Nuclear envelope impairment is facilitated by the herpes simplex virus 1 Us3 kinase

Background: Capsids of herpes simplex virus 1 (HSV-1) are assembled in the nucleus, translocated either to the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope, or released to the cytosol in a “naked” state via impaired nuclear pores that finally results in impairment of the nuclear envelope. The Us3 gene encodes a protein acting as a kinase, which is responsible for phosphorylation of numerous viral and cellular substrates. The Us3 kinase plays a crucial role in nucleus to cytoplasm capsid translocation. We thus investigate the nuclear surface in order to evaluate the significance of Us3 in maintenance of the nuclear envelope during HSV-1 infection.Methods: To address alterations of the nuclear envelope and capsid nucleus to cytoplasm translocation related to the function of the Us3 kinase we investigated cells infected with wild type HSV-1 or the Us3 deletion mutant R7041(∆Us3) by transmission electron microscopy, focused ion-beam electron scanning microscopy, cryo-field emission scanning electron microscopy, confocal super resolution light microscopy, and polyacrylamide gel electrophoresis.Results: Confocal super resolution microscopy and cryo-field emission scanning electron microscopy revealed decrement in pore numbers in infected cells. Number and degree of pore impairment was significantly reduced after infection with R7041(∆Us3) compared to infection with wild type HSV-1. The nuclear surface was significantly enlarged in cells infected with any of the viruses. Morphometric analysis revealed that additional nuclear membranes were produced forming multiple folds and caveolae, in which virions accumulated as documented by three-dimensional reconstruction after ion-beam scanning electron microscopy. Finally, significantly more R7041(∆Us3) capsids were retained in the nucleus than wild-type capsids whereas the number of R7041(∆Us3) capsids in the cytosol was significantly lower.Conclusions: The data indicate that Us3 kinase is involved in facilitation of nuclear pore impairment and, concomitantly, in capsid release through impaired nuclear envelope.

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High-resolution scanning electron microscopy of the nuclear surface in Herpes Simplex Virus 1 infected cells

Scanning Electron Microscopy for the Life Sciences ◽

10.1017/cbo9781139018173.009 ◽

2013 ◽

pp. 115-136 ◽

Cited By ~ 4

Author(s):

Peter Wild ◽

Andres Kaech ◽

Miriam S. Lucas

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

High Resolution ◽

Nuclear Surface ◽

Herpes Simplex Virus 1 ◽

Infected Cells ◽

Simplex Virus ◽

Scanning Electron

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Endoplasmic reticulum-to-Golgi transitions upon herpes virus infection

F1000Research ◽

10.12688/f1000research.12252.1 ◽

2017 ◽

Vol 6 ◽

pp. 1804 ◽

Cited By ~ 3

Author(s):

Peter Wild ◽

Andres Kaech ◽

Elisabeth M. Schraner ◽

Ladina Walser ◽

Mathias Ackermann

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Endoplasmic Reticulum ◽

Nuclear Envelope ◽

Golgi Complex ◽

Progeny Virus ◽

Perinuclear Space ◽

Exit Route ◽

Scanning Electron ◽

Hsv 1

Background: Herpesvirus capsids are assembled in the nucleus before they are translocated to the perinuclear space by budding, acquiring tegument and envelope, or releasing to the cytoplasm in a “naked” state via impaired nuclear envelope. One model proposes that envelopment, “de-envelopment” and “re-envelopment” are essential steps for production of infectious virus. Glycoproteins gB/gH were reported to be essential for de-envelopment, by fusion of the “primary” envelope with the outer nuclear membrane. Yet, a high proportion of enveloped virions generated from genomes with deleted gB/gH were found in the cytoplasm and extracellular space, suggesting the existence of an alternative exit route.Methods: We investigated the relatedness between the nuclear envelope and membranes of the endoplasmic reticulum and Golgi complex, in cells infected with either herpes simplex virus 1 (HSV-1) or a Us3 deletion mutant thereof, or with bovine herpesvirus 1 (BoHV-1) by transmission and scanning electron microscopy, employing freezing technique protocols that lead to improved spatial and temporal resolution.Results: Scanning electron microscopy showed the Golgi complex as a compact entity in a juxtanuclear position covered by a membrane on thecisface. Transmission electron microscopy revealed that Golgi membranes merge with membranes of the endoplasmic reticulum forming an entity with the perinuclear space. All compartments contained enveloped virions. After treatment with brefeldin A, HSV-1 virions aggregated in the perinuclear space and endoplasmic reticulum, while infectious progeny virus was still produced.Conclusions: The data strongly suggest that virions are intraluminally transported from the perinuclear space via Golgi complex-endoplasmic reticulum transitions into Golgi cisternae for packaging into transport vacuoles. Furthermore, virions derived by budding at nuclear membranes are infective as has been shown for HSV-1 Us3 deletion mutants, which almost entirely accumulate in the perinuclear space. Therefore, de-envelopment followed by re-envelopment is not essential for production of infective progeny virus.

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Ultrastructural Features of Normal and Diseased Hair Revealed by Ion Beam Etching and Freeze Fracture

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115419 ◽

1975 ◽

Vol 33 ◽

pp. 358-359

Author(s):

M. Spector ◽

A. C. Brown

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Ectodermal Dysplasia ◽

Ion Beam ◽

Freeze Fracture ◽

Ion Current ◽

Ion Beam Etching ◽

Pili Torti ◽

Argon Ion ◽

Scanning Electron

Ion beam etching and freeze fracture techniques were utilized in conjunction with scanning electron microscopy to study the ultrastructure of normal and diseased human hair. Topographical differences in the cuticular scale of normal and diseased hair were demonstrated in previous scanning electron microscope studies. In the present study, ion beam etching and freeze fracture techniques were utilized to reveal subsurface ultrastructural features of the cuticle and cortex.Samples of normal and diseased hair including monilethrix, pili torti, pili annulati, and hidrotic ectodermal dysplasia were cut from areas near the base of the hair. In preparation for ion beam etching, untreated hairs were mounted on conducting tape on a conducting silicon substrate. The hairs were ion beam etched by an 18 ky argon ion beam (5μA ion current) from an ETEC ion beam etching device. The ion beam was oriented perpendicular to the substrate. The specimen remained stationary in the beam for exposures of 6 to 8 minutes.

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Microscopy Methods for Biofilm Imaging: Focus on SEM and VP-SEM Pros and Cons

Biology ◽

10.3390/biology10010051 ◽

2021 ◽

Vol 10 (1) ◽

pp. 51

Author(s):

Michela Relucenti ◽

Giuseppe Familiari ◽

Orlando Donfrancesco ◽

Maurizio Taurino ◽

Xiaobo Li ◽

...

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Ion Beam ◽

Ruthenium Red ◽

Variable Pressure ◽

Sem Images ◽

Advantages And Disadvantages ◽

Pros And Cons ◽

Microscopy Techniques ◽

Scanning Electron

Several imaging methodologies have been used in biofilm studies, contributing to deepening the knowledge on their structure. This review illustrates the most widely used microscopy techniques in biofilm investigations, focusing on traditional and innovative scanning electron microscopy techniques such as scanning electron microscopy (SEM), variable pressure SEM (VP-SEM), environmental SEM (ESEM), and the more recent ambiental SEM (ASEM), ending with the cutting edge Cryo-SEM and focused ion beam SEM (FIB SEM), highlighting the pros and cons of several methods with particular emphasis on conventional SEM and VP-SEM. As each technique has its own advantages and disadvantages, the choice of the most appropriate method must be done carefully, based on the specific aim of the study. The evaluation of the drug effects on biofilm requires imaging methods that show the most detailed ultrastructural features of the biofilm. In this kind of research, the use of scanning electron microscopy with customized protocols such as osmium tetroxide (OsO4), ruthenium red (RR), tannic acid (TA) staining, and ionic liquid (IL) treatment is unrivalled for its image quality, magnification, resolution, minimal sample loss, and actual sample structure preservation. The combined use of innovative SEM protocols and 3-D image analysis software will allow for quantitative data from SEM images to be extracted; in this way, data from images of samples that have undergone different antibiofilm treatments can be compared.

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