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Structural Studies of Large dsDNA Viruses using Single Particle Methods
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Structural studies of large biological assemblies pose a unique problem due to their size, complexity and heterogeneity. Conventional methods like x-ray crystallography, NMR, etc. are limited in their ability to address these issues. To overcome some of these limitations, single particle methods were used. In these methods, each particle image is manipulated individually to find the best possible set of images to reconstruct the 3D structure. The structural studies in this thesis, exploit the advantages of single particle methods. 

The large data set generated by the SPI study of PR772 provides better statistics about the sample quality due to the use of GDVN, a container-free sample delivery method. By analyzing the diffusion map, we see that the use of GDVNs as a sample delivery method produces wide range of particle sizes owing to the large droplet that are created. 

The high-resolution structure of bacteriophage PR772 confirmed the speculation about the heteropentameric nature of the penton and revealed the new architecture of the vertex complex consisting of a hetero-pentameric penton formed with three copies of P5 and two copies of P31. The beta propeller region of P2, formed by domains I and II is bound to the N-terminal domain of P5. The structure also reveals new conformations of N-terminal and C-terminal region of P3 which play an important role in particle assembly and structural stability. 

The study of Melbournevirus revealed the protein composition in a packed particle. The CryoEM structure of Melbournevirus reveals a T=309 capsid with an inner lipid membrane. A dense body was found in the viral particle, a feature not observed in other viruses of the Marseilleviridae family. The density of this body is similar to a nucleic acid-protein complex. This observation, along with the histone-like protein identified during study, suggest genome organization in the viral particle, similar to higher organisms.

The soft X-ray microscope operated in the water-window shows the progression of the Cedratvirus lurbo infection in the host cell without the use of chemical fixation, staining, sample dehydration or polymer embedding. The study revealed a significant bioconversion from the host cell to the viral particle at later stages of infection.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 72
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1847
Keywords [en]
PR772, phage, PRD1, Bacteriophage, coliphage, Melbournevirus, Cedratvirus, lurbo, Pithovirus, CryoEM, Single particle imaging, Coherent, Diffractive, Imaging, Soft X-ray, Microscopy, Microscope, GDVN, High resolution, XFEL, aerosol, Injection, electrospray, gas dynamic virtual nozzle, CDI, CXI, FEL
National Category
Structural Biology Biophysics
Research subject
Chemistry with specialization in Biophysics
Identifiers
URN: urn:nbn:se:uu:diva-391671ISBN: 978-91-513-0732-9 (print)OAI: oai:DiVA.org:uu-391671DiVA, id: diva2:1345500
Public defence
2019-10-11, Room C2:301, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2019-09-20 Created: 2019-08-25 Last updated: 2019-10-15
List of papers
1. Coherent soft X-ray diffraction imaging of Coliphage PR772 at the Linac coherent light source
Open this publication in new window or tab >>Coherent soft X-ray diffraction imaging of Coliphage PR772 at the Linac coherent light source
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2017 (English)In: Scientific Data, E-ISSN 2052-4463, Vol. 4, article id 170079Article in journal (Refereed) Published
National Category
Biophysics
Identifiers
urn:nbn:se:uu:diva-328536 (URN)10.1038/sdata.2017.79 (DOI)000404232100001 ()28654088 (PubMedID)
Projects
eSSENCE
Available from: 2017-06-27 Created: 2017-08-25 Last updated: 2019-08-25Bibliographically approved
2. CryoEM of coliphage PR772 reveals the composition & structure of the elusive vertex complex and the capsid architecture.
Open this publication in new window or tab >>CryoEM of coliphage PR772 reveals the composition & structure of the elusive vertex complex and the capsid architecture.
(English)In: eLIFE, E-ISSN 2050-084XArticle in journal (Refereed) Submitted
Abstract [en]

Bacteriophage PR772, a member of the Tectiviridae family, has a 70-nm diameter icosahedral protein capsid that encapsulates a lipid membrane, dsDNA, and various internal proteins. An icosahedrally averaged CryoEM reconstruction of the wild-type virion and a localized reconstruction of the vertex region reveal the composition and the structure of the vertex complex along with new protein conformations that play a vital role in maintaining the capsid architecture of the virion. The overall resolution of the virion is 2.75 Å, while the resolution of the protein capsid is 2.3 Å. The conventional penta-symmetron formed by the capsomeres is replaced by a large vertex complex in the pseudo T=25 capsid. All the vertices contain the host-recognition protein, P5; two of these vertices show the presence of the receptor-binding protein, P2. The 3D structure of the vertex complex shows interactions with the viral membrane, indicating a possible mechanism for viral infection.

Keywords
PR772, Phage, Bacteriophage, PRD1, Vertex Complex, Penton, heteropentamer
National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-391669 (URN)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationEU, European Research Council
Available from: 2019-08-25 Created: 2019-08-25 Last updated: 2019-08-25
3. Cryo-EM structure of a Marseilleviridae virus particle reveals a large internal microassembly
Open this publication in new window or tab >>Cryo-EM structure of a Marseilleviridae virus particle reveals a large internal microassembly
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2018 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 516, p. 239-245, article id S0042-6822(18)30028-XArticle in journal (Refereed) Published
Abstract [en]

Nucleocytoplasmic large DNA viruses (NCLDVs) blur the line between viruses and cells. Melbournevirus (MelV, family Marseilleviridae) belongs to a new family of NCLDVs. Here we present an electron cryo-microscopy structure of the MelV particle, with the large triangulation number T = 309 constructed by 3080 pseudo-hexagonal capsomers. The most distinct feature of the particle is a large and dense body (LDB) consistently found inside all particles. Electron cryo-tomography of 147 particles shows that the LDB is preferentially located in proximity to the probable lipid bilayer. The LDB is 30 nm in size and its density matches that of a genome/protein complex. The observed LDB reinforces the structural complexity of MelV, setting it apart from other NCLDVs.

Keywords
Amoeba, Capsid, Cryo-electron microscopy, Marseilleviridae, Melbournevirus, NCLDV, Protein complex, Structure, Tomography, Virus
National Category
Biophysics
Identifiers
urn:nbn:se:uu:diva-370071 (URN)10.1016/j.virol.2018.01.021 (DOI)000428004800025 ()29407382 (PubMedID)
Funder
Swedish Research Council, 628-20081109 822-2010-6157 822-2012-5260 828-2012-108Knut and Alice Wallenberg Foundation, KAW-2011.081EU, European Research Council, ERC-291602The Swedish Foundation for International Cooperation in Research and Higher Education (STINT), JA2014-5721
Available from: 2018-12-18 Created: 2018-12-18 Last updated: 2019-08-25Bibliographically approved
4. Giant DNA virus infection reveals high bioconversion from amoeba to virus
Open this publication in new window or tab >>Giant DNA virus infection reveals high bioconversion from amoeba to virus
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

During the last couple of decades, new discoveries of giant DNA viruses visible under a light microscope and with genome larger than 500 kbp are becoming more and more frequent. Interestingly, about two-thirds of their predicted genes correspond to open reading frames without recognizable database homologs. Herein, we quantitatively investigate viral replication of the newly discovered Lurbovirus to understand what cellular function is retained through the unknown open reading frames. We apply high-resolution soft x-ray microscopy to intact cell systems in their near-native state with high carbon-to-water contrast. New virions produced inside the cell are visible from 12 hours post infection and increase to several hundreds after 48 hours post infection. Due to the large size of the virion, this corresponds to a high bioconversion of 6-12 % from amoebal host to virus. We associate the high bioconversion of large DNA viruses with their large genome that enables complex functionality. The vacuolated structure of the amoebal host disappears when virions are starting to be produced at 12 hours post infection, whereas large circular x-ray-lucent cytoplasmic areas persist that are attributed to viral factories. The nucleus and nucleolus appear unaffected throughout the whole replication cycle, which suggests that nuclear functions are needed for viral replication to occur, whereas other functions are retained in the viral factories in the cytoplasm of the host cell.

Keywords
dsDNA, lurbo, Cedratvirus, x-ray, microscope
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-391670 (URN)
Available from: 2019-08-25 Created: 2019-08-25 Last updated: 2019-08-25

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