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Protein & Cell May 2019Many viruses, enveloped or non-enveloped, remodel host membrane structures for their replication, assembly and escape from host cells. Herpesviruses are important human... (Review)
Review
Many viruses, enveloped or non-enveloped, remodel host membrane structures for their replication, assembly and escape from host cells. Herpesviruses are important human pathogens and cause many diseases. As large enveloped DNA viruses, herpesviruses undergo several complex steps to complete their life cycles and produce infectious progenies. Firstly, herpesvirus assembly initiates in the nucleus, producing nucleocapsids that are too large to cross through the nuclear pores. Nascent nucleocapsids instead bud at the inner nuclear membrane to form primary enveloped virions in the perinuclear space followed by fusion of the primary envelopes with the outer nuclear membrane, to translocate the nucleocapsids into the cytoplasm. Secondly, nucleocapsids obtain a series of tegument proteins in the cytoplasm and bud into vesicles derived from host organelles to acquire viral envelopes. The vesicles are then transported to and fuse with the plasma membrane to release the mature virions to the extracellular space. Therefore, at least two budding and fusion events take place at cellular membrane structures during herpesviruses assembly and egress, which induce membrane deformations. In this review, we describe and discuss how herpesviruses exploit and remodel host membrane structures to assemble and escape from the host cell.
Topics: Animals; Herpesviridae; Herpesviridae Infections; Host Microbial Interactions; Humans; Nuclear Envelope; Nucleocapsid; Viral Proteins; Virus Assembly; Virus Release
PubMed: 30242641
DOI: 10.1007/s13238-018-0577-9 -
Microbiology Spectrum Aug 2022Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is responsible for the COVID-19 pandemic that has caused unprecedented loss of life and economic trouble all...
Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is responsible for the COVID-19 pandemic that has caused unprecedented loss of life and economic trouble all over the world, though the mechanism of its replication remains poorly understood. In this study, antibodies were generated and used to systematically determine the expression profile and subcellular distribution of 11 SARS-CoV-2 nonstructural replicase proteins (nsp1, nsp2, nsp3, nsp5, nsp7, nsp8, nsp9, nsp10, nsp13, nsp14, and nsp15) by Western blot and immunofluorescence assay. Nsp3, nsp5, and nsp8 were detected in perinuclear foci at different time points, with diffusion and stronger fluorescence observed over time. In particular, colocalization of nsp8 and nsp13 with different replicase proteins suggested viral protein-protein interaction, which may be key to understanding their functions and potential molecular mechanisms. Viral intermediate dsRNA was detected in perinuclear foci as early as 2-h postinfection, indicating the initiation of virus replication. With the passage of time, these perinuclear dsRNA foci became larger and brighter, and nearly all colocalized with N protein, consistent with viral growth over time. Thus, the development of these anti-nsp antibodies provides basic tools for the further study of replication and diagnosis of SARS-CoV-2. The intracellular localization of SARS-CoV-2 replicase nonstructural proteins (nsp) during infection has not been fully elucidated. In this study, we systematically analyzed the expression and subcellular localization of 11 distinct viral nsp and dsRNA over time in SARS-CoV-2-infected cells by using individual antibody against these replicase proteins. The data indicated that nsp gene expression is highly regulated in space and time, which could be useful to understand the function of viral replicases and future development of diagnostics and potential antiviral strategies against SARS-CoV-2.
Topics: COVID-19; Humans; Open Reading Frames; Pandemics; RNA-Dependent RNA Polymerase; SARS-CoV-2
PubMed: 35730969
DOI: 10.1128/spectrum.00744-22 -
Journal of Virology May 2023Adeno-associated viruses (AAVs) are small, helper-dependent, single-stranded DNA viruses that exploit a broad spectrum of host factors for cell entry. During the course...
Adeno-associated viruses (AAVs) are small, helper-dependent, single-stranded DNA viruses that exploit a broad spectrum of host factors for cell entry. During the course of infection, several AAV serotypes have been shown to transit through the -Golgi network within the host cell. In the current study, we investigated whether the Golgi-localized, calcium-dependent protease furin influences AAV transduction. While CRISPR/Cas9-mediated knockout (KO) of the gene minimally affected the transduction efficiency of most recombinant AAV serotypes tested, we observed a striking increase in transgene expression (~2 log orders) for the African green monkey isolate AAV4. Interrogation of different steps in the infectious pathway revealed that AAV4 binding, uptake, and transcript levels are increased in furin KO cells, but postentry steps such as uncoating or nuclear entry remain unaffected. Recombinant furin does not cleave AAV4 capsid proteins nor alter cellular expression levels of essential factors such as AAVR or GPR108. Interestingly, fluorescent lectin screening revealed a marked increase in 2,3--linked sialoglycan staining on the surface and perinuclear space of furin KO cells. The essential nature of increased sialoglycan expression in furin KO cells in enhancing AAV4 transduction was further corroborated by (i) increased transduction by the closely related isolates AAVrh.32.33 and sea lion AAV and (ii) selective blockade or removal of cellular 2,3--linked sialoglycans by specific lectins or neuraminidase, respectively. Based on the overall findings, we postulate that furin likely plays a key role in regulating expression of cellular sialoglycans, which in turn can influence permissivity to AAVs and possibly other viruses. Adeno-associated viruses (AAVs) are a proven recombinant vector platform for gene therapy and have demonstrated success in the clinic. Continuing to improve our knowledge of AAV-host cell interactions is critical for improving the safety and efficacy. The current study dissects the interplay between furin, a common intracellular protease, and certain cell surface sialoglycans that serve as viral attachment factors for cell entry. Based on the findings, we postulate that differential expression of furin in host cells and tissues is likely to influence gene expression by certain recombinant AAV serotypes.
Topics: Animals; Chlorocebus aethiops; Dependovirus; Virus Internalization; Furin; Genetic Vectors; Capsid Proteins; Transduction, Genetic
PubMed: 37097176
DOI: 10.1128/jvi.00093-23 -
Rheumatology (Oxford, England) Aug 2021To evaluate the prevalence and meaning of antineutrophil cytoplasmic antibodies (ANCA) positivity in a cohort of IgG4-related disease (IgG4-RD).
OBJECTIVES
To evaluate the prevalence and meaning of antineutrophil cytoplasmic antibodies (ANCA) positivity in a cohort of IgG4-related disease (IgG4-RD).
METHODS
We identified patients with ANCA determination from a retrospective cohort of 69 patients with IgG4-RD. ANCA were measured by indirect immunofluorescence microscopy (IIF) and/or proteinase 3 (PR3)-ANCA and MPO-ANCA by ELISA. IIF patterns were classified as perinuclear (P-ANCA), cytoplasmic (C-ANCA) and atypical (X-ANCA). We compared the ANCA-positive vs the ANCA-negative IgG4-RD group.
RESULTS
Out of 69 patients, 31 IgG4-RD patients had an ANCA determination. Four patients with concomitant systemic autoimmune diseases were excluded. We found positive ANCA by IIF in 14 (56%) of 25 patients tested. The most common IIF pattern was C-ANCA in eight (57.1%), followed by dual C-ANCA/X-ANCA in four (28.6%) and P-ANCA and dual C-ANCA/P-ANCA in one each (7.1%). Of the 20 patients with ANCA determination by both IIF and ELISA, four have positive ANCA by ELISA (three for MPO-ANCA and one for PR3-ANCA). Of the two patients with only ELISA determination, one was positive for MPO-ANCA. The prevalence of ANCA positivity by ELISA was 22.7% (5 out of 22 patients). ANCA was more frequent in the Mikulizc/systemic phenotype (42.9%) compared with other phenotypes (P = 0.04). ANCA-positive IgG4-RD patients had more frequently lymph node and kidney involvement, high IgG1 levels and erythrocyte sedimentation rate, and positive antinuclear antibodies.
CONCLUSION
ANCA are found in a significant number of patients with IgG4-RD and differed from the ANCA-negative group in terms of clinical and serological features.
Topics: Adult; Aged; Antibodies, Antineutrophil Cytoplasmic; Aortic Diseases; Biliary Tract Diseases; Case-Control Studies; Female; Humans; Immunoglobulin G4-Related Disease; Kidney Diseases; Lacrimal Apparatus Diseases; Liver Diseases; Lymph Nodes; Male; Microscopy, Fluorescence; Middle Aged; Myeloblastin; Pancreatic Diseases; Peroxidase; Retroperitoneal Space; Retrospective Studies; Salivary Gland Diseases
PubMed: 33547775
DOI: 10.1093/rheumatology/keab124 -
Methods in Enzymology 2019Three-prime Repair Exonuclease (TREX1) degrades ssDNA and dsDNA. TREX1 localizes to the perinuclear space in cells and degrades cytosolic DNA to prevent aberrant nucleic...
Three-prime Repair Exonuclease (TREX1) degrades ssDNA and dsDNA. TREX1 localizes to the perinuclear space in cells and degrades cytosolic DNA to prevent aberrant nucleic acid sensing and immune activation in humans and mice. Mutations in the TREX1 gene cause a spectrum of human autoimmune diseases including Aicardi-Goutières syndrome, familial chilblain lupus, retinal vasculopathy with cerebral leukodystrophy, and are associated with systemic lupus erythematosus. More than 60 disease-causing TREX1 variants have been identified including dominant and recessive, missense, and frameshift mutations that map to the catalytic core region and to the C-terminal cell localization region. The TREX1-disease causing mutations affect exonuclease activity at varied levels. In this chapter, we describe methods to purify variant recombinant TREX1 enzymes and measure the exonuclease activity using ssDNA and dsDNA substrates. The relationships between TREX1 activities, types of TREX1 mutations, and TREX1-associated autoimmune diseases are considered.
Topics: Animals; Autoimmunity; Exodeoxyribonucleases; Humans; Phosphoproteins
PubMed: 31455522
DOI: 10.1016/bs.mie.2019.05.004 -
Nagoya Journal of Medical Science Feb 2017A 65-year-old man was referred to our hospital for the treatment of a lesion on the medial lacrimal canthus of both eyes. He had a history of perinuclear anti-neutrophil...
A 65-year-old man was referred to our hospital for the treatment of a lesion on the medial lacrimal canthus of both eyes. He had a history of perinuclear anti-neutrophil cytoplasmic antibodies, i.e., pANCA-positive interstitial pneumonia. Orbital magnetic resonance imaging excluded space occupying lesions, and laboratory testing excluded thyroid-related diseases. The masses were excised, and histopathological examinations showed sebaceous gland hyperplasia and inflammatory changes around the gland. In addition, the specimen from the left eye showed a retention cyst possibly caused by an infection. It was also possible that the use of steroid was involved in the development of the lesions. A relationship between the ANCA and the lesions was not completely eliminated.
Topics: Aged; Antibodies, Antineutrophil Cytoplasmic; Humans; Inflammation; Lacrimal Apparatus Diseases; Lung Diseases, Interstitial; Male; Sebaceous Glands
PubMed: 28303065
DOI: 10.18999/nagjms.79.1.85 -
F1000Research 2017: Herpesvirus capsids are assembled in the nucleus, translocated to the perinuclear space by budding, acquiring tegument and envelope, or released to the cytoplasm via...
: 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. : 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. : The Golgi complex is a compact entity in a juxtanuclear position covered by a membrane on the face. 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. : 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.
PubMed: 30135710
DOI: 10.12688/f1000research.12252.2 -
Redox Biology Jan 2021Mitochondria are strategically trafficked throughout the cell by the action of microtubule motors, the actin cytoskeleton and adapter proteins. The intracellular...
Mitochondria are strategically trafficked throughout the cell by the action of microtubule motors, the actin cytoskeleton and adapter proteins. The intracellular positioning of mitochondria supports subcellular levels of ATP, Ca and reactive oxygen species (ROS, i.e. hydrogen peroxide, HO). Previous work from our group showed that deletion of the mitochondrial adapter protein Miro1 leads to perinuclear clustering of mitochondria, leaving the cell periphery devoid of mitochondria which compromises peripheral energy status. Herein, we report that deletion of Miro1 significantly restricts subcellular HO levels to the perinuclear space which directly affects intracellular responses to elevated mitochondrial ROS. Using the genetically encoded HO-responsive fluorescent biosensor HyPer7, we show that the highest levels of subcellular HO map to sites of increased mitochondrial density. Deletion of Miro1 or disruption of microtubule dynamics with Taxol significantly reduces peripheral HO levels. Following inhibition of mitochondrial complex 1 with rotenone we observe elevated spikes of HO in the cell periphery and complementary oxidation of mitochondrial peroxiredoxin 3 (PRX3) and cytosolic peroxiredoxin 2 (PRX2). Conversely, in cells lacking Miro1, rotenone did not increase peripheral HO or PRX2 oxidation but rather lead to increased nuclear HO and an elevated DNA-damage response. Lastly, local levels of HyPer7 oxidation correlate with the size and abundance of focal adhesions (FAs) in MEFs and cells lacking Miro1 have significantly smaller focal adhesions and reduced phosphorylation levels of vinculin and p130Cas compared to Miro1 MEFs. Together, we present evidence that the intracellular distribution of mitochondria influences subcellular HO levels and local cellular responses dependent on mitochondrial ROS.
Topics: Hydrogen Peroxide; Mitochondria; Mitochondrial Proteins; Oxidation-Reduction; Reactive Oxygen Species
PubMed: 33341544
DOI: 10.1016/j.redox.2020.101818 -
Cell Calcium Mar 2020Numerous lines of evidence indicate that nuclear calcium concentration ([Ca]) may be controlled independently from cytosolic events by a local machinery. In particular,... (Review)
Review
Numerous lines of evidence indicate that nuclear calcium concentration ([Ca]) may be controlled independently from cytosolic events by a local machinery. In particular, the perinuclear space between the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) of the nuclear envelope (NE) likely serves as an intracellular store for Ca ions. Since ONM is contiguous with the endoplasmic reticulum (ER), the perinuclear space is adjacent to the lumen of ER thus allowing a direct exchange of ions and factors between the two organelles. Moreover, INM and ONM are fused at the nuclear pore complex (NPC), which provides the only direct passageway between the nucleoplasm and cytoplasm. However, due to the presence of ion channels, exchangers and transporters, it has been generally accepted that nuclear ion fluxes may occur across ONM and INM. Within the INM, the Na/Ca exchanger (NCX) isoform 1 seems to play an important role in handling Ca through the different nuclear compartments. Particularly, nuclear NCX preferentially allows local Ca flowing from nucleoplasm into NE lumen thanks to the Na gradient created by the juxtaposed Na/K-ATPase. Such transfer reduces abnormal elevation of [Ca] within the nucleoplasm thus modulating specific transductional pathways and providing a protective mechanism against cell death. Despite very few studies on this issue, here we discuss those making major contribution to the field, also addressing the pathophysiological implication of nuclear NCX malfunction.
Topics: Animals; Calcium; Calcium Signaling; Cell Nucleus; Disease; Humans; Models, Biological; Sodium-Calcium Exchanger
PubMed: 31865040
DOI: 10.1016/j.ceca.2019.102143 -
Frontiers in Endocrinology 2021Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants that have become globally ubiquitous in humans and the environment. PFAS exposure is...
BACKGROUND
Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants that have become globally ubiquitous in humans and the environment. PFAS exposure is associated with neurodevelopmental effects; however, the mechanism is poorly understood. Brain-derived neurotrophic factor (BDNF) signaling is critical to fetal neurodevelopment during pregnancy and maintains important regulatory roles later in life. This study aims to characterize placental BDNF signaling and investigate whether PFAS exposure disrupts the signaling pathway in placental trophoblast cells.
METHODS
The expression and localization of BDNF receptors-p75 and TrkB-in first trimester and term human placentas and trophoblast cells were investigated by immunofluorescence staining. To assess the effects of PFAS exposure on the BDNF pathway, BeWo cells were treated with PFAS mixtures that mimicked blood levels in a highly exposed population and major PFAS compounds in the mixture at 0.01, 0.1, 1, and 10 µM concentrations. Changes in pro-BDNF levels and phosphorylation of TrkB receptors were examined by Western blot.
RESULTS
In first trimester human placentas, TrkB and p75 receptors were primarily localized to syncytiotrophoblast and cytotrophoblast cells. At term, TrkB and p75 receptors were primarily observed in the placental villous stroma. TrkB receptor staining in trophoblasts was reduced at term, while p75 receptor staining was negative. TrkB receptors were confined to the nuclear and perinuclear spaces, and phosphorylation occurred at the Tyr816 residue in BeWo cells. Exposure to PFOS, PFOA, PFBS, and the six-PFAS mixture did not significantly affect BDNF levels or activation (phosphorylation) of TrkB. Treating cells with 1 μM and 10 μM of PFNA resulted in increased TrkB phosphorylation compared to unexposed controls, but BDNF levels were unchanged.
CONCLUSIONS
BDNF receptors are present in different regions of human placental villi, indicating diverse functions of BDNF signaling in placental development. Our findings suggest that the BDNF pathway in placental trophoblast cells is not disrupted by exposures to PFOS, PFOA, PFBS, and a PFAS mixture, but may be affected by PFNA exposures. Further investigation is needed on how PFAS affects other critical signaling pathways during fetal neurodevelopment.
Topics: Brain-Derived Neurotrophic Factor; Female; Fluorocarbons; Humans; Maternal Exposure; Membrane Glycoproteins; Phosphorylation; Placenta; Placentation; Pregnancy; Pregnancy Trimester, First; Receptor, trkB; Signal Transduction; Trophoblasts; Tumor Cells, Cultured
PubMed: 34394001
DOI: 10.3389/fendo.2021.694885