Crystals of rhinovirus 14 have been grown reproducibly. They diffract x-rays to a resolution of at least 3.5 A. The orthorhombic crystal unit cell contains two virions, each situated on a crystallographic twofold axis. At less than 30-A resolution, the space group approximates to 1222 with the particles possessing 222 pseudo crystallographic symmetry.
C'Human rhinoviruses cause the common cold (1). They are a subgroup, with more than 113 characterized serotypes, of the picornaviruses, a family of viruses that cause a variety of infectious diseases of man and other mammals, including livestock. Although picornaviruses have been subclassified according to antigenic specificity, pH stability, and buoyant density, the architecture, assembly, and molecular biology of these viruses appear to be fundamentally similar (1). To further our understanding of the molecular etiology ofpicornaviruses in general, and of the common cold virus in particular, we have embarked on a high-resolution structural study of human rhinovirus 14 (R14). Hexagonal crystals of the 1A strain had been reported (2), and these we had earlier examined for their diffraction properties. However, the present crystals of R14 (Fig. 1) diffract to better than 3.5-A resolution (Fig. 2).The R14 viruses were grown in rhinovirus-sensitive HeLa cells and purified by a modification of the procedure described for encephalomyocarditis virus (3). Viruses were allowed to attach at room temperature (210C), usually at a multiplicity of infection of around 10 plaque-forming units per cell. This was followed by an 8-to 10-hr incubation, at 350C, with agitation. Rhinovirions remained about 90-95% associated with the infected cells, which were recovered by centrifugation (5 min at 800 X g). After three cycles of freezing and thawing, followed by centrifugation (1,000 X g) to remove debris, the virus-containing supernatant was supplemented with MgCl2 and deoxyribonuclease I to attain concentrations of 5 mM and 10 mg/ml, respectively. This solution was incubated for 30 min at room temperature, warmed to 370C (at which time trypsin was added to a final concentration of 0.5 mg/ml), and then incubated for an additional 10 min. One-tenth volume of 10% Sarkosyl along with EDTA to 10 mM was then added to the solution, which was clarified by centrifugation (10,000 rpm, Sorvall SS34 rotor, 10 min). Virus was pelleted by centrifuging in a Beckman type 30 rotor at 10TC for 4 hr at 27,000 rpm. Pellets were resuspended in 10 mM Tris HCI buffer, pH 7.5, containing 1 mM EDTA and 0.1% 2-mercaptoethanol (TEM). The virus was then banded in 7.5-45% sucrose gradients, in TEM buffer, by centrifuging at 100C for 90 min at 38,000 rpm in a Beckman SW 41 swinging-bucket rotor and collected by using an ISCO density gradient fractionation system. Virus-containing fractions were pooled, diluted with a minimum of 2 vol of TEM, and repelleted by centrifuging in a Beckman Ti 65 rotor at 100C for 90 min at 48,000 rpm. Virus was resuspended in TEM and stored at 4TC or -70TC prior to use. Yield...
