Assembly of a nucleus-like structure during viral replication in bacteria

Chaikeeratisak, Vorrapon; Nguyen, Katrina; Khanna, K. N.; Brilot, Axel F.; Erb, Marcella L.; Coker, Joanna; Vavilina, Anastasia; Newton, Gerald L.; Buschauer, Robert; Pogliano, Kit; Villa, Elizabeth; Agard, David A.; Pogliano, Joe

doi:10.1126/science.aal2130

Cited by 232 publications

(431 citation statements)

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“…These structures can be nuclear or cytoplasmic, and they compartmentalize viral and cellular factors required for DNA and RNA synthesis (1, 2). With regard to bacterial viruses, a few examples of the organization of viral replication have been reported (3, 4, 25), with one recent study showing that the phage 2012-1 replication compartment can display the complexity of the viral factories formed by eukaryal viruses (5). We demonstrate the formation of a viral replication focus at the periphery of Sulfolobus cells, after infection with SIRV2, where viral DNA synthesis takes place and where replication-related proteins of the virus (gp17) and host (PCNA and Dpo1) are concentrated.…”

Section: Discussionmentioning

confidence: 99%

Formation of a Viral Replication Focus in Sulfolobus Cells Infected by the Rudivirus Sulfolobus islandicus Rod-Shaped Virus 2

Martínez-Álvarez

Deng

Peng

2017

J Virol

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Viral factories are compartmentalized centers for viral replication and assembly in infected eukaryotic cells. Here, we report the formation of a replication focus by prototypical archaeal Sulfolobus islandicus rod-shaped virus 2 (SIRV2) in the model archaeon Sulfolobus. This rod-shaped virus belongs to the viral family Rudiviridae, carrying linear double-stranded DNA (dsDNA) genomes, which are very common in geothermal environments. We demonstrate that SIRV2 DNA synthesis is confined to a focus near the periphery of infected cells. Moreover, viral and cellular replication proteins are recruited to, and concentrated in, the viral replication focus. Furthermore, we show that of the four host DNA polymerases (DNA polymerase I [Dpo1] to Dpo4), only Dpo1 participates in viral DNA synthesis. This constitutes the first report of the formation of a viral replication focus in archaeal cells, suggesting that organization of viral replication in foci is a widespread strategy employed by viruses of the three domains of life. IMPORTANCEThe organization of viral replication in foci or viral factories has been mostly described for different eukaryotic viruses and for several bacteriophages. This work constitutes the first report of the formation of a viral replication center by a virus infecting members of the Archaea domain.KEYWORDS replication focus, archaeal virus, SIRV2, DNA polymerase T he compartmentalization of viral genome replication is a well-known feature of many eukaryotic viruses, where replication is confined to specific subcellular microenvironments termed viral factories, viroplasms, viral replication compartments, or viral replication centers. In general, viral factories function as a scaffold containing viral genomes and proteins involved in viral replication and assembly, and this is hypothesized to increase the efficiency of viral replication and confer protection from the cellular antiviral immune responses (1, 2).The formation of viral factories has been observed for DNA, double-stranded RNA (dsRNA), positive-sense single-stranded RNA (ssRNA), and negative-sense ssRNA viruses, and it has been extensively studied for nucleocytoplasmic large DNA viruses (e.g., poxviruses and asfarviruses) and positive-sense ssRNA viruses, including flaviviruses, coronaviruses, picornaviruses, and togaviruses (1). Although these viruses infect a wide range of hosts and their corresponding viral factories differ in their morphologies, components, and intracellular locations, their modes of formation share some similarities. First, formation of viral factories usually involves extensive organizational changes in the host cell membranes and/or cytoskeleton. Second, viral factories constitute a scaffold for the concentration of viral genomes and of viral and cellular proteins required for viral replication and assembly (1, 2).To date, the formation of viral factories has been studied mainly for eukaryotic viruses, although the organization of viral replication has been reported for bacteriophages 29, PRD1, SSP1, and 20...

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Section: Discussionmentioning

confidence: 99%

Formation of a Viral Replication Focus in Sulfolobus Cells Infected by the Rudivirus Sulfolobus islandicus Rod-Shaped Virus 2

Martínez-Álvarez

Deng

Peng

2017

J Virol

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“…We recently described a nucleus-like structure in bacteria, assembled by phage 201Φ2-1 in Pseudomonas chlororaphis cells during infection (Chaikeeratisak et al, 2017). This “phage nucleus” is bound by a proteinaceous barrier composed of gp105 that encloses viral DNA and separates cellular functions.…”

Section: Introductionmentioning

confidence: 99%

The Phage Nucleus and Tubulin Spindle Are Conserved among Large Pseudomonas Phages

et al. 2017

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Summary We recently demonstrated that the large Pseudomonas chlororaphis bacteriophage 201φ2-1 assembles a nucleus-like structure that encloses phage DNA and segregates proteins according to function, with DNA processing proteins inside and metabolic enzymes and ribosomes outside the nucleus. Here we investigate the replication pathway of the Pseudomonas aeruginosa bacteriophages φKZ and φPA3. Bacteriophages φKZ and φPA3 encode a proteinaceous shell that assembles a nucleus-like structure that compartmentalizes proteins and DNA during viral infection. We show that the tubulin-like protein PhuZ encoded by each phage assembles a bipolar spindle that displays dynamic instability and positions the nucleus at midcell. Our results suggest that the phage spindle and nucleus play the same functional role in all three phages, 201φ2-1, φKZ and φPA3, demonstrating that these key structures are conserved among large Pseudomonas phages.

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“…Focused Ion Beam (FIB-SEM) erosion under cryogenic temperatures offers researchers a way of producing damage and artefact free lamella which can be utilized in high end tomography tools such as the FEI Titan Krios™. Several Cryo-FIB prototype tools exist and have been producing significant results utilizing the 'in situ' approach [2,3,4].Correct preparation of samples for in situ lamella prep is critical for making use of this emerging technique. There are many steps where the sample can be significantly contaminated or even completely damaged which would prevent from collection of high-quality cryo-electron tomography data.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Sample Optimization for In Situ Lamella Preparation for Cryo Electron Tomography

2017

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Cryo Electron Tomography is emerging as a technique which enables the extension of structural biology from solely purified structures, to the dynamic complexity of the cell [1]. Focused Ion Beam (FIB-SEM) erosion under cryogenic temperatures offers researchers a way of producing damage and artefact free lamella which can be utilized in high end tomography tools such as the FEI Titan Krios™. Several Cryo-FIB prototype tools exist and have been producing significant results utilizing the 'in situ' approach [2,3,4].Correct preparation of samples for in situ lamella prep is critical for making use of this emerging technique. There are many steps where the sample can be significantly contaminated or even completely damaged which would prevent from collection of high-quality cryo-electron tomography data. Standardization of the whole process of ablation of cellular material and sample handling is required to provide sufficient yield of high-quality samples for TEM imaging.Prior to these steps, the protocols for adhesion of the cells on the TEM grids and its subsequent vitrification require standardization. Here, we provide a protocol for preparation of samples for cryofocused ion beam micromachining (FIB-milling) which in our hands provides high reproducibility and quality of the input material for FIB-milling. An example of standardization based on Saccharomyces cerevisiae and A9 are presented, showing the critical steps and success indicators required to reproduce high quality samples. It is hoped that these insights will help others to quickly adapt to this new method of sample preparation.Many existing users of cryo preparation for structural biology have developed methods which suit single particle type preparation; this can involve many grid chemistries, buffer types and preparation stages. Cells require a lengthier grid preparation stage. Here cells must be encouraged to attach with a grid surface either in the plunge freezer or under culture conditions over the course of 24 hours or so. Figure 1 shows the culture method for TEM grids. Using live cells also requires the user to work aseptically and utilize biocompatible processes; ensuring that the environmental conditions are kept controlled, the cells are in a biologically relevant condition and that the materials used in the preparation are biocompatible. Being able to control the deposition process gives better morphological presentation of the cells but also ensures more uniform freezing characteristics.Cells can be upwards of 500nm thick. Cells of greater than 10µm will unlikely experience uniform freezing and vitrification via plunge freezing. The method of blotting, the buffer, temperature, humidity, cryogen selection and transfer methods also play important roles. All these factors can greatly affect the success rate of the method. In this presentation the approaches to sample preparation which enable successful result in the FIB-SEM and TEM are discussed. Figure 2 shows a typical view of cells before freezing and after optimum freezing on the same TE...

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Assembly of a nucleus-like structure during viral replication in bacteria

Cited by 232 publications

References 21 publications

Formation of a Viral Replication Focus in Sulfolobus Cells Infected by the Rudivirus Sulfolobus islandicus Rod-Shaped Virus 2

Formation of a Viral Replication Focus in Sulfolobus Cells Infected by the Rudivirus Sulfolobus islandicus Rod-Shaped Virus 2

The Phage Nucleus and Tubulin Spindle Are Conserved among Large Pseudomonas Phages

Sample Optimization for In Situ Lamella Preparation for Cryo Electron Tomography

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