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An atomic model of brome mosaic virus using direct electron detection and real-space optimization.

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.

SUBMITTER: Wang Z

PROVIDER: S-EPMC4155512 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Brome mosaic virus

Project description:Brome mosaic virus complete genome sequencing

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Project description:Viral particles are biological machines that have evolved to package, protect, and deliver the viral genome into the host via regulated conformational changes of virions. We have developed a procedure to modify lysine residues with S-methylthioacetimidate across the pH range from 5.5 to 8.5. Lysine residues that are not completely modified are involved in tertiary or quaternary structural interactions, and their extent of modification can be quantified as a function of pH. This procedure was applied to the pH-dependent structural transitions of brome mosaic virus (BMV). As the reaction pH increases from 5.5 to 8.5, the average number of modified lysine residues in the BMV capsid protein increases from 6 to 12, correlating well with the known pH-dependent swelling behavior of BMV virions. The extent of reaction of each of the capsid protein's lysine residues has been quantified at eight pH values using coupled liquid chromatography-tandem mass spectrometry. Each lysine can be assigned to one of three structural classes identified by inspection of the BMV virion crystal structure. Several lysine residues display reactivity that indicates their involvement in dynamic interactions that are not obvious in the crystal structure. The influence of several capsid protein mutants on the pH-dependent structural transition of BMV has also been investigated. Mutant H75Q exhibits an altered swelling transition accompanying solution pH increases. The H75Q capsids show increased reactivity at lysine residues 64 and 130, residues distal from the dimer interface occupied by H75, across the entire pH range.

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Seeing tobacco mosaic virus through direct electron detectors.

Project description:With the introduction of direct electron detectors (DED) to the field of electron cryo-microscopy, a wave of atomic-resolution structures has become available. As the new detectors still require comparative characterization, we have used tobacco mosaic virus (TMV) as a test specimen to study the quality of 3D image reconstructions from data recorded on the two direct electron detector cameras, K2 Summit and Falcon II. Using DED movie frames, we explored related image-processing aspects and compared the performance of micrograph-based and segment-based motion correction approaches. In addition, we investigated the effect of dose deposition on the atomic-resolution structure of TMV and show that radiation damage affects negative carboxyl chains first in a side-chain specific manner. Finally, using 450,000 asymmetric units and limiting the effects of radiation damage, we determined a high-resolution cryo-EM map at 3.35Å resolution. Here, we provide a comparative case study of highly ordered TMV recorded on different direct electron detectors to establish recording and processing conditions that enable structure determination up to 3.2Å in resolution using cryo-EM.

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Full virus map of Brome Mosaic Virus (picked particles)

Project description: Not available

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Modeling the helicase domain of Brome mosaic virus 1a replicase.

Project description:Brome mosaic virus (BMV) is a representative member of positive-strand RNA viruses. The 1a replicase from BMV is a membrane protein of unknown structure with a methyltransferase N-terminal domain and a putative helicase activity in the C-terminal domain. In order to make a functional prediction of the helicase activity of the BMV 1a C-terminal domain, we have built a model of its structure. The use of fold recognition servers hinted at two different superfamilies of helicases [superfamily 1 (SF1) and superfamily 2 (SF2)] as putative templates for the C-terminal fragment of BMV 1a. A structural model of BMV 1a in SF2 was obtained by means of a fold recognition server (3D-PSSM). On the other hand, we used the helicase motifs described in the literature to construct a model of the structure of the BMV 1a C-terminal domain as a member of the SF1. The biological functionality and statistic potentials were used to discriminate between the two models. The results illustrate that the use of sequence profiles and patterns helps modeling. Accordingly, the C-terminal domain of BMV 1a is a potential member of the SF1 of helicases, and it can be modeled with the structure of a member of the UvrD family of helicases. The helicase mechanism was corroborated by the model and this supports the hypothesis that BMV 1a should have helicase activity.

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Full virus map of Brome Mosaic Virus (micrographs and particle coordinates)

Project description: Not available

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Brome mosaic virus-like particles as siRNA nanocarriers for biomedical purposes.

Project description:There is an increasing interest in the use of plant viruses as vehicles for anti-cancer therapy. In particular, the plant virus brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are novel potential nanocarriers for different therapies in nanomedicine. In this work, BMV and CCMV were loaded with a fluorophore and assayed on breast tumor cells. The viruses BMV and CCMV were internalized into breast tumor cells. Both viruses, BMV and CCMV, did not show cytotoxic effects on tumor cells in vitro. However, only BMV did not activate macrophages in vitro. This suggests that BMV is less immunogenic and may be a potential carrier for therapy delivery in tumor cells. Furthermore, BMV virus-like particles (VLPs) were efficiently loaded with small interfering RNA (siRNA) without packaging signal. The gene silencing was demonstrated by VLPs loaded with siGFP and tested on breast tumor cells that constitutively express the green fluorescent protein (GPF). After VLP-siGFP treatment, GFP expression was efficiently inhibited corroborating the cargo release inside tumor cells and the gene silencing. In addition, BMV VLP carring siAkt1 inhibited the tumor growth in mice. These results show the attractive potential of plant virus VLPs to deliver molecular therapy to tumor cells with low immunogenic response.

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Duplex-RT-PCR assay for the simultaneous detection and discrimination of Brome mosaic virus and Cocksfoot mottle virus in cereal plants.

Project description:Brome mosaic virus (BMV) and cocksfoot mottle virus (CfMV) are pathogens of grass species including all economically important cereals. Both viruses have been identified in Poland therefore they create a potential risk to cereal crops. In this study, a duplex-reverse transcription-polymerase chain reaction (duplex-RT-PCR) was developed and optimized for simultaneous detection and differentiation of BMV and CfMV as well as for confirmation of their co-infection. Selected primers CfMVdiag-F/CfMVdiag-R and BMV2-F/BMV2-R amplified 390 bp and 798 bp RT-PCR products within coat protein (CP) region of CfMV and replicase gene of BMV, respectively. Duplex-RT-PCR was successfully applied for the detection of CfMV-P1 and different Polish BMV isolates. Moreover, one sample was found to be co-infected with BMV-ML1 and CfMV-ML1 isolates. The specificity of generated RT-PCR products was verified by sequencing. Duplex-RT-PCR, like conventional RT-PCR, was able to detect two viruses occurring in plant tissues in very low concentration (as low as 4.5 pg/µL of total RNA). In contrast to existing methods, newly developed technique offers a significant time and cost-saving advantage. In conclusion, duplex-RT-PCR is a useful tool which can be implemented by phytosanitary services to rapid detection and differentiation of BMV and CfMV.

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Enzyme-mediated binding of tyrosine to brome-mosaic-virus ribonucleic acid.

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