Principles of Virology. Jane Flint
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the trigger for fusion is acid pH. In contrast, herpesviruses carry multiple envelope proteins on their surface that might contribute to entry in different ways depending on the target cell (Fig. 5.9). It is unclear how conformational changes induced in these different surface proteins upon engagement of their various targets trigger fusion by gB.
Figure 5.19 Conformational changes in class III proteins during fusion. Structure of part of the ectodomain of vesicular stomatitis virus G protein at neutral and acid pH (PDB ID: 5I2S, 5MDM). For simplicity, only one monomer of the trimer is depicted. Domain I is colored green, domain II black, domain III orange, and domain IV (the β-sandwich core) cyan, with the two fusion loops in red. Upon exposure to acid pH, the protein flips to extend the fusion loops toward the target membrane and bring the viral membrane closer. In contrast to other fusion proteins, the conformational changes induced by acid pH in the vesicular stomatitis virus G protein are reversible in solution.
Intracellular Trafficking and Uncoating
Following entry, viral and subviral particles travel within the cell to compartments appropriate for virus genome replication. Transport relies on cellular networks. For enveloped virus particles that fuse at the plasma membrane, the transport cargo is subviral particles, whereas for those that fuse in internal cellular compartments, transport, at least in part, is achieved by processes that move vesicles. At some point during or after transport, uncoating occurs so that the viral genome is released from the viral capsid and can replicate. The genomes of nonenveloped viruses are transferred across the cell membrane by mechanisms different than membrane fusion. For these viruses, the processes of entry and uncoating are tightly linked.
Movement of Viral and Subviral Particles within Cells
Movement of molecules larger than 500 kDa does not occur by passive diffusion, because the cytoplasm is crowded with organelles, high concentrations of proteins, and the cytoskeleton. Rather, viral particles and their components are transported via the cytoskeleton. Such movement can be visualized in live cells by using fluorescently labeled viral proteins (Chapter 2).
The cytoskeleton is a dynamic network of protein filaments that extends throughout the cytoplasm. It is composed of microtubules and actin filaments. Microtubules are organized in a polarized manner, with minus ends situated at the microtubule-organizing center near the nucleus, and plus ends located at the cell periphery (Fig. 5.11). This arrangement permits directed movement of cellular and viral components over long distances. Actin filaments typically assist in virus movement close to the plasma membrane. Techniques to follow the movement of virus particles after entry continue to improve. For example, a combination of technologies such as real-time quantum dots-based single particle tracking with biochemical assays was used to track reovirus particles that enter cells via clathrin-mediated endocytosis. Following internalization, movement of individual particles was slow and dependent on actin, while movement became faster toward the cell interior and dependent on microtubules.
Transport along actin filaments is accomplished by myosin motors, and movement on microtubules is via kinesin and dynein motors. Hydrolysis of adenosine triphosphate (ATP) provides the energy for the motors to move their cargo along cytoskeletal tracks. There are two basic ways for viral or subviral particles to travel within the cell: within a membrane vesicle such as an endosome, which interacts with the cytoskeletal transport machinery; or directly (Fig. 5.11). In the latter case, some form of the virus particle must bind to the transport machinery. After leaving endosomes, the subviral particles derived from adenoviruses and parvoviruses are transported along microtubules to the nucleus. Although adenovirus particles exhibit bidirectional plus- and minus-end-directed microtubule movement, their net movement is toward the nucleus. Adenovirus binding to cells activates two different signal transduction pathways that increase the net velocity of minus-end-directed motility. The signaling pathways are therefore required for efficient delivery of the viral genome to the nucleus. Adenovirus subviral particles are loaded onto microtubules by interaction of the capsid protein, hexon, with dynein. The particles move toward the centrosome and are then released and dock onto nuclear pores, prior to viral genome entry into the nucleus.
A number of different viruses enter the peripheral nervous system and spread to the central nervous system via axons. As no viral genome 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