds three to six molecules of C protein, disrupting the nucleocapsid. This process occurs while the nucleocapsid is attached to the cytoplasmic side of the endosomal membrane and ultimately results in disassembly. The uncoated viral RNA remains associated with cellular membranes, where translation and replication begin.
Uncoating of Nonenveloped Viruses
Disrupting the Endosomal Membrane
Adenoviruses comprise a double-stranded DNA genome packaged in an icosahedral capsid (Chapter 4). Adenovirus uncoating is a sequential, multistep process that was determined using multiple techniques that include live-cell, atomic force, and cryo-electron microscopy and X-ray crystallography. Internalization of most adenovirus serotypes by receptor-mediated endocytosis requires attachment of viral fibers to an Ig-like cell surface receptor and binding of the penton base to a second cell receptor, an integrin (Fig. 5.5). Uncoating begins with this initial attachment; the interaction of two viral capsid proteins with two different receptors promotes the dissociation of the fiber from the capsid and disrupts its structure. Additionally, it has been proposed that the interaction with multiple integrin molecules might induce conformational changes to the penton base. Uncoating continues as the virus particle is transported via the endosomes from the cell surface toward the nuclear membrane (Fig. 5.21). Endosome acidification promotes the release of protein VI, which induces disruption of the endosomal membrane, thereby delivering the remainder of the particle into the cytoplasm. An N-terminal amphipathic α-helix of protein VI is probably responsible for disrupting the membrane in a pH-dependent manner. Like the fusion peptides of class I fusion proteins, this region of the protein is exposed following cleavage during particle maturation and appears to be masked in the native capsid by the hexon protein until capsid destabilization. The liberated subviral particle then docks onto the nuclear pore complex, where uncoating is completed (see “Nuclear Import of DNA Genomes” below).
Figure 5.20 Entry of Semliki Forest virus into cells. Semliki Forest virus enters cells by clathrin-dependent receptor-mediated endocytosis, and membrane fusion is catalyzed by acidification of late endosomes. Fusion results in the exposure of nucleocapsid to the cytoplasm. Cellular ribosomes bind and disassemble the capsid, rendering the viral RNA accessible to translation. Adapted from Marsh M, Helenius A. 1989. Adv Virus Res 36:107–151, 1989, with permission.
Forming a Pore in the Endosomal Membrane
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 αvβ3 and αvβ5 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