encodes the molecular motors or cytoskeletal structures needed for long-distance axonal transport, viral adapter proteins are required to allow movement within nerves. An example is the axonal transport of alphaherpesvirus subviral particles. After fusion at the plasma membrane, the viral nucleocapsid is carried by retrograde transport to the neuronal cell body. Such transport is accomplished by the interaction of a major component of the tegument, viral protein VP1/2, with minus-end-directed dynein motors. In contrast, other virus particles are carried to the nerve cell body within endocytic vesicles. For example, after endocytosis of poliovirus, virus particles remain attached to the cellular receptor CD155. The cytoplasmic domain of the receptor engages the dynein light chain TCTEX-1 to allow retrograde transport of virus-containing vesicles.
Uncoating of Enveloped Virus Particles
Release of Viral Ribonucleoprotein
The genomes of many enveloped RNA viruses are present as ribonucleoproteins (vRNP) in the virus particle. In the case of influenza virus, each vRNP is composed of a segment of the RNA genome bound by nucleoprotein (NP) molecules and the viral RNA polymerase, which must be released into the cytoplasm and enter the nucleus, where mRNA synthesis takes place. The vRNP structures interact with viral M1 protein, an abundant protein in virus particles that underlies the envelope and provides rigidity (Fig. 5.13). The M1 protein also contacts the internal tails of the HA and neuraminidase transmembrane proteins. This arrangement presents problems. Unless M1-vRNP interactions are disrupted, vRNPs might not be released into the cytoplasm. Furthermore, the vRNPs cannot enter the nucleus, because M1 masks a nuclear localization signal (see “Import of Influenza Virus Ribonucleoprotein” below).
The influenza virus M2 protein, the first viral protein identified as an ion channel, provides the solution to both problems. The envelope of the virus particle contains a small number of molecules of M2 protein, which form a homotetramer. When purified M2 was reconstituted into synthetic lipid bilayers, ion channel activity was observed, indicating that this property requires only the M2 protein. The M2 protein channel is structurally much simpler than other ion channels and is the smallest channel discovered to date.
The M2 ion channel is activated by the low pH of the endosome before HA-catalyzed membrane fusion occurs. As a result, protons enter the interior of the virus particle. It has been suggested that the reduced pH of the particle interior leads to conformational changes in the M1 protein, thereby disrupting M1-vRNP interactions. When fusion between the viral envelope and the endosomal membrane takes place, vRNPs are released into the cytoplasm free of M1 and can then be imported into the nucleus (Fig. 5.13). Support for this model comes from studies with the anti-influenza virus drug amantadine, which specifically inhibits M2 ion channel activity (Volume II, Fig. 8.12). In the presence of this drug, influenza virus particles can bind to cells, enter endosomes, and undergo HA-mediated membrane fusion, but vRNPs are not released from endosomes.
Uncoating by Ribosomes in the Cytoplasm
Some enveloped RNA-containing viruses, such as Semliki Forest virus, contain nucleocapsids that are disassembled in 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
