Endoplasmic reticulum-to-Golgi transitions upon herpes virus infection

Background: Herpesvirus capsids are assembled in the nucleus, translocated to the perinuclear space by budding, acquiring tegument and envelope, or released to the cytoplasm via impaired nuclear envelope. One model proposes that envelopment, “de-envelopment” and “re-envelopment” is essential 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 alternative exit routes.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.Results: The Golgi complex is a compact entity in a juxtanuclear position covered by a membrane on thecisface. 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 suggest that virions derived by budding at nuclear membranes are intraluminally transported from the perinuclear space via Golgi -endoplasmic reticulum transitions into Golgi cisternae for packaging. Virions derived by budding at nuclear membranes are infective like Us3 deletion mutants, which accumulate in the perinuclear space. Therefore, i) de-envelopment followed by re-envelopment is not essential for production of infective progeny virus, ii) the process taking place at the outer nuclear membrane is budding not fusion, and iii) naked capsids gain access to the cytoplasmic matrix via impaired nuclear envelope as reported earlier.

Download Full-text

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.

Download Full-text

Integrity of the Linker of Nucleoskeleton and Cytoskeleton Is Required for Efficient Herpesvirus Nuclear Egress

Journal of Virology ◽

10.1128/jvi.00330-17 ◽

2017 ◽

Vol 91 (19) ◽

Cited By ~ 8

Author(s):

Barbara G. Klupp ◽

Teresa Hellberg ◽

Harald Granzow ◽

Kati Franzke ◽

Beatriz Dominguez Gonzalez ◽

...

Keyword(s):

Nuclear Membrane ◽

Dominant Negative ◽

Progeny Virus ◽

Linc Complex ◽

Outer Nuclear Membrane ◽

Inner Nuclear Membrane ◽

Nuclear Membranes ◽

Nuclear Egress ◽

Virion Envelope ◽

In Cells

ABSTRACT Herpesvirus capsids assemble in the nucleus, while final virion maturation proceeds in the cytoplasm. This requires that newly formed nucleocapsids cross the nuclear envelope (NE), which occurs by budding at the inner nuclear membrane (INM), release of the primary enveloped virion into the perinuclear space (PNS), and subsequent rapid fusion with the outer nuclear membrane (ONM). During this process, the NE remains intact, even at late stages of infection. In addition, the spacing between the INM and ONM is maintained, as is that between the primary virion envelope and nuclear membranes. The linker of nucleoskeleton and cytoskeleton (LINC) complex consists of INM proteins with a luminal SUN (Sad1/UNC-84 homology) domain connected to ONM proteins with a KASH (Klarsicht, ANC-1, SYNE homology) domain and is thought to be responsible for spacing the nuclear membranes. To investigate the role of the LINC complex during herpesvirus infection, we generated cell lines constitutively expressing dominant negative (dn) forms of SUN1 and SUN2. Ultrastructural analyses revealed a significant expansion of the PNS and the contiguous intracytoplasmic lumen, most likely representing endoplasmic reticulum (ER), especially in cells expressing dn-SUN2. After infection, primary virions accumulated in these expanded luminal regions, also very distant from the nucleus. The importance of the LINC complex was also confirmed by reduced progeny virus titers in cells expressing dn-SUN2. These data show that the intact LINC complex is required for efficient nuclear egress of herpesviruses, likely acting to promote fusion of primary enveloped virions with the ONM. IMPORTANCE While the viral factors for primary envelopment of nucleocapsids at the inner nuclear membrane are known to the point of high-resolution structures, the roles of cellular components and regulators remain enigmatic. Furthermore, the machinery responsible for fusion with the outer nuclear membrane is unsolved. We show here that dominant negative SUN2 interferes with efficient herpesvirus nuclear egress, apparently by interfering with fusion between the primary virion envelope and outer nuclear membrane. This identifies a new cellular component important for viral egress and implicates LINC complex integrity in nonconventional nuclear membrane trafficking.

Download Full-text

NUCLEAR MEMBRANES FROM MAMMALIAN LIVER

The Journal of Cell Biology ◽

10.1083/jcb.46.2.379 ◽

1970 ◽

Vol 46 (2) ◽

pp. 379-395 ◽

Cited By ~ 142

Author(s):

Werner W. Franke ◽

Barbara Deumling ◽

Baerbel Ermen ◽

Ernst-Dieter Jarasch ◽

Hans Kleinig

Keyword(s):

Endoplasmic Reticulum ◽

Nuclear Envelope ◽

Nuclear Membrane ◽

Cytochrome B5 ◽

Buoyant Density ◽

Nuclear Pore ◽

Nuclear Membranes ◽

Dna And Rna ◽

Mammalian Liver ◽

Order Of Magnitude

Nuclear membranes were isolated from rat and pig liver by sonication of highly purified nuclear fractions and subsequent removal of adhering nucleoproteins in a high salt medium. The fractions were examined in the electron microscope by both negative staining and thin sectioning techniques and were found to consist of nuclear envelope fragments of widely varying sizes. Nuclear pore complex constituents still could frequently be recognized. The chemical composition of the nuclear membrane fractions was determined and compared with those of microsomal fractions prepared in parallel. For total nuclei as well as for nuclear membranes and microsomes, various enzyme activities were studied. The results indicate that a similarity exists between both fractions of cytomembranes, nuclear envelope, and endoplasmic reticulum, with respect to their RNA:protein ratio and their content of polar and nonpolar lipids. Both membranous fractions had many proteins in common including some membrane-bound enzymes. Activities in Mg-ATPase and the two examined cytochrome reductases were of the same order of magnitude. The content of cytochrome b5 as well as of P-450 was markedly lower in the nuclear membranes. The nuclear membranes were found to have a higher buoyant density and to be richer in protein. The glucose-6-phosphatase and Na-K-ATPase activities in the nuclear membrane fraction were very low. In the gel electrophoresis, in addition to many common protein bands, some characteristic ones for either microsomal or nuclear membranous material were detected. Significant small amounts of DNA and RNA were found to remain closely associated with the nuclear envelope fragments. Our findings indicate that nuclear and endoplasmic reticulum membranes which are known to be in morphological continuity have, besides a far-reaching similarity, some characteristic differences.

Download Full-text

Host Vesicle Fusion Proteins VAPB, Rab11b and Rab18 Contribute to HSV-1 Infectivity by Facilitating Egress through the Nuclear Membrane

10.1101/088633 ◽

2016 ◽

Author(s):

Natalia Saiz-Ros ◽

Rafal Czapiewski ◽

Andrew Stevenson ◽

Ilaria Epifano ◽

Selene K. Swanson ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Life Cycle ◽

Nuclear Envelope ◽

Host Cell ◽

Nuclear Membrane ◽

Fusion Proteins ◽

Vesicle Fusion ◽

Endoplasmic Reticulum Membrane ◽

Nuclear Egress ◽

Hsv 1

AbstractThe herpesvirus process of primary envelopment and de-envelopment as viral particles exit the nucleus has been for many years one of the least understood steps in the virus life cycle. Though viral proteins such as pUL31, pUL34, pUS3 and others are clearly important, these are likely insufficient for efficient fusion with the nuclear membrane. We postulated that host nuclear membrane proteins involved in virus nuclear egress would move from the inner to outer nuclear membranes due to membrane fusion events in primary envelopment and de-envelopment and then diffuse into the endoplasmic reticulum. Membrane fractions were prepared enriched in the nuclear envelope or the endoplasmic reticulum with and without HSV-1 infection and analyzed by mass spectrometry, revealing several vesicle fusion proteins as candidates in the viral nuclear egress pathway. Knockdown of three of these, VAPB, Rab11b, and Rab18, significantly reduced titers of released virus while yielding nuclear accumulation of encapsidated particles. Antibody staining revealed that VAPB visually accumulates in the inner nuclear membrane during HSV-1 infection. VAPB also co-localizes at early time points with the viral pUL34 protein known to be involved in nuclear egress. Most strikingly, VAPB was also observed on HSV-1 virus particles by immunogold labelling electron microscopy. Thus, these data reveal several new host cell vesicle fusion proteins involved in viral nuclear egress.Author SummaryHuman herpesviruses are associated with common human diseases such as chicken pox, shingles and mononucleosis and infect a wide range of animals making them economically important pathogens for livestock. Herpes simplex virus 1 (HSV-1) is most commonly associated with cold sores, but is also the leading cause of blindness by infection in the Western world. All herpesviruses share many aspects of infection. As large nuclear replicating dsDNA viruses with capsid sizes too large to use the nuclear pores to exit the nucleus, they have evolved a complex mechanism for envelopment and de-envelopment of primary herpesvirus particles, but this critical step in the virus lifecycle remains poorly understood. We have identified several host cell vesicle fusion proteins, VAPB, Rab11b and Rab18 that appear to contribute to this step in the HSV-1 life cycle. VAPB accumulates at the nuclear envelope with the HSV-1 pUL34 protein important for viral nuclear egress. Knockdown of any of these vesicle fusion proteins reduces viral titers, further arguing that they are important for nuclear egress. As there appears to be a specific subset of vesicle fusion proteins involved in viral egress, they could possibly represent novel targets for therapeutic interventions.

Download Full-text

Biogenesis of the crystalloid endoplasmic reticulum in UT-1 cells: evidence that newly formed endoplasmic reticulum emerges from the nuclear envelope.

The Journal of Cell Biology ◽

10.1083/jcb.102.6.2158 ◽

1986 ◽

Vol 102 (6) ◽

pp. 2158-2168 ◽

Cited By ~ 83

Author(s):

R K Pathak ◽

K L Luskey ◽

R G Anderson

Keyword(s):

Endoplasmic Reticulum ◽

Nuclear Envelope ◽

Nuclear Membrane ◽

Cholesterol Biosynthesis ◽

Integral Membrane Protein ◽

Enzyme Synthesis ◽

Hmg Coa Reductase ◽

Outer Nuclear Membrane ◽

Sequence Of Events ◽

Coa Reductase

The crystalloid endoplasmic reticulum (ER), a specialized smooth ER of the compactin-resistant UT-1 cell, is composed of multiple membrane tubules packed together in a hexagonal pattern. This membrane contains large amounts of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, an integral membrane protein that enzymatically regulates endogenous cholesterol biosynthesis. Using morphological and immunocytochemical techniques, we have traced the sequence of events in the biogenesis of this ER when compactin-withdrawn UT-1 cells, which do not have a crystalloid ER, are incubated in the presence of compactin. After 15 h of incubation in the presence of compactin, many cells had profiles of ER cisternae that were juxtaposed to the nuclear envelope and studded with ribosomes on their outer membrane. Both the outer nuclear membrane and the ER membrane contained HMG CoA reductase; however, there was little or no detectable enzyme in rough ER that was free in the cytoplasm. With longer times of incubation in the presence of compactin, these cells had lamellar stacks of smooth ER next to the nuclear envelope that contained HMG CoA reductase. Coordinate with the appearance of the smooth ER, crystalloid ER appeared in the same cell. Often regions of continuity were found between the membrane of the smooth ER and the membrane of the crystalloid ER tubules. These studies suggest that HMG CoA reductase is synthesized along the outer nuclear membrane and in response to increased enzyme synthesis, a membrane emerges from the outer nuclear membrane as smooth ER cisternae, which then transforms into crystalloid ER tubules.

Download Full-text

The role of nesprins as multifunctional organizers in the nucleus and the cytoskeleton

Biochemical Society Transactions ◽

10.1042/bst20110668 ◽

2011 ◽

Vol 39 (6) ◽

pp. 1725-1728 ◽

Cited By ~ 7

Author(s):

Angelika A. Noegel ◽

Sascha Neumann

Keyword(s):

Nuclear Envelope ◽

Lipid Bilayers ◽

Nuclear Membrane ◽

Envelope Protein ◽

Membrane System ◽

Outer Nuclear Membrane ◽

Spectrin Repeat ◽

Nuclear Membranes ◽

Repeat Proteins

Nesprins (nuclear envelope spectrin repeat proteins), also known as SYNE (synaptic nuclear envelope protein), MYNE (myocyte nuclear envelope protein), ENAPTIN and NUANCE, are proteins that are primarily components of the nuclear envelope. The nuclear envelope is a continuous membrane system composed of two lipid bilayers: an inner and an outer nuclear membrane. Nesprins are components of both nuclear membranes and reach into the nucleoplasm and the cytoplasm, where they undergo different interactions and have the potential to influence transcriptional processes and cytoskeletal activities.

Download Full-text

The herpes simplex virus 1 Us3 kinase is involved in assembly of membranes needed for viral envelopment and in distribution of glycoprotein K

F1000Research ◽

10.12688/f1000research.19194.1 ◽

2019 ◽

Vol 8 ◽

pp. 727 ◽

Cited By ~ 1

Author(s):

Kurt Tobler ◽

Claudia Senn ◽

Elisabeth M. Schraner ◽

Mathias Ackermann ◽

Cornel Fraefel ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Progeny Virus ◽

Herpes Simplex Virus 1 ◽

Nuclear Membranes ◽

Golgi Membranes ◽

Perinuclear Space ◽

Glycoprotein K ◽

Simplex Virus ◽

Hsv 1

Background:Capsids of herpes simplex virus 1 (HSV-1) are assembled in cell nuclei, released into the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope. Alternatively, capsids gain access to the cytoplasm via dilated nuclear pores. They are enveloped by Golgi membranes. Us3 is a non-essential viral kinase that is involved in nucleus-to-cytoplasm translocation, preventing apoptosis and regulation of phospholipid-biosynthesis. Us3-deletion mutants(HSV-1∆Us3) accumulate in the perinuclear space. Nuclear and Golgi membranes proliferate, and homogeneous, proteinaceous structures of unknown identity are deposited in nuclei and cytoplasm. Glycoprotein K (gK), a highly hydrophobic viral protein, is essential for production of infectious progeny virus but, according to the literature, exclusively vital for envelopment of capsids by Golgi membranes. In the absence of Us3, virions remain stuck in the perinuclear space but mature to infectivity without reaching Golgi membranes, suggesting further function of gK than assumed.Methods:We constructed a HSV-1∆Us3 mutant designated CK177∆Us3gK-HA, in which gK was hemagglutinin (HA) epitope-tagged in order to localize gK by immunolabeling using antibodies against HA for light and electron microscopy.Results:CK177∆Us3gK-HA-infected Vero cells showed similar alterations as those reported for other HSV-1∆Us3, including accumulation of virions in the perinuclear space, overproduction of nuclear and Golgi membranes containing electron dense material with staining property of proteins. Immunolabeling using antibodies against HA revealed that gK is overproduced and localized at nuclear membranes, perinuclear virions stuck in the perinuclear space, Golgi membranes and on protein deposits in cytoplasm and nuclei.Conclusions:Us3 is involved in proper assembly of membranes needed for envelopment and incorporation of gK. Without Us3, virions derived by budding at nuclear membranes remain stuck in the perinuclear space but incorporate gK into their envelope to gain infectivity.

Download Full-text

POM121 and Sun1 play a role in early steps of interphase NPC assembly

The Journal of Cell Biology ◽

10.1083/jcb.201012154 ◽

2011 ◽

Vol 194 (1) ◽

pp. 27-37 ◽

Cited By ~ 90

Author(s):

Jessica A. Talamas ◽

Martin W. Hetzer

Keyword(s):

Cell Cycle ◽

Nuclear Envelope ◽

Nuclear Membrane ◽

Molecular Mechanisms ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Outer Nuclear Membrane ◽

Nuclear Membranes ◽

Distinct Cell ◽

Pore Complexes

Nuclear pore complexes (NPCs) assemble at the end of mitosis during nuclear envelope (NE) reformation and into an intact NE as cells progress through interphase. Although recent studies have shown that NPC formation occurs by two different molecular mechanisms at two distinct cell cycle stages, little is known about the molecular players that mediate the fusion of the outer and inner nuclear membranes to form pores. In this paper, we provide evidence that the transmembrane nucleoporin (Nup), POM121, but not the Nup107–160 complex, is present at new pore assembly sites at a time that coincides with inner nuclear membrane (INM) and outer nuclear membrane (ONM) fusion. Overexpression of POM121 resulted in juxtaposition of the INM and ONM. Additionally, Sun1, an INM protein that is known to interact with the cytoskeleton, was specifically required for interphase assembly and localized with POM121 at forming pores. We propose a model in which POM121 and Sun1 interact transiently to promote early steps of interphase NPC assembly.

Download Full-text

THE FINE STRUCTURAL ORGANISATION OF ROUS TUMOUR CELLS

The Journal of Cell Biology ◽

10.1083/jcb.3.6.851 ◽

1957 ◽

Vol 3 (6) ◽

pp. 851-858 ◽

Cited By ~ 61

Author(s):

M. A. Epstein

Keyword(s):

Endoplasmic Reticulum ◽

Fine Structure ◽

Electron Microscope ◽

Cell Membrane ◽

Nuclear Membrane ◽

Tumour Cells ◽

Thin Sections ◽

Outer Nuclear Membrane ◽

Structural Organisation ◽

Perinuclear Space

The fibroblast-like tumour cells of Rous sarcomata have been studied in thin sections with the electron microscope. A description is given of the fine structure of the cells which includes some features not hitherto recorded. The tightly packed piles of smooth cisternae usually found only in the centrosome region have been observed, in individual Rous cells, in two separate areas of cytoplasm at opposite poles of the nucleus. Continuity between the perinuclear space and the lumen of rough surfaced cisternae of the endoplasmic reticulum has frequently been found; a similar continuity between the cisternae and the exterior of the cell has also been seen. In some cases, the cell membrane has been shown to have an unbroken connection with the outer nuclear membrane through continuity with the limiting membranes of elements of the endoplasmic reticulum. These findings are discussed.

Download Full-text

A Cellular Reaction to Antibody in Tissue Culture Studied with Electron Microscopy

The Journal of Cell Biology ◽

10.1083/jcb.5.3.405 ◽

1959 ◽

Vol 5 (3) ◽

pp. 405-410 ◽

Cited By ~ 25

Author(s):

Harrison Latta

Keyword(s):

Electron Microscopy ◽

Tissue Culture ◽

Endoplasmic Reticulum ◽

Nuclear Membrane ◽

Normal Serum ◽

Butyl Methacrylate ◽

Heart Cells ◽

Plasma Clot ◽

Outer Nuclear Membrane ◽

Cell Components

The reaction of embryonic chick heart cells grown in tissue culture to specific guinea pig antiserum has been studied with electron microscopy. Heart fragments from chick embryos were cultured with a plasma clot. After being tested with antiserum or normal serum, they were fixed with buffered osmium tetroxide and embedded in butyl methacrylate before removal from the glass culture chamber. Thin cells found by phase microscopy to have reacted were sectioned in a plane parallel to the glass surface on which they had grown. The results confirm and extend observations made previously while the reactions were occurring. The plasma membrane, like that of the red cell, becomes disrupted or less resistant to trauma following the action of antiserum. The membranes of mitochondria and endoplasmic reticulum vesiculate and swell. Before nuclear shrinkage becomes prominent, the outer nuclear membrane separates over a large portion of the nuclear envelope and forms one or more large swollen blebs. Thus, the outer nuclear membrane shows a reactivity similar to endoplasmic reticulum. It is suggested that the various physical and chemical changes observed to follow the action of antibody and complement on fibroblasts may be explained by osmotic pressure differences between various cell components. Some basic similarities to the action of hemolytic agents on red cells are noted.

Download Full-text