Quantitative Electron Microscopy to Study HCMV Morphogenesis

Read, Clarissa; Walther, Paul; Einem, Jens von

doi:10.1007/978-1-0716-1111-1_14

Cited by 8 publications

(7 citation statements)

References 67 publications

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“…In eukaryotes, some viruses acquire envelopes inside the cell by budding through organelles, such as endoplasmic reticulum, nuclear envelope or Golgi complex (56)(57)(58)(59). However, internal, membranebound compartments have never been observed in Saccharolobus or any other archaeal cells, rendering the possibility that SIFV virions are enveloped by budding through intracellular membranes highly unlikely.…”

Section: Discussionmentioning

confidence: 99%

A filamentous archaeal virus is enveloped inside the cell and released through pyramidal portals

Baquero

Gazi

Sachse

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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The majority of viruses infecting hyperthermophilic archaea display unique virion architectures and are evolutionarily unrelated to viruses of bacteria and eukaryotes. The lack of relationships to other known viruses suggests that the mechanisms of virus–host interaction in Archaea are also likely to be distinct. To gain insights into archaeal virus–host interactions, we studied the life cycle of the enveloped, ∼2-μm-long Sulfolobus islandicus filamentous virus (SIFV), a member of the family Lipothrixviridae infecting a hyperthermophilic and acidophilic archaeon Saccharolobus islandicus LAL14/1. Using dual-axis electron tomography and convolutional neural network analysis, we characterize the life cycle of SIFV and show that the virions, which are nearly two times longer than the host cell diameter, are assembled in the cell cytoplasm, forming twisted virion bundles organized on a nonperfect hexagonal lattice. Remarkably, our results indicate that envelopment of the helical nucleocapsids takes place inside the cell rather than by budding as in the case of most other known enveloped viruses. The mature virions are released from the cell through large (up to 220 nm in diameter), six-sided pyramidal portals, which are built from multiple copies of a single 89-amino-acid-long viral protein gp43. The overexpression of this protein in Escherichia coli leads to pyramid formation in the bacterial membrane. Collectively, our results provide insights into the assembly and release of enveloped filamentous viruses and illuminate the evolution of virus–host interactions in Archaea.

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Section: Discussionmentioning

confidence: 99%

A filamentous archaeal virus is enveloped inside the cell and released through pyramidal portals

Baquero

Gazi

Sachse

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…For TEM coupled to focused ion beam-scanning electron microscopy (FIB-SEM), freshly released gametes were collected in cellulose capillaries and cultivated in the capillaries in Provasoli-enriched seawater for 3-5 days at 14°C in the dark or approximately 1 day at 14°C in a 12 h light/12 h dark regime to produce two- to five-cell stage partheno-sporophytes. Cells in capillaries were frozen at high-pressure (HPF Compact 03, Engineering Office M. Wohlwend), freeze-substituted (AFS2, Leica Microsystems) with 0.2% OsO 4 and 0.1% uranyl acetate in acetone containing 5% H 2 O as substitution medium ( Read et al, 2021 ) and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and analysed with a Tecnai Spirit (Thermo Fisher Scientific) operated at 120 kV.…”

Section: Methodsmentioning

confidence: 99%

The baseless mutant links protein phosphatase 2A with basal cell identity in the brown alga Ectocarpus

et al. 2023

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The first mitotic division of the initial cell is a key event in all multicellular organisms and is associated with the establishment of major developmental axes and cell fates. The brown alga Ectocarpus has a haploid-diploid life cycle that involves the development of two multicellular generations: the sporophyte and the gametophyte. Each generation deploys a distinct developmental programme autonomously from an initial cell, the first cell division of which sets up the future body pattern. Here, we show that mutations in the BASELESS (BAS) gene result in multiple cellular defects during the first cell division and subsequent failure to produce basal structures during both generations. BAS encodes a type B″ regulatory subunit of protein phosphatase 2A (PP2A), and transcriptomic analysis identified potential effector genes that may be involved in determining basal cell fate. The bas mutant phenotype is very similar to that observed in distag (dis) mutants, which lack a functional Tubulin-binding co-factor Cd1 (TBCCd1) protein, indicating that TBCCd1 and PP2A are two essential components of the cellular machinery that regulates the first cell division and mediates basal cell fate determination.

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“…TEM and FIB-SEM For transmission electron microscopy (TEM) and focused ion beam-scanning electron microscopy (FIB-SEM), freshly released gametes were collected in cellulose capillaries and cultivated in the capillaries in Provasoli-enriched seawater for 3 to 5 days at 14°C in the dark or approximately 1 day at 14°C in 12 h light/12 h dark regime to produce two-to five-cell stage partheno-sporophytes. Cells in capillaries were frozen at high-pressure (HPF Compact 03, Engineering Office M. Wohlwend GmbH), freeze-substituted (AFS2, Leica Microsystems) with 0.2% OsO4 and 0.1% uranyl acetate in acetone containing 5% H2O as substitution medium (Read et al, 2021) and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and analysed with a Tecnai Spirit (Thermo Fisher Scientific) operated at 120 kV.…”

Section: Confocal Microscopymentioning

confidence: 99%

The baseless mutant links protein phosphatase 2A with basal cell identity in the brown alga Ectocarpus

Zheng

Yao

Henschen

et al. 2022

Preprint

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The first mitotic division of the initial cell is a key event in all multicellular organisms and is usually concomitant with the establishment of major developmental axes and cell fates. The brown alga Ectocarpus has a haploid-diploid life cycle that involves the development of two multicellular and independent generations, the sporophyte and the gametophyte. Each generation deploys a distinct developmental program autonomously from an initial cell, whose first cell division sets up the future body pattern. Here, we show that mutations in the BASELESS (BAS) gene result in multiple cellular defects during the first division of the initial cell and subsequently failure to produce basal structures (rhizoids and prostrate filaments) during both generations of the life cycle. Cloning-by-sequencing revealed that BAS encodes a type B" regulatory subunit of protein phosphatase 2A, and transcriptomic analysis of early developmental stages uncovered potential effector genes involved in basal cell fate determination in this organism. The bas mutant phenotype is very similar to that observed in the distag (dis) mutants, which lack a functional TBCCd1 protein, at both the cellular and morphological levels. The high level of similarity of the dis and bas mutant phenotypes indicate that TBCCd1 and PP2A are two critical components of the cellular machinery that regulates the division of the initial cell and mediates the establishment of basal cell fate in the developing thallus.

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Quantitative Electron Microscopy to Study HCMV Morphogenesis

Cited by 8 publications

References 67 publications

A filamentous archaeal virus is enveloped inside the cell and released through pyramidal portals

A filamentous archaeal virus is enveloped inside the cell and released through pyramidal portals

The baseless mutant links protein phosphatase 2A with basal cell identity in the brown alga Ectocarpus

The baseless mutant links protein phosphatase 2A with basal cell identity in the brown alga Ectocarpus

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