Cryo Electron Microscopy Reconstructions of the Leviviridae Unveil the Densest Icosahedral RNA Packing Possible

doi:10.1016/j.jmb.2006.08.053

Journal of Molecular Biology

Volume 363, Issue 4, 3 November 2006, Pages 858-865

https://doi.org/10.1016/j.jmb.2006.08.053 Get rights and content

Abstract

We solved the structures of the single-stranded RNA bacteriophages Qβ, PP7 and AP205 by cryo-electron microscopy. On the outside, the symmetrized electron density maps resemble the previously described cryo-electron microscopy structure of MS2. RNA density is present inside the capsids, suggesting that the genomic RNA of Qβ, PP7 and AP205, analogous to MS2, contains many coat protein-binding sites in addition to the hairpin on which assembly and packaging are initiated. All four bacteriophages harbour the same overall arrangement of the RNA, which is a unique combination of both triangles and pentagons. This combination has not been found in other icosahedral viruses, in which the RNA structures are either triangular or pentagonal. Strikingly, the unique RNA packing of the Leviviridae appears to deploy the most efficient method of RNA storage by obeying icosahedral symmetry.

Introduction

The single-stranded RNA bacteriophages from the Leviviridae family, which are divided into leviviruses and alloleviviruses, infect many Gram-negative bacteria, such as Escherichia, Pseudomonas, Caulobacter and Acinetobacter. The positive-sense genome of the leviviruses (∼3600 nt) encodes four proteins (Figure 1): maturation protein (MP), coat protein (CP), replicase subunit (R) and lysis protein (L). The genome of the alloleviviruses (∼4200 nt) codes for MP, CP, R and a read-through protein (RT), which is a C-terminal extension of CP (Figure 1). CP acts as a switch in the life-cycle of the bacteriophages. At sufficiently high concentrations, CP dimers bind to the operator hairpin, harbouring the start of the replicase gene, thereby repressing translation of the replicase subunit. In due course, encapsidation is started by this initial interaction.

The crystal structure of the recombinant wild-type capsid in complex with a 19 nt operator showed the atomic details of the precise recognition.¹ Recently, a cryo-electron microscopy (cryo-EM) reconstruction of infectious virus particles was determined.² It was suggested that interactions between the CP dimer and stem-loop structures of the highly structured genome are common in bacteriophage MS2, extending beyond the operator-CP dimer interaction.² It has been shown that non-specific RNA sequences are able to interact with CP dimers and contribute substantially to the overall assembly reaction.³ In addition, it has been suggested that the RNA actively templates the assembly pathway.⁴ This supports the idea that the phages utilize additional binding sites that would assist the operator in making the capsid grow.⁵^,⁶ The secondary binding sites probably consist of hairpins resembling the operator, and they are likely to bind CP dimers in a similar manner.⁷^,⁸

These secondary binding sites in the RNA may exist in other bacteriophages. To investigate whether the overall RNA organization is conserved and whether secondary CP-binding sites may exist, we determined the three-dimensional, icosahedrally symmetrized structures of three members of the Leviviridae (Qβ, PP7 and AP205) by cryo-EM and compared them to the previous cryo-EM structure determination of MS2.² Firstly, the reconstructions show the expected icosahedral capsid enclosing the genomic RNA. Secondly, the general RNA organization inside the capsids indeed resembles that found in MS2, giving an important clue to the existence of secondary CP-binding sites.² The details of the RNA densities inside the capsids of PP7 and AP205, however, differ significantly from MS2 and Qβ, which are nearly indistinguishable. In addition, the characteristic combination of triangles and pentagons is not observed in other virus structures determined so far, where the overall RNA pattern is formed by either pentagons building a dodecahedron,⁹^,¹⁰ or triangles building an icosahedron.11., 12., 13. The combined structure in the Leviviridae seems to be unique.

Section snippets

Protein in the reconstruction

Cryo-EM and icosahedral object reconstruction of bacteriophages Qβ, PP7 and AP205 were used to solve the three-dimensional structures (Figure 2). The resolution of the models was estimated to be between 17 Å and 24 Å by both Fourier shell correlation (cut-off at 0.5 Å; data not shown) and comparison with truncated atomic models for Qβ¹⁴ and PP7.¹⁵

The models display starfish-like features around the 5-fold axes formed by the C-terminal α-helices in all structures (not shown). Holes are found in

Discussion

The principal objective of this work was to study the overall RNA structure inside the small bacteriophages using cryo-EM and object reconstruction. The previously determined cryo-EM structure of MS2² was compared to the symmetrized cryo-EM maps of Qβ, PP7 and AP205. The icosahedral object reconstructions of the bacteriophages Qβ, PP7 and AP205 reveal similar packing of RNA inside the capsids, which resembles packing in MS2 as determined by cryo-EM.² All electron density maps show the

Purification of Qβ, PP7 and AP205 virus particles

Qβ, PP7 and AP205 were propagated in Escherichia coli GM-1,²⁵ Pseudomonas aeruginosa PA01,²⁶ and Acinetobacter genospecies 16,²⁷ respectively. Infection, propagation and purification of virus particles were performed essentially as described.²

Specimen preparation and cryo electron microscopy

Holey carbon grids were glow-discharged for 2 min at 30 mA in a turbo-coater equipped with a glow-discharge unit (EMITECH K950X) immediately before use. A suspension of purified bacteriophage (Qβ, PP7 or AP205) was flash-frozen in a thin layer of vitreous

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^†: S.H.E.v.d.W. and R.I.K. contributed equally to this work.

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Journal of Molecular Biology

Cryo Electron Microscopy Reconstructions of the Leviviridae Unveil the Densest Icosahedral RNA Packing Possible

Abstract

Introduction

Section snippets

Protein in the reconstruction

Discussion

Purification of Qβ, PP7 and AP205 virus particles

Specimen preparation and cryo electron microscopy

J. Mol. Biol.

J. Mol. Biol.

Virology

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Virology

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Structure

J. Mol. Biol.

J. Mol. Biol.

J. Biol. Chem.

J. Mol. Biol.