Early Events in Poliovirus Infection
The protein capsid that surrounds many animal viruses plays an important role in viral infections. Upon encountering a target cell, the capsid must interact with a cell surface receptor and release the viral nucleic acid into the cell. In the infected cell, newly synthesized virion proteins encapsidate the viral genome and protect it as it travels to a new cell target. The viral capsid must therefore be stable to the extracellular environment, yet flexible enough to discharge the nucleic acid into the cell. Poliovirus is a particularly good model for studying how the capsid mediates these functions. The three dimensional structure of poliovirus has been determined by X-ray crystallography, the cell receptor for poliovirus has been identified, and genetic manipulation of the virus is possible with infectious cDNA copies of the viral genome. One of the goals of our research is to use these experimental tools to understand how the poliovirus capsid controls early events in poliovirus infection.
Poliovirus is a member of the Picornaviridae, a family of viruses that cause a variety of human and animal diseases. The virion consists of 60 copies each of four different polypeptides, VP1-VP4, arranged in icosahedral symmetry, which enclose the 7.5 kb positive sense RNA genome. Poliovirus initiates infection of cells by binding to a cell surface receptor, PVR, which is a novel member of the immunoglobulin superfamily of proteins. Resolution of the atomic structures of poliovirus type 1 and human rhinovirus 14 (HRV14), another picornavirus, has provided insight into how these viruses might recognize their cell receptors. It has been proposed that a deep depression on the surface of these viruses, called the canyon, is the receptor binding site. The canyon encircles each five fold axis of symmetry on the virion particle.
One of our approaches to this problem has been to study poliovirus mutants selected for their resistance to neutralization with soluble PVR (srr mutants). These mutants have reduced binding affinity for HeLa cells and contain single amino acid changes in the capsid. The study of such mutants provides considerable information on capsid residues that control the interaction of poliovirus with PVR.
To study the interaction of poliovirus with PVR, we have mutagenized the receptor to identify putative contact points. The results indicate that the C'-C" ridge on PVR is likely to be the main contact point with poliovirus. Selection of poliovirus mutants adapted to utilize these mutant receptors has identified additional capsid residues that control receptor recognition.
After binding to cell surface PVR, poliovirus must release its RNA genome into the cell. The mechanism of this uncoating step remains an enigma. If poliovirus is bound to cells at temperatures below 33 degrees C, the attached viruses can be released in an infectious form by exposure to 6M LiCl, 8M urea, low pH, or detergents. When poliovirus is bound to cells at 37 degrees C, a substantial fraction of the bound viruses release their copies of VP4 and are converted into a conformationally altered form known as the A particle. A particles sediment more slowly than native viruses, are noninfectious (although they contain infectious RNA), and are sensitive to detergent and proteinases. The N-terminus of VP1, which is on the interior of the native particle, is on the surface of A particles.
Experimental evidence suggests that A particles release their RNA into the cytoplasm, although this uncoating process is not well understood. A particles can be found within cells early after infection. They are more hydrophobic than native virions as a result of exposure of the amino terminus of VP1, the first 20 residues of which resemble an amphipathic helix. It has been suggested that five copies of the VP1 N-terminus might create a pore in the cell membrane that could lead to passage of the viral genome into the cytoplasm. Consistent with this hypothesis is the observation that a mutant virus lacking VP1 amino acids 8 and 9 is defective in uncoating. Because the infectivity of typical preparations of poliovirus is often only 0.5% of the physical population, it is difficult to assess the role of intermediates such as A particles in infectivity. However, the finding that the antiviral WIN compounds block the formation of A particles strongly suggests an important role for these particles in infection.
A model for the early stages of poliovirus entry is presented in Figure 1, below. Virus attaches to cell surface PVR and undergoes a receptor-mediated conformational transition to A particles. While many A particles elute from the cell, a small fraction remains bound and releases RNA into the cell. The trigger for this uncoating step is not known; however, acidification of endosomes does not appear to be required for uncoating.