Structural Assembly of Qβ Virion and Its Diverse Forms of Virus-like Particles

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.

Keywords: genomic RNA; single-particle; ssRNA virus; virus assembly.

Figure 1

The complete model of the dominant gRNA fold in Qβ. (A) The cut-open view of the Qβ virion to display the gRNA, shown as ribbons and rainbow-colored from the 5′ end (blue) to the 3′ end (red). The CP shell and Mat are shown as electron density, colored light grey, and dark blue, respectively. (B) The view from Panel A vertically rotated 90° with half of the CP shell removed. (C) Cross-section from between the marks in Panel B and viewed from the black arrows. The internal CP dimer (Internal CP2), and the 5′ and 3′ gRNA domains are light blue, blue, and red, respectively. (D) Left: zoomed-in view within the blue box in Panel C showing the gRNA 5-way junction domain (red) interacting with the Mat (dark blue), an exposed CP dimer (CP2, grey), and the internal CP dimer (Internal CP2, light blue). The 5′ and 3′ ends of the 5-way junction domain are labeled as green diamond and orange triangle, respectively. Right: secondary structure of the gRNA 5-way junction domain. The dark blue, grey, and light blue arcs denote the interactions with the Mat, and the exposed and internal CP dimers, respectively. (E) Left: zoomed-in views within the orange and red boxes in Panel C showing the two kissing loops that link helices R22, RT4, R23, and the bulge R24. The orange and red arrows indicate the locations of the kissing loops. Right: Models of the two kissing loops and their secondary structures showing the base pairing within the sequence.

Figure 2

Operator-like RNA stem-loops in the gRNA that interact with the capsid shell. (A) The near icosahedral capsid of the Qβ is illustrated as a cage with the Mat labeled as a magenta oval. The twelve pentamers are dark grey, and labeled from I to XII. (B) The cage is unwrapped to display the capsid as a 2D map, with each thick line representing a CP dimer. The asymmetric A/B dimer is between a grey pentamer and a white hexamer. The symmetric C/C dimer is between two white hexamers. The rainbow-colored circles label the operator-like stem-loops from the 5′ (blue) to 3′ (red) ends of the gRNA. (C) A model showing an operator-like RNA stem-loop interacting with a CP dimer. (D) The sequence of the Qβ gRNA is rainbow-colored. The UTRs, the genes of maturation protein (mat), coat protein (cp), replicase (rep), and A₁ protein (read-through or A₁) are labeled. The 59 stem-loops, which have a distance of less than 5 Å to the capsid proteins, are numbered and labeled on the sequence with operator-like (bold black), CP-sandwiched (black), and CP-anchored (grey) stem-loops labeled. The actual translational operator and the stem-loop that interacts with the internal CP dimer are labeled by the underlined numbers 34 and 57, respectively. The underlined 59 indicates the stem-loop which interacts with the Mat. (E) The operator-like RNA stem-loops (rainbow-colored) interact with the CPs in Pentamers I, II, and III. The red star labels one CP-sandwiched RNA stem-loop (number 15 in Pentamer II).

Figure 3

Structures of the Qβ VLPs. (A) Top: cryo-EM structures of the Qβ VLPs of T = 4, prolate, T = 3, oblate, and small prolate symmetries from left to right. The corresponding symmetry cages are drawn with pentamers and hexamers colored grey and white, respectively. Bottom: the same structure horizontally rotated 90°. (B) The table shows the number of CPs, pentamers, and hexamers for each form of the VLPs. (C) The population for the T = 3, oblate, and small prolate VLPs produced by the wild-type Qβ infection (light blue) and over-expressing the cp/A₁ gene (deep blue). Samples were purified by gel-filtration chromatography.

Figure 4

Plasticity in the hexamers of different Qβ VLPs. (A) Different forms of the VLPs with the pentamers in white and hexamers colored. The conformations of the hexamers are classified to be “straight”, “less arched”, “normal arched”, and “over arched”. (B) The model of a hexamer with the six subunits labeled from a to f. The black dot indicates the relative orientation of the hexamer in Panel A. The curvature of the hexamer can be defined by three arch angles, θ (between subunits a and d), ϕ (between subunits b and e), and Ψ (between subunits c and f). (C–F) Structural overlay of the hexamers in each type (colored as in Panel A) against the regular T = 3 hexamer (grey). The models are aligned based on subunit a of the hexamers as labeled by a black dot in Panels A and B. (G) The table showing the values of θ, ϕ, and Ψ for each form of the VLPs. ϕ and Ψ have the same value for each VLP form due to the symmetry. The names of the hexamers are highlighted with the corresponding color in Panel A. (H) The relative motion between the C-terminus (dark grey) of one subunit and the CD/DE-loops of its neighbor. The black arrows indicate the movements of the CD/DE-loops between the over arched (red) and straight (green) hexamer conformations. The location of the C-terminus and the CD/DE-loops are labeled in Panel B with the corresponding colors, grey and red, respectively.

Figure 5

Proposed model for the co-replicational assembly of the Qβ virion, and the geometrical relationship between the oblate, T = 3, and prolate VLPs. (A) Proposed model for assembly of the Qβ virion along with the gRNA replication. The gRNA synthesized by the replicase (Rep) presents several high-affinity stem-loops for CP dimers, which, upon binding to CP dimers, change their conformations from C/C to A/B. The protein–protein interactions of CP dimers on the growing capsid facilitate RNA collapse, and form the first pentamer, then other pentamers and hexamers. Almost towards the end of the assembly (after the last stem-loop, U1, has been synthesized), the Mat binds to the 3′ UTR, and is incorporated into the capsid to form a complete virion. (B) The geometrical relationship between the oblate, T = 3, and prolate VLPs. The conversion from the oblate to T = 3 VLPs can be achieved through the division of the oblate VLP, perpendicular to the 5-fold axis, into two hemispheres, with one hemisphere rotated 36° relative to the other, followed by the insertion of five hexamers (red). The elongation from the T = 3 to prolate VLP follows the same protocol with an addition of five more hexamers (green).