Molecular tectonic model of virus structural transitions: the putative cell entry states of poliovirus.

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 approximately 22-A resolution by cryo-electron microscopy. The 135S and 80S particles are both approximately 4% 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 A 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.

Three-dimensional structure of physalis mottle virus: implications for the viral assembly.

The structure of the T=3 single stranded RNA tymovirus, physalis mottle virus (PhMV), has been determined to 3.8 A resolution. PhMV crystals belong to the rhombohedral space group R 3, with one icosahedral particle in the unit cell leading to 20-fold non-crystallographic redundancy. Polyalanine coordinates of the related turnip yellow mosaic virus (TYMV) with which PhMV coat protein shares 32 % amino acid sequence identity were used for obtaining the initial phases. Extensive phase refinement by real space molecular replacement density averaging resulted in an electron density map that revealed density for most of the side-chains and for the 17 residues ordered in PhMV, but not seen in TYMV, at the N terminus of the A subunits. The core secondary and tertiary structures of the subunits have a topology consistent with the capsid proteins of other T=3 plant viruses. The N-terminal arms of the A subunits, which constitute 12 pentamers at the icosahedral 5-fold axes, have a conformation very different from the conformations observed in B and C subunits that constitute hexameric capsomers with near 6-fold symmetry at the icosahedral 3-fold axes. An analysis of the interfacial contacts between protein subunits indicates that the hexamers are held more strongly than pentamers and hexamer-hexamer contacts are more extensive than pentamer-hexamer contacts. These observations suggest a plausible mechanism for the formation of empty capsids, which might be initiated by a change in the conformation of the N-terminal arm of the A subunits. The structure also provides insights into immunological and mutagenesis results. Comparison of PhMV with the sobemovirus, sesbania mosaic virus reveals striking similarities in the overall tertiary fold of the coat protein although the capsid morphologies of these two viruses are very different.

Expression and purification of recombinant hRPABC25, hRPABC17, and hRPABC14.4, three essential subunits of human RNA polymerases I, II, and III.

Transcription of eukaryotic genes is performed by RNA polymerases I, II, and III, which synthesize ribosomal, messenger, and transfer RNAs, respectively. Eukaryotic RNA polymerases are large macromolecular complexes composed of multiple subunits. Among these subunits, five are shared by all RNA polymerases and are essential for cell growth and viability. Remarkably, the human common subunits are structurally conserved and functionally interchangeable with their yeast homologues and are believed to play an important role in the assembly of the three transcription complexes. To understand the structure and function of human RNA polymerases, we overexpressed the common subunits hRPABC25, hRPABC17, and hRPABC14.4 as hexahistidine fusions in Escherichia coli. The recombinant proteins were purified using metal-chelate affinity chromatography on Ni-NTA resin and gel filtration. Depending on the subunit, the yield was 5-17 mg of purified recombinant protein per liter of culture medium. The purified proteins were of high quality and sufficient quantity for structural studies, as demonstrated by the successful crystallization of hRPABC17 and hRPABC14.4. The expression and purification of the common subunits hRPABC25, hRPABC17, and hRPABC14. 4 will make possible their structural analysis with X-ray crystallography and nuclear magnetic resonance, providing important insights into the structure and function of the three human RNA polymerases.

Ligand-induced conformational changes in poliovirus-antiviral drug complexes.

Crystal structures of the Mahoney strain of type 1 poliovirus complexed with the antiviral compounds R80633 and R77975 were determined at 2.9 A resolution. These compounds block infection by preventing conformational changes required for viral uncoating. In various drug-poliovirus complexes reported earlier, no significant conformational changes were found in the structures of the capsid proteins. In the structures reported here, the strain of virus is relatively insensitive to these antivirals. Correspondingly, significant conformational changes are necessary to accommodate the drug. These conformational changes affect both the immediate vicinity of the drug binding site, and more distant loops located near the fivefold axis. In addition, small but concerted shifts of the centers of mass of the major capsid proteins consistently have been detected whose magnitudes are correlated inversely with the effectiveness of the drugs. Collectively, the drug complexes appear to sample the conformational repertoire of poliovirus near equilibrium, and thus provide a possible model for the earliest stages of viral uncoating during infection.

Binding of the antiviral drug WIN51711 to the sabin strain of type 3 poliovirus: structural comparison with drug binding in rhinovirus 14.

The crystal structure of the Sabin strain of type 3 poliovirus (P3/Sabin) complexed with the antiviral drug WIN51711 has been determined at 2.9 A resolution. Drugs of this kind are known to inhibit the uncoating of the virus during infection, by stabilizing the capsid against receptor-induced conformational changes. The electron density for the bound drug is very well defined so that its position and orientation are unambiguous. The drug binds in a nearly extended conformation, slightly bent in the middle, in a blind pocket formed predominantly by hydrophobic residues in the core of the beta-barrel of capsid protein VP1. Comparisons between this structure, the corresponding drug complex in human rhinovirus 14 (HRV 14), and the native structures of both viruses demonstrate that the binding of WIN51711 has markedly different effects on the structures of these two viruses. Unlike HRV14, wherein large conformational changes are observed in the coat protein after drug binding, the binding of this drug in poliovirus does not induce any significant conformational changes in the structure of the capsid protein, though the drug has a greater inhibitory effect in P3/Sabin than in HRV14. The implications of this result for the mechanism of capsid stabilization are discussed.

Structures of poliovirus complexes with anti-viral drugs: implications for viral stability and drug design.

BACKGROUND: Picornaviruses, such as the structurally related polioviruses and rhinoviruses, are important human pathogens which have been the target of major drug development efforts. Receptor-mediated uncoating and thermal inactivation of poliovirus and rhinovirus are inhibited by agents that bind to each virus by inserting into a pocket in the beta barrel of the viral capsid protein, VP1. This pocket, which is normally empty in human rhinovirus-14 (HRV14), is occupied by an unknown natural ligand in poliovirus. Structural studies of HRV14-drug complexes have shown that drug binding causes large, localized changes in the conformation of VP1. RESULTS: We report the crystal structures of six complexes between poliovirus and capsid-binding, antiviral drugs, including complexes of four different drugs with the Sabin vaccine strain of type 3 poliovirus, and complexes of one of these drugs with two other poliovirus strains that contain sequence differences in the drug-binding site. In each complex, the changes in capsid structure associated with drug binding are limited to minor adjustments in the conformations of a few side chains lining the binding site. CONCLUSIONS: The minor structural changes caused by drug binding suggest a model of drug action in which it is the conformational changes prevented by the bound drug, rather than obvious conformational changes induced by drug binding, which exert the biological effect. Our results, along with additional structures of rhinovirus-drug complexes, suggest possible improvements in drug design, and provide important clues about the nature of the conformational changes that are involved in the uncoating process.