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Asymmetric cryo-EM structure of the canonical Allolevivirus Qβ reveals a single maturation protein and the genomic ssRNA in situ

Significance Single-stranded (ss) RNA viruses have ribonucleic acid as their genetic material and infect animals, plants, and bacteria. Here we used cryo-electron microscopy to reveal the genomic RNA (gRNA) of the ssRNA virus Qβ. The asymmetric gRNA adopts a single dominant structure in all virions and binds the capsid of Qβ at each coat protein. At the same time, we determined the structure of the maturation protein, A2, which functions both as the virion’s “tail” and its lysis protein. We see the gRNA is more ordered when interacting with A2. These results provide structural insights into gRNA packaging and host infection in ssRNA viruses. 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. Qβ, 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 Qβ 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.