Greg Kamer

The structure determination of a common cold virus, human rhinovirus 14

The methods used to solve the structure of human rhinovirus 14 at 3.0 A resolution are described in detail. The crystals are cubic, space group P213, a = 445.1 A with 20-fold non-crystallographic redundancy and with approximately 55% solvent and RNA content. The data used to solve the structure were collected at the Cornell High Energy Synchrotron Source (CHESS) using oscillation photography. Most of the computations were performed on Purdue Universitys Cyber 205 supercomputer. Two heavy-atom derivative data sets from crystals soaked in 1 and 5 mM KAu(CN)2 were used to provide isomorphous phasing to 4 A resolution, although it was subsequently shown that phases beyond 5 A resolution were random. The phases were refined at 5 A resolution by five cycles of real-space molecular replacement. Phase extension from 5 to 3 A was then performed using 60 cycles of real-space molecular replacement while extending the resolution in steps of three reciprocal-lattice points at a time once every three cycles. The 3.5 A skew-averaged map was easily interpreted and showed 811 of the 855 amino acids in the four distinct viral polypeptide chains. A complete atomic model has been built using FRODO on an Evans & Sutherland PS300 graphics system with respect to the 3.08 A resolution electron density map. The roles of the non-crystallographic symmetry, solvent content, errors in amplitudes, orientation and translation in the molecular replacement process are discussed.

The Structure of a Human Common Cold Virus (Rhinovirus 14) and its Functional Relations to Other Picornaviruses

Picornaviruses are associated with serious diseases in humans and other animals, and they comprise one of the largest families of viral pathogens. For example, the common cold, poliomyelitis, foot-and-mouth disease and hepatitis can be caused by these viruses. They are among the smallest RNA-containing animal viruses (1–3). Their molecular weight is a round 8.5 x 106 and they contain about 30% by weight RNA. Their external diameter is roughly 300 A and they form icosahedral shells. Picornaviridae have been subdivided into four genera on the basis of their buoyant density, pH stability and sedimentation coefficients: enterovirus (e . g . polio, hepatitis A and coxsackie viruses), cardiovirus (e .g . encephalomyocarditis and Mengo viruses), aphthovirus (e.g. foot-and-mouth disease virus) and rhinovirus . They differ also in the number of known serotypes. For instance there are three known serotypes for polioviruses, seven for foot-and- mouth disease viruses (FMDV) and at least 89 for human rhinoviruses (HRV). Accordingly, it has been possible to produce effective vaccines for poliomyelitis and, with greater difficulty , for foot-and-mouth disease, but not for the common cold.

Common cold viruses

Abstract The structure of a human common cold (rhinovirus) serotype, human rhinovirus 14 (HRV14) is providing extensive information on viral assembly, stability, neutralization by antibodies and antiviral agents as well as the site of receptor attachment.

Structure determination of Mengo virus.

The structure of Mengo virus was determined to 3.0 A resolution using human rhinovirus 14 as an initial phasing model at 8.0 A resolution. Oscillation diffraction photographs were collected at the Cornell High Energy Synchrotron Source using orthorhombic Mengo virus crystals. The crystal space group was P2(1)2(1)2(1), a = 441.4, b = 427.3 and c = 421.9 A, with one icosahedral particle per asymmetric unit, giving 60-fold noncrystallographic redundancy. The orientations of the four viral particles in the unit cell were determined with a rotation function. Their positions relative to the crystallographic symmetry axes were found by a combination of Patterson-function analysis and a subsequent R-factor search using human rhinovirus 14 atomic coordinates as a model. The initial phases to 8.0 A resolution were then computed by placing human rhinovirus 14 particles in the orientations and positions of Mengo virus particles. These phases were improved by ten cycles of real-space molecular replacement averaging. Phases between 8.0 and 3.0 A resolution were obtained by molecular replacement phase extension. One or two reciprocal-space lattice points were used for each extension followed by two cycles of averaging.

Viral Particles at Atomic Resolution

Crick and Watson (ref. 1) first recognized that spherical viruses had to be regular polyhedra. Of these, the icosahedron has the largest number (60) of asymmetric units and was subsequently found to be the preferred envelope. The coding capacity of the enclosed genetic material could therefore be limited to coding only a relatively limited structural protein(s) for one-sixth of the virion shell. The assembly of viral particles from smaller, repeated subunit s presents several defined advantages: such strategy of replication reduces considerably the amount of genetic information needed to code for the structural protein(s), and minimizes the risks of incurring in fatal errors, as faulty subunits inaccurately synthesized can be discarded at assembly time. The entire replication cycle of a virus, therefore, can be visualized as a two-step process: a) the synthesis of viral components (nucleic acid and proteins), i.e: a template dependent, energy consuming process of polymerization of preformed blocks (nucleotides or amino acids), and b) the self-assembly of the subunits into more complex structures, a process that does not involve the formation of stable chemical bonds but brings the the subunits (or the intermediate structures) to a thermodynamically stable configuration.