Abstract
The coat proteins (CPs) of single-stranded RNA bacteriophages (ssRNA phages) directly assemble around the genomic RNA (gRNA) to form a near-icosahedral capsid with a single maturation protein (Mat) that binds the gRNA and interacts with the retractile pilus during infection of the host. Understanding the assembly of ssRNA phages is essential for their use in biotechnology, such as RNA protection and delivery. Here, we present the complete gRNA model of the ssRNA phage Qβ, revealing that the 3′ untranslated region binds to the Mat and the 4127 nucleotides fold domain-by-domain, and is connected through long-range RNA–RNA interactions, such as kissing loops. Thirty-three operator-like RNA stem-loops are located and primarily interact with the asymmetric A/B CP-dimers, suggesting a pathway for the assembly of the virions. Additionally, we have discovered various forms of the virus-like particles (VLPs), including the canonical T = 3 icosahedral, larger T = 4 icosahedral, prolate, oblate forms, and a small prolate form elongated along the 3-fold axis. These particles are all produced during a normal infection, as well as when overexpressing the CPs. When overexpressing the shorter RNA fragments encoding only the CPs, we observed an increased percentage of the smaller VLPs, which may be sufficient to encapsidate a shorter RNA.
Highlights
Single-stranded RNA bacteriophages infect a variety of Gram-negative bacteria [1–5]
The canonical ssRNA phage Qβ has a genomic RNA of 4217 nucleotides, from the 50 to the 30 end, encoding the maturation protein (Mat); the major coat protein (CP); the minor capsid protein A1, which is a read-through extension caused by a leaky stop codon for the major coat proteins (CPs); and the β-subunit of the RNA-dependent RNA replicase (Rep) [1]
In packaging a nascent genomic RNA (gRNA) that is the gRNA, which are synthesized at the beginning, bind CP dimers to coordinate the forstill coming out of the replicase, the first few operator-like stem-loops at the 50 region of the mation of
Summary
Single-stranded RNA bacteriophages (ssRNA phages) infect a variety of Gram-negative bacteria [1–5]. Inside the Qβ capsid, there is a sequestered dimer of CPs interacting with an RNA stem-loop originated from a 5-way junction domain, which presents a stem-loop to interact with the Mat [6]. Rather than purifying the virus based on infectious activity, we purified the particles by identifying the CPs in different steps, and identified a variety of alternative forms of VLPs produced directly from a wild-type phage infection for the first time. Two forms of these VLPs, namely, the oblate and the D3 symmetrical small-prolate forms, have not been observed before. These data show the versatility of the CP of Qβ, and provide a better understanding of viral assembly for ssRNA phages
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