Graduate Student, UCLA
Reconstructing the RNA genome inside a single-stranded RNA virus using cryo-electron microscopy
The capsid protein structure of many spherical, single-stranded (ss)RNA viruses have been determined using both x-ray crystallography and cryo-electron microscopy (cryoEM). However, these structures have generally been determined by imposing symmetry necessarily obscuring any asymmetrical structures, particularly the RNA genome inside the particle. This work aims to use cryoEM and asymmetric reconstruction to investigate the structure of the asymmetric RNA genome inside Brome Mosaic Virus (BMV). The power of asymmetric reconstruction has already been established for ssRNA viruses with an asymmetric capsid, namely MS2 and bacteriophage Q-beta, which benefit from having the asymmetric capsid shell imparting its asymmetry on the internal ssRNA genome. We find that the capsid shell agrees well with structures determined previously, as does the RNA density seen at the hexamers of the symmetric structure. Our results illustrate a fundamental difference between the ssRNA organization in the symmetric BMV viral capsid and the ssRNA organization in the asymmetric capsids of the bacteriophages MS2 and Q-beta. For these bacteriophages, it has been shown that a dominant RNA conformation is found inside the assembled viral capsids, and RNA density is conserved even at the center of the particle. We find in BMV, on the other hand, that the RNA is only organized at the capsid shell, and our results indicate that there is not a single dominant conformation for the RNA inside the virus but rather an ensemble of RNA structures that interact strongly with the capsid protein shell.
Abstract: Advances in electron cryo-microscopy have enabled structure determination of macromolecules at near-atomic resolution. However, structure determination, even using de novo methods, remains susceptible to model bias and overfitting. Here we describe a complete workflow for data acquisition, image processing, all-atom modelling and validation of brome mosaic virus, an RNA virus. Data were collected with a direct electron detector in integrating mode and an exposure beyond the traditional radiation damage limit. The final density map has a resolution of 3.8 Å as assessed by two independent data sets and maps. We used the map to derive an all-atom model with a newly implemented real-space optimization protocol. The validity of the model was verified by its match with the density map and a previous model from X-ray crystallography, as well as the internal consistency of models from independent maps. This study demonstrates a practical approach to obtain a rigorously validated atomic resolution electron cryo-microscopy structure.
Pub.: 05 Sep '14, Pinned: 09 Jun '17
Abstract: The four Brome mosaic virus (BMV) RNAs are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidates each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of post-translational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1, but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. CryoEM reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion production. The degree of phosphorylation affected CP-RNA interaction to modulate interaction with the encapsidated RNA, the release of three of the BMV RNAs. CLIP-seq experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contained regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection.How icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome mosaic virus (BMV) is an RNA virus with its three genomic RNAs encapsidated in separate virions. Through proteomic structural, and biochemical analyses this work shows that post-translational modifications, specifically phosphorylation, on the capsid protein regulates capsid-RNA interaction, the stability of the virions, and affected viral gene expression. Mutational analysis confirmed that changes in modification affected virion stability and the timing of viral infection. The mechanism for modification of the virion has striking parallels to the regulation of chromatin packaging by nucleosomes.
Pub.: 24 Jun '16, Pinned: 09 Jun '17
Abstract: Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-coat protein interactions among 15 of these stem-loops with diverse sequences. The stem-loop at the 3' end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome-capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.
Pub.: 20 Dec '16, Pinned: 09 Jun '17