Amino acid sequence data from 57 different enzymes were used to determine the divergence times of the major biological groupings. Deuterostomes and protostomes split about 670 million years ago and plants, animals, and fungi last shared a common ancestor about a billion years ago. With regard to these protein sequences, plants are slightly more similar to animals than are the fungi. In contrast, phylogenetic analysis of the same sequences indicates that fungi and animals shared a common ancestor more recently than either did with plants, the greater difference resulting from the fungal lineage changing faster than the animal and plant lines over the last 965 million years. The major protist lineages have been changing at a somewhat faster rate than other eukaryotes and split off about 1230 million years ago. If the rate of change has been approximately constant, then prokaryotes and eukaryotes last shared a common ancestor about 2 billion years ago, archaebacterial sequences being measurably more similar to eukaryotic ones than are eubacterial ones.

Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicated in cell entry, including the externalization of the viral protein VP4 and the N terminus of VP1. We have determined the structures of native virions and of two putative cell entry intermediates, the 135S and 80S particles, at ϳ22-Å resolution by cryo-electron microscopy. The 135S and 80S particles are both ϳ4% larger than the virion. Pseudoatomic models were constructed by adjusting the beta-barrel domains of the three capsid proteins VP1, VP2, and VP3 from their known positions in the virion to fit the 135S and 80S reconstructions. Domain movements of up to 9 Å were detected, analogous to the shifting of tectonic plates. These movements create gaps between adjacent subunits. The gaps at the sites where VP1, VP2, and VP3 subunits meet are plausible candidates for the emergence of VP4 and the N terminus of VP1. The implications of these observations are discussed for models in which the externalized components form a transmembrane pore through which viral RNA enters the infected cell.Poliovirus, like related picornaviruses, is a small nonenveloped virus consisting of a plus-sense RNA genome enclosed within a protein shell or capsid (reviewed in reference 53). The capsid consists of 60 copies each of four proteins (VP1, VP2, VP3, and VP4) arranged on an icosahedral lattice. VP1, VP2, and VP3 have similar wedge-shaped cores, each an eightstranded beta-barrel (the strands are designated B, I, D, G, C, H, E, and F), but each protein has unique loops connecting the strands and unique N and C termini (31). VP4 is small, myristylated (15), and has an extended structure.The wedge-shaped cores of the subunits form the closed protein shell of the virion, with five copies of VP1 packing around the fivefold axes and VP2 and VP3 alternating around the threefold axes of the particle. The virion is stabilized by interactions among the wedge-shaped cores and by a network-on the inner surface of the protein shell-that is formed by VP4 and the N-terminal extensions of VP1, VP2, and VP3. The high-resolution structure of an empty capsid assembly intermediate shows that formation of the internal network is dependent on a late proteolytic cleavage of the capsid protein precursor, VP0, to yield VP4 and VP2 (5). This "maturation cleavage" is associated with the encapsidation of the viral RNA and is required for virion stability. Unfortunately, the viral RNA (which lacks the icosahedral symmetry of the protein coat) is not visible in the high-resolution structure of the virus, and its role in stabilizing the virus structure is unknown.Despite extensive chemical and molecular characterization, the mechanism of picornavirus cell entry is known only in outline (14,48,49,(51)(52)(53)59). In order to initiate a productive infection, the viral RNA must be externalized, cross a membrane, and be delivered to the cytoplasm. Infection begins with binding of the virion to a receptor that is a member of the immunoglobulin superfamily (37, 45), whereup...

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We examined the role of soluble poliovirus receptor on the transition of native poliovirus (160S or N particle) to an infectious intermediate (135S or A particle). The viral receptor behaves as a classic transition state theory catalyst, facilitating the N-to-A conversion by lowering the activation energy for the process by 50 kcal/mol. In contrast to earlier studies which demonstrated that capsid-binding drugs inhibit thermally mediated N-to-A conversion through entropic stabilization alone, capsid-binding drugs are shown to inhibit receptor-mediated N-to-A conversion through a combination of enthalpic and entropic effects.Poliovirus is a nonenveloped virus of the family Picornaviridae. Picornaviruses share an icosahedral capsid architecture consisting of 60 copies of four proteins, VP1, VP2, VP3, and VP4. The surface of the virion is dominated by prominent star-shaped mesas at the fivefold axes and three-bladed propeller-like features at the threefold axes. These surface features are separated by deep canyons encircling the fivefold axes. These canyons are involved in many essential aspects of capsid function. Structural studies have shown that the receptor footprints for major group rhinoviruses (19) and poliovirus (2, 10, 27) map to the canyon. At the base of the canyon underneath the receptor footprint, there is an entry to a long, narrow hydrophobic pocket within the ␤-barrel core of VP1. For most entero-and rhinoviruses, crystallographic studies have revealed that this pocket is occupied by an unidentified fatty acid-like moiety, or pocket factor (6,12,17,18,23), which can be displaced by a family of capsid-binding antiviral drugs (9,22). Interestingly, the pocket factor and the antiviral drugs can exert large-scale, global effects on the capsid's conformational dynamics, which play a critical role in the viral life cycle.When poliovirus attaches to its receptor, the particle converts irreversibly from the N (native or 160S) to the A (infectious [4] intermediate, or 135S) conformation. In the course of this uncoating transition, normally internal components, including VP4 and the N-terminal extension of VP1, are externalized. Externalization of these components has been shown to facilitate the attachment of the A particle to liposomes in vitro (7), suggesting a mechanism for the entry of virus or viral RNA to the cell (1, 2). Transient and reversible exposure of portions of VP4 and the N terminus of VP1 also occurs naturally at physiological temperatures (14). This "breathing" process suggests that the particle is primed to undergo the N-to-A transition but cannot complete the transition in the absence of a trigger, i.e., the receptor. We have previously proposed that the receptor acts like an enzyme, accelerating the rate of the N-to-A transition at physiological temperature by lowering the activation energy (E a ) for the transition. Later in the cell entry process, the A particle undergoes further changes, which result in the release of the viral RNA and formation of an empty particle that sediments at...

Computational methods were used to design structure-based combinatorial libraries of antipicornaviral capsid-binding ligands. The multiple copy simultaneous search (MCSS) program was employed to calculate functionality maps for many diverse functional groups for both the poliovirus and rhinovirus capsid structures in the region of the known drug binding pocket. Based on the results of the MCSS calculations, small combinatorial libraries consisting of 10s or 100s of three-monomer compounds were designed and synthesized. Ligand binding was demonstrated by a noncell-based mass spectrometric assay, a functional immuno-precipitation assay, and crystallographic analysis of the complexes of the virus with two of the candidate ligands. The P1/Mahoney poliovirus strain was used in the experimental studies. A comparison showed that the MCSS calculations had correctly identified the observed binding site for all three monomer units in one ligand and for two out of three in the other ligand. The correct central monomer position in the second ligand was reproduced in calculations in which the several key residues lining the pocket were allowed to move. This study validates the computational methodology. It also illustrates that subtle changes in protein structure can lead to differences in docking results and points to the importance of including target flexibility, as well as ligand flexibility, in the design process.