RESUMEN
Most rhinoviruses, which are the leading cause of the common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. To release their genomes, rhinoviruses convert to activated particles that contain pores in the capsid, lack minor capsid protein VP4, and have an altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here, we present the structures of virion of rhinovirus 14 and its complex with ICAM-1 determined to resolutions of 2.6 and 2.4 Å, respectively. The cryo-electron microscopy reconstruction of rhinovirus 14 virions contains the resolved density of octanucleotide segments from the RNA genome that interact with VP2 subunits. We show that the binding of ICAM-1 to rhinovirus 14 is required to prime the virus for activation and genome release at acidic pH. Formation of the rhinovirus 14-ICAM-1 complex induces conformational changes to the rhinovirus 14 capsid, including translocation of the C termini of VP4 subunits, which become poised for release through pores that open in the capsids of activated particles. VP4 subunits with altered conformation block the RNA-VP2 interactions and expose patches of positively charged residues. The conformational changes to the capsid induce the redistribution of the virus genome by altering the capsid-RNA interactions. The restructuring of the rhinovirus 14 capsid and genome prepares the virions for conversion to activated particles. The high-resolution structure of rhinovirus 14 in complex with ICAM-1 explains how the binding of uncoating receptors enables enterovirus genome release.
Asunto(s)
Cápside/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , ARN Viral/metabolismo , Rhinovirus/metabolismo , Activación Viral/fisiología , Desencapsidación Viral/fisiología , Secuencia de Aminoácidos , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Infecciones por Enterovirus/metabolismo , Infecciones por Enterovirus/virología , Genoma Viral/genética , Células HeLa , Humanos , Molécula 1 de Adhesión Intercelular/química , Molécula 1 de Adhesión Intercelular/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , ARN Viral/química , ARN Viral/genética , Rhinovirus/genética , Rhinovirus/fisiología , Homología de Secuencia de Aminoácido , Virión/genética , Virión/metabolismo , Virión/ultraestructuraRESUMEN
Cryo-electron microscopy (cryo-EM) is one of the primary methods used to determine the structures of macromolecules and their complexes. With the increased availability of cryo-electron microscopes, the preparation of high-quality samples has become a bottleneck in the cryo-EM structure-determination pipeline. Macromolecules can be damaged during the purification or preparation of vitrified samples for cryo-EM, making them prone to binding to the grid support, to aggregation or to the adoption of preferential orientations at the air-water interface. Here, it is shown that coating cryo-EM grids with a negatively charged polyelectrolyte, such as single-stranded DNA, before applying the sample reduces the aggregation of macromolecules and improves their distribution. The single-stranded DNA-coated grids enabled the determination of high-resolution structures from samples that aggregated on conventional grids. The polyelectrolyte coating reduces the diffusion of macromolecules and thus may limit the negative effects of the contact of macromolecules with the grid support and blotting paper, as well as of the shear forces on macromolecules during grid blotting. Coating grids with polyelectrolytes can readily be employed in any laboratory dealing with cryo-EM sample preparation, since it is fast, simple, inexpensive and does not require specialized equipment.