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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 [en]
dsDNA, lurbo, Cedratvirus, x-ray, microscope
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-391670OAI: oai:DiVA.org:uu-391670DiVA, id: diva2:1345495
Available from: 2019-08-25 Created: 2019-08-25 Last updated: 2019-08-25
In thesis
1. Structural Studies of Large dsDNA Viruses using Single Particle Methods
Open this publication in new window or tab >>Structural Studies of Large dsDNA Viruses using Single Particle Methods
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
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:nbn:se:uu:diva-391671 (URN)978-91-513-0732-9 (ISBN)
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

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