RESUMEN
Single-stranded (ss) RNA viruses infect all domains of life. To date, for most ssRNA virions, only the structures of the capsids and their associated protein components have been resolved to high resolution. Qß, an ssRNA phage specific for the conjugative F-pilus, has a T = 3 icosahedral lattice of coat proteins assembled around its 4,217 nucleotides of genomic RNA (gRNA). In the mature virion, the maturation protein, A2, binds to the gRNA and is required for adsorption to the F-pilus. Here, we report the cryo-electron microscopy (cryo-EM) structures of Qß with and without symmetry applied. The icosahedral structure, at 3.7-Å resolution, resolves loops not previously seen in the published X-ray structure, whereas the asymmetric structure, at 7-Å resolution, reveals A2 and the gRNA. A2 contains a bundle of α-helices and replaces one dimer of coat proteins at a twofold axis. The helix bundle binds gRNA, causing denser packing of RNA in its proximity, which asymmetrically expands the surrounding coat protein shell to potentially facilitate RNA release during infection. We observe a fixed pattern of gRNA organization among all viral particles, with the major and minor grooves of RNA helices clearly visible. A single layer of RNA directly contacts every copy of the coat protein, with one-third of the interactions occurring at operator-like RNA hairpins. These RNA-coat interactions stabilize the tertiary structure of gRNA within the virion, which could further provide a roadmap for capsid assembly.
Asunto(s)
Allolevivirus/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/ultraestructura , Microscopía por Crioelectrón , Genoma Viral , ARN Viral/química , ARN Viral/ultraestructura , Cápside/química , Cristalografía por Rayos X , Motivos EF Hand , Modelos Moleculares , Multimerización de ProteínaRESUMEN
The coat proteins of single-stranded RNA bacteriophages specifically recognize and bind to a hairpin structure in their genome at the beginning of the replicase gene. The interaction serves to repress the synthesis of the replicase enzyme late in infection and contributes to the specific encapsidation of phage RNA. While this mechanism is conserved throughout the Leviviridae family, the coat protein and operator sequences from different phages show remarkable variation, serving as prime examples for the co-evolution of protein and RNA structure. To better understand the protein-RNA interactions in this virus family, we have determined the three-dimensional structure of the coat protein from bacteriophage Qß bound to its cognate translational operator. The RNA binding mode of Qß coat protein shares several features with that of the widely studied phage MS2, but only one nucleotide base in the hairpin loop makes sequence-specific contacts with the protein. Unlike in other RNA phages, the Qß coat protein does not utilize an adenine-recognition pocket for binding a bulged adenine base in the hairpin stem but instead uses a stacking interaction with a tyrosine side chain to accommodate the base. The extended loop between ß strands E and F of Qß coat protein makes contacts with the lower part of the RNA stem, explaining the greater length dependence of the RNA helix for optimal binding to the protein. Consequently, the complex structure allows the proposal of a mechanism by which the Qß coat protein recognizes and discriminates in favor of its cognate RNA.