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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 -
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 -
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 -
Nucleus (Austin, Tex.) Dec 2019LINC complexes (Linker of Nucleoskeleton and Cytoskeleton), consisting of inner nuclear membrane SUN (Sad1, UNC-84) proteins and outer nuclear membrane KASH (Klarsicht,... (Review)
Review
LINC complexes (Linker of Nucleoskeleton and Cytoskeleton), consisting of inner nuclear membrane SUN (Sad1, UNC-84) proteins and outer nuclear membrane KASH (Klarsicht, ANC-1, and Syne Homology) proteins, are essential for nuclear positioning, cell migration and chromosome dynamics. To test the in vivo functions of conserved interfaces revealed by crystal structures, Cain et al used a combination of Caenorhabditis elegans genetics, imaging in cultured NIH 3T3 fibroblasts, and Molecular Dynamic simulations, to study SUN-KASH interactions. Conserved aromatic residues at the -7 position of the C-termini of KASH proteins and conserved disulfide bonds in LINC complexes play important roles in force transmission across the nuclear envelope. Other properties of LINC complexes, such as the helices preceding the SUN domain, the longer coiled-coils spanning the perinuclear space and higher-order organization may also function to transmit mechanical forces generated by the cytoskeleton across the nuclear envelope.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Cycle Proteins; Mice; NIH 3T3 Cells; Nuclear Envelope; Nuclear Proteins; Receptors, Cytoplasmic and Nuclear
PubMed: 30888237
DOI: 10.1080/19491034.2019.1595313