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F1000Research 2019Capsids 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...
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. 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. 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. 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.
Topics: Animals; Chlorocebus aethiops; Glycoproteins; Herpesvirus 1, Human; Vero Cells; Viral Proteins; Virion
PubMed: 31448105
DOI: 10.12688/f1000research.19194.1 -
The Journal of Clinical Investigation Aug 2019Deciphering novel pathways regulating liver lipid content has profound implications for understanding the pathophysiology of nonalcoholic fatty liver disease and...
Deciphering novel pathways regulating liver lipid content has profound implications for understanding the pathophysiology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Recent evidence suggests that the nuclear envelope is a site of regulation of lipid metabolism but there is limited appreciation of the responsible mechanisms and molecular components within this organelle. We showed that conditional hepatocyte deletion of the inner nuclear membrane protein lamina-associated polypeptide 1 (LAP1) caused defective VLDL secretion and steatosis, including intranuclear lipid accumulation. LAP1 binds to and activates torsinA, an AAA+ ATPase that resides in the perinuclear space and continuous main ER. Deletion of torsinA from mouse hepatocytes caused even greater reductions in VLDL secretion and profound steatosis. Both of these mutant mouse lines developed hepatic steatosis and subsequent steatohepatitis on a regular chow diet in the absence of whole-body insulin resistance or obesity. Our results establish an essential role for the nuclear envelope-localized torsinA-LAP1 complex in hepatic VLDL secretion and suggest that the torsinA pathway participates in the pathophysiology of nonalcoholic fatty liver disease.
Topics: Animals; Carrier Proteins; Hepatocytes; Lipid Metabolism; Lipoproteins, VLDL; Membrane Proteins; Mice; Mice, Knockout; Molecular Chaperones; Non-alcoholic Fatty Liver Disease; Nuclear Envelope
PubMed: 31408437
DOI: 10.1172/JCI129769 -
Journal of Virology Nov 2019During nuclear egress of nascent progeny herpesvirus nucleocapsids, the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane of...
During nuclear egress of nascent progeny herpesvirus nucleocapsids, the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane of infected cells into the perinuclear space between the inner and outer nuclear membranes. Herpes simplex virus 1 (HSV-1) U34 and U31 proteins form a nuclear egress complex (NEC) and play critical roles in this budding process, designated primary envelopment. To clarify the role of NEC binding to progeny nucleocapsids in HSV-1 primary envelopment, we established an assay system for HSV-1 NEC binding to nucleocapsids and capsid proteins Using this assay system, we showed that HSV-1 NEC bound to nucleocapsids and to capsid protein U25 but not to the other capsid proteins tested (i.e., VP5, VP23, and U17) and that HSV-1 NEC binding of nucleocapsids was mediated by the interaction of NEC with U25. U31 residues arginine-281 (R281) and aspartic acid-282 (D282) were required for efficient NEC binding to nucleocapsids and U25. We also showed that alanine substitution of U31 R281 and D282 reduced HSV-1 replication, caused aberrant accumulation of capsids in the nucleus, and induced an accumulation of empty vesicles that were similar in size and morphology to primary envelopes in the perinuclear space. These results suggested that NEC binding via U31 R281 and D282 to nucleocapsids, and probably to U25 in the nucleocapsids, has an important role in HSV-1 replication by promoting the incorporation of nucleocapsids into vesicles during primary envelopment. Binding of HSV-1 NEC to nucleocapsids has been thought to promote nucleocapsid budding at the inner nuclear membrane and subsequent incorporation of nucleocapsids into vesicles during nuclear egress of nucleocapsids. However, data to directly support this hypothesis have not been reported thus far. In this study, we have present data showing that two amino acids in the membrane-distal face of the HSV-1 NEC, which contains the putative capsid binding site based on the solved NEC structure, were in fact required for efficient NEC binding to nucleocapsids and for efficient incorporation of nucleocapsids into vesicles during primary envelopment. This is the first report showing direct linkage between NEC binding to nucleocapsids and an increase in nucleocapsid incorporation into vesicles during herpesvirus primary envelopment.
Topics: Active Transport, Cell Nucleus; Binding Sites; Capsid Proteins; Cell Nucleus; Herpes Simplex; Herpesvirus 1, Human; Humans; Nuclear Proteins; Nucleocapsid; Protein Binding; Viral Proteins; Virion; Virus Assembly; Virus Release
PubMed: 31391274
DOI: 10.1128/JVI.01290-19 -
Autophagy May 2020In the adult mammalian skin, cells are constantly renewing, differentiating and moving upward, to finally die in a yet not fully understood manner. Here, we provide...
In the adult mammalian skin, cells are constantly renewing, differentiating and moving upward, to finally die in a yet not fully understood manner. Here, we provide evidence that macroautophagy/autophagy has a dual role in the skin. In addition to its known catabolic protective role as an evolutionary conserved upstream regulator of lysosomal degradation, we show that autophagy induced cell death (CDA) occurs in epithelial lineage-derived organs, such as the inter-follicular epidermis, the sebaceous- and the Harderian gland. By utilizing GFP-LC3 transgenic and ATG7-deficient mice, we show that CDA is initiated during terminal differentiation at a stage when the cells have become highly resistant to apoptosis. In these transitional cells, the Golgi compartment expands, which accounts for the formation of primary lysosomes, and the nucleus starts to condense. During CDA a burst of autophagosome formation is observed, first the endoplasmic reticulum (ER) is phagocytosed followed by autophagy of the nucleus. By this selective form of cell death, most of the cytoplasmic organelles are degraded, but structural proteins remain intact. In the absence of autophagy, consequently, parts of the ER, ribosomes, and chromatin remain. A burst of autophagy was stochastically observed in single cells of the epidermis and collectively in larger areas of ductal cells, arguing for a coordinated induction. We conclude that autophagy is an integral part of cell death in keratinocyte lineage cells and participates in their terminal cell fate. Atg7: autophagy related 7; BECN1: beclin 1; CDA: cell death-induced autophagy; Cre: Cre-recombinase; DAPI: 4',6-diamidino-2-phenylindole; ER: endoplasmatic reticulum; GFP: green fluorescent protein; HaGl: haderian gland; IVL: involucrin; KRT14: keratin 14; LD: lipid droplet; LSM: laser scanning microscope; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PN: perinuclear space; RB: residual body; rER: rough endoplasmatic reticulum; SB: sebum; SG-SC: stratum granulosum - stratum corneum; SGl: sebaceous gland; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling.
Topics: Animals; Apoptosis; Autophagosomes; Autophagy; Cell Differentiation; Epithelial Cells; Lysosomes; Mice, Transgenic; Skin
PubMed: 31379249
DOI: 10.1080/15548627.2019.1646552 -
F1000Research 2019: 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...
: 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. : 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. : 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. : The data indicate that Us3 kinase is involved in facilitation of nuclear pore impairment and, concomitantly, in capsid release through impaired nuclear envelope.
Topics: Capsid; Herpes Simplex; Herpesvirus 1, Human; Humans; Nuclear Envelope; Protein Serine-Threonine Kinases; Viral Proteins
PubMed: 31249678
DOI: 10.12688/f1000research.17802.1 -
Journal of Virology Sep 2019Varicella-zoster virus (VZV) is an alphaherpesvirus that lacks the herpesviral neurovirulence protein ICP34.5. The underlying hypothesis of this project was that...
Varicella-zoster virus (VZV) is an alphaherpesvirus that lacks the herpesviral neurovirulence protein ICP34.5. The underlying hypothesis of this project was that inhibitors of autophagy reduce VZV infectivity. We selected the vacuolar proton ATPase inhibitor bafilomycin A1 for analysis because of its well-known antiautophagy property of impeding acidification during the late stage of autophagic flux. We documented that bafilomycin treatment from 48 to 72 h postinfection lowered VZV titers substantially ( ≤ 0.008). Because we were unable to define the site of the block in the infectious cycle by confocal microscopy, we turned to electron microscopy. Capsids were observed in the nucleus, in the perinuclear space, and in the cytoplasm adjacent to Golgi apparatus vesicles. Many of the capsids had an aberrant appearance, as has been observed previously in infections not treated with bafilomycin. In contrast to prior untreated infections, however, secondary envelopment of capsids was not seen in the -Golgi network, nor were prototypical enveloped particles with capsids (virions) seen in cytoplasmic vesicles after bafilomycin treatment. Instead, multiple particles with varying diameters without capsids (light particles) were seen in large virus assembly compartments near the disorganized Golgi apparatus. Bafilomycin treatment also led to increased numbers of multivesicular bodies in the cytoplasm, some of which contained remnants of the Golgi apparatus. In summary, we have defined a previously unrecognized property of bafilomycin whereby it disrupted the site of secondary envelopment of VZV capsids by altering the pH of the -Golgi network and thereby preventing the correct formation of virus assembly compartments. This study of VZV assembly in the presence of bafilomycin A1 emphasizes the importance of the Golgi apparatus/-Golgi network as a platform in the alphaherpesvirus life cycle. We have previously shown that VZV induces levels of autophagy far above the basal levels of autophagy in human skin, a major site of VZV assembly. The current study documented that bafilomycin treatment led to impaired assembly of VZV capsids after primary envelopment/de-envelopment but before secondary reenvelopment. This VZV study also complemented prior herpes simplex virus 1 and pseudorabies virus studies investigating two other inhibitors of endoplasmic reticulum (ER)/Golgi apparatus function: brefeldin A and monensin. Studies with porcine herpesvirus demonstrated that primary enveloped particles accumulated in the perinuclear space in the presence of brefeldin A, while studies with herpes simplex virus 1 documented an impaired secondary assembly of enveloped viral particles in the presence of monensin.
Topics: Autophagy; Capsid; Cell Line; Cell Nucleus; Cytoplasm; Herpesvirus 3, Human; Humans; Macrolides; Microscopy, Electron; Varicella Zoster Virus Infection; Viral Load; Virulence; Virus Assembly; trans-Golgi Network
PubMed: 31217243
DOI: 10.1128/JVI.00505-19 -
Redox Biology Jun 2019Mountainous evidence suggests that inflammation, cardiomyocyte apoptosis and pyroptosis are involved in the development of sepsis and sepsis-induced cardiomyopathy...
Mountainous evidence suggests that inflammation, cardiomyocyte apoptosis and pyroptosis are involved in the development of sepsis and sepsis-induced cardiomyopathy (SIC). Stimulator of interferon genes (STING) is an indispensable molecule that could regulate inflammation and immune response in multiple diseases. However, the role of STING in cardiovascular disease, especially SIC remains unclear. This study was designed to investigate the potential molecular mechanisms of STING in lipopolysaccharide (LPS)-induced cardiac injury using STING global knockout mice. In wild type mice and cardiomyocytes, LPS stimulation triggered the perinuclear translocation of STING, which further bound to Type-I interferons (IFN) regulatory factor 3 (IRF3) and phosphorylated IRF3. Phosphorylated (P-) IRF3 subsequently translocated into nucleus and increased the expression of NOD-like receptor protein 3 (NLRP3). Knockout of STING in mice significantly improved survival rate and cardiac function, apart from suppressing myocardial and serum inflammatory cytokines, apoptosis, as well as cardiomyocyte pyroptosis. In vitro experiments revealed that NLRP3 overexpression by adenovirus could offset protective effects of STING knockdown in LPS-induced cardiomyocytes. Additionally, LPS stimulation also promoted the production of intracellular reactive oxygen (ROS), which further induced the NLRP3 translocation to the cytoplasm from the nucleus. Dissociative TXNIP could directly interact with cytoplasmic NLRP3 and form inflammasome, eventually triggering cardiomyocyte injury. Collectively, our findings disclose that STING deficiency could alleviate LPS-induced SIC in mice. Hence, targeting STING in cardiomyocytes may be a promising therapeutic strategy for preventing SIC.
Topics: Animals; Apoptosis; Cytokines; Disease Models, Animal; Echocardiography; Heart Diseases; Interferon Regulatory Factor-3; Intracellular Space; Lipopolysaccharides; Male; Membrane Proteins; Mice; Myocytes, Cardiac; NLR Family, Pyrin Domain-Containing 3 Protein; Pyroptosis; Reactive Oxygen Species
PubMed: 31121492
DOI: 10.1016/j.redox.2019.101215 -
Cell Stress & Chaperones Jul 2019Skeletal myogenesis is a coordinated sequence of events associated with dramatic changes in cell morphology, motility, and metabolism, which causes cellular stress and...
Skeletal myogenesis is a coordinated sequence of events associated with dramatic changes in cell morphology, motility, and metabolism, which causes cellular stress and alters proteostasis. Chaperones, such as heat-shock proteins (HSPs), play important roles in limiting cellular stresses and maintaining proteostasis, but whether HSPs are specifically involved in myogenesis is not well understood. Here, we characterized gene and protein expression and subcellular localization of various HSPs in proliferating C2C12 myoblasts and differentiating myotubes under control conditions and in response to heat stress. Hsp25, Hsp40, and Hsp60 protein expression declined by 48, 35, and 83%, respectively, during differentiation. In contrast, Hsp70 protein levels doubled during early differentiation. Hsp25 was predominantly localized to the cytoplasm of myoblasts and myotubes but formed distinct aggregates in perinuclear spaces of myoblasts after heat-shock. Hsp40 was distributed diffusely throughout the cytoplasm and nucleus and, after heat-shock, translocated to the nucleus of myoblasts but formed aggregates in myotubes. Hsp60 localized to the perinuclear space in myoblasts but was distributed more diffusely across the cytoplasm in myotubes. Hsp70 was expressed diffusely throughout the cytoplasm and nucleus and translocated to the nucleus after heat-shock in myoblasts, but not in myotubes. Hsp90 was expressed diffusely across the cytoplasm in both myoblasts and myotubes under control conditions and did not change in response to heat-shock. These findings reveal distinct and different roles for HSPs in the regulation of myogenic cell proliferation and differentiation.
Topics: Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Heat-Shock Proteins; Heat-Shock Response; Muscle Development; Muscle Fibers, Skeletal; Myoblasts
PubMed: 31098840
DOI: 10.1007/s12192-019-01001-2 -
Journal of Virology Jul 2019During the nuclear export of nascent nucleocapsids of herpes simplex virus 1 (HSV-1), the nucleocapsids acquire a primary envelope by budding through the inner nuclear...
During the nuclear export of nascent nucleocapsids of herpes simplex virus 1 (HSV-1), the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes. This unique budding process, termed primary envelopment, is initiated by the nuclear egress complex (NEC), composed of the HSV-1 UL31 and UL34 proteins. Earlier biochemical approaches have shown that the NEC has an intrinsic ability to vesiculate membranes through the formation of a hexagonal lattice structure. The significance of intrahexamer interactions of the NEC in the primary envelopment of HSV-1-infected cells has been reported. In contrast, the contribution of lattice formation of the NEC hexamer to primary envelopment in HSV-1-infected cells remains to be elucidated. Therefore, we constructed and characterized a recombinant HSV-1 strain carrying an amino acid substitution in a UL31 residue that is an interhexamer contact site for the lattice formation of the NEC hexamer. This mutation was reported to destabilize the interhexamer interactions of the HSV-1 NEC. Here, we demonstrate that the mutation causes the aberrant accumulation of nucleocapsids in the nucleus and reduces viral replication in Vero and HeLa cells. Thus, the ability of HSV-1 to form the hexagonal lattice structure of the NEC was linked to an increase in primary envelopment and viral replication. Our results suggest that the lattice formation of the NEC hexamer has an important role in HSV-1 replication by regulating primary envelopment. The scaffolding proteins of several envelope viruses required for virion assembly form high-order lattice structures. However, information on the significance of their lattice formation in infected cells is limited. Herpesviruses acquire envelopes twice during their viral replication. The first envelop acquisition (primary envelopment) is one of the steps in the vesicle-mediated nucleocytoplasmic transport of nascent nucleocapsids, which is unique in biology. HSV-1 NEC, thought to be conserved in all members of the family, is critical for primary envelopment and was shown to form a hexagonal lattice structure. Here, we investigated the significance of the interhexamer contact site for hexagonal lattice formation of the NEC in HSV-1-infected cells and present evidence suggesting that the lattice formation of the NEC hexamer has an important role in HSV-1 replication by regulating primary envelopment. Our results provide insights into the mechanisms of the envelopment of herpesviruses and other envelope viruses.
Topics: Animals; Cell Nucleus; Chlorocebus aethiops; HeLa Cells; Herpes Simplex; Herpesvirus 1, Human; Humans; Multiprotein Complexes; Nuclear Proteins; Rabbits; Vero Cells; Viral Proteins; Virus Replication
PubMed: 31043535
DOI: 10.1128/JVI.00498-19 -
Biology of Reproduction Jun 2019The perinuclear theca (PT) is a cytosolic protein capsule that surrounds the nucleus of eutherian spermatozoa. Compositionally, it is divided into two regions: the...
The perinuclear theca (PT) is a cytosolic protein capsule that surrounds the nucleus of eutherian spermatozoa. Compositionally, it is divided into two regions: the subacrosomal layer (SAL) and the postacrosomal sheath (PAS). In falciform spermatozoa, a third region of the PT emerges that extends beyond the nuclear apex called the perforatorium. The formation of the SAL and PAS differs, with the former assembling early in spermiogenesis concomitant with acrosome formation, and the latter dependent on manchette descent during spermatid elongation. The perforatorium also forms during the elongation phase of spermiogenesis, suggesting that like the PAS, its assembly is facilitated by the manchette. The temporal similarity in biogenesis between the PAS and perforatorium led us to compare their molecular composition using cell fractionation and immunodetection techniques. Although the perforatorium is predominantly composed of its endemic protein FABP9/PERF15, immunolocalization indicates that it also shares proteins with the PAS. These include WBP2NL/PAWP, WBP2, GSTO2, and core histones, which have been implicated in early fertilization and zygotic events. The compositional homogeny between the PAS and perforatorium supports our observation that their development is linked. Immunocytochemistry indicates that both PAS and perforatorial biogenesis depend on the transport and deposition of cytosolic proteins by the microtubular manchette. Proteins translocated from the manchette pass ventrally along the spermatid head into the apical perforatorial space prior to PAS deposition in the wake of manchette descent. Our findings demonstrate that the perforatorium and PAS share a mechanism of developmental assembly and thereby contain common proteins that facilitate fertilization.
Topics: Acrosome; Animals; Cattle; Cytoskeleton; Humans; Male; Proteins; Rats; Rats, Sprague-Dawley; Semen Analysis; Sperm Head; Spermatogenesis; Spermatozoa; Swine
PubMed: 30939204
DOI: 10.1093/biolre/ioz052