In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus

Abstract

Summary Genome packaging into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike dsDNA viruses which pump their genome into a preformed capsid1-3, ssRNA viruses, such as bacteriophage MS2, co-assemble their capsid with genome4-7; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via host “sex” pilus (F-pilus)8 and is the first fully-sequenced organism9 and a model system for studies of gene translational regulations10,11, RNA-protein interactions12-14, and RNA virus assembly15-17. Its positive-sense ssRNA genome of 3569 bases is enclosed in a capsid with one maturation protein (MP) monomer and 89 coat protein (CP) dimers arranged in a T=3 icosahedral lattice18,19. MP is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection8, but how the genome is organized and delivered are not known. Here we show the MS2 structure at 3.6Å resolution determined by electron-counting cryo electron microscopy (cryoEM) and asymmetric reconstruction. We traced ~80% backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-CP interactions among 15 of these stem-loops with diverse sequences. The stem-loop at 3’ end of the genome interacts extensively with the MP, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus, and joins 89 CP-dimers to form a capsid. This first atomic description of genome-capsid interactions in a spherical ssRNA virus provides insights into genome delivery via host “sex” pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires imaginations about links between nucleoprotein complexes and the origin of viruses.

Bacteriophage MS2 displays unreported capsid variability assembling T = 4 and mixed capsids

“…Here, we report two cryo‐EM structures of the MS2 capsid: the previously reported T = 3 symmetry form; as well as a novel T = 4 icosahedral symmetry variant, both loaded with a 155 bp sequence containing the MS2 capsid interacting stem‐loop. Our results support the notion that MS2 exhibits structural variability, in the absence of the highly ordered viral genome, accommodated by slight structural changes in coat proteins (Dai et al , ). Importantly, analysis of the non‐standard T = 4 variant structure shows that this capsid architecture changes the environment of the maturation protein‐binding site, thus making less plausible its inclusion into the capsid without major structural changes.…”

“…As a spherical ssRNA virus, bacteriophage MS2 co‐assembles its capsid around its genome (Stockley et al , ), forming a T = 3 icosahedral capsid containing 90 coat protein dimers (Valegard et al , ; Golmohammadi et al , ). This conformation allows the binding of the maturation protein during the assembly process, which replaces one of these coat protein dimers on a twofold axis (leaving 89), and breaks the capsid symmetry (Dent et al , ; Koning et al , ; Dai et al , ). During infection, this maturation protein interacts with the F‐pilus of E. coli to introduce the viral genome into a new host (Valentine and Strand, ).…”

Alphavirus Nucleocapsid Packaging and Assembly

“…Each PS acts as a staple on a path, which influences the path itself as well as future CP-CP interactions. The most accurate structure of MS2 was published in January 2017 by a group not associated with this hypothesis [96]. There has yet to be any formal comparison between the RNA interactions identified in the new structure and the predictions made in previous studies.…”

Atomic cryo-EM structures of viruses

“…X-ray crystallographic studies showed that fractions of viral genome were ordered as a result of interactions with capsid proteins, but most of the viral genome was not resolved due to non-crystallographic symmetry averaging and averaging during crystallization. In last two years, cryo-EM has dramatically contributed to our understanding of genome organization in dsRNA [32**,33**] and ssRNA viruses [34,35**]. In the recent 3.6 Å resolution cryo-EM asymmetric reconstruction of (+)RNA bacteriophage, MS2, ~80% of the 3,569 nucleotides in the viral genome could be traced [35**].…”

“…In last two years, cryo-EM has dramatically contributed to our understanding of genome organization in dsRNA [32**,33**] and ssRNA viruses [34,35**]. In the recent 3.6 Å resolution cryo-EM asymmetric reconstruction of (+)RNA bacteriophage, MS2, ~80% of the 3,569 nucleotides in the viral genome could be traced [35**]. …”