scholarly journals Herpesvirus Nuclear Egress across the Outer Nuclear Membrane

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2356
Author(s):  
Richard J. Roller ◽  
David C. Johnson
Keyword(s):  
Membrane Fusion ◽  
Nuclear Membrane ◽  
Membrane Curvature ◽  
Fusion Pore ◽  
Second Step ◽  
Second Phase ◽  
Viral Fusion ◽  
Outer Nuclear Membrane ◽  
Viral Fusion Protein ◽  
Nuclear Egress

Herpesvirus capsids are assembled in the nucleus and undergo a two-step process to cross the nuclear envelope. Capsids bud into the inner nuclear membrane (INM) aided by the nuclear egress complex (NEC) proteins UL31/34. At that stage of egress, enveloped virions are found for a short time in the perinuclear space. In the second step of nuclear egress, perinuclear enveloped virions (PEVs) fuse with the outer nuclear membrane (ONM) delivering capsids into the cytoplasm. Once in the cytoplasm, capsids undergo re-envelopment in the Golgi/trans-Golgi apparatus producing mature virions. This second step of nuclear egress is known as de-envelopment and is the focus of this review. Compared with herpesvirus envelopment at the INM, much less is known about de-envelopment. We propose a model in which de-envelopment involves two phases: (i) fusion of the PEV membrane with the ONM and (ii) expansion of the fusion pore leading to release of the viral capsid into the cytoplasm. The first phase of de-envelopment, membrane fusion, involves four herpes simplex virus (HSV) proteins: gB, gH/gL, gK and UL20. gB is the viral fusion protein and appears to act to perturb membranes and promote fusion. gH/gL may also have similar properties and appears to be able to act in de-envelopment without gB. gK and UL20 negatively regulate these fusion proteins. In the second phase of de-envelopment (pore expansion and capsid release), an alpha-herpesvirus protein kinase, US3, acts to phosphorylate NEC proteins, which normally produce membrane curvature during envelopment. Phosphorylation of NEC proteins reverses tight membrane curvature, causing expansion of the membrane fusion pore and promoting release of capsids into the cytoplasm.

scholarly journals Reevaluating Herpes Simplex Virus Hemifusion

2010 ◽  
Vol 84 (22) ◽  
pp. 11814-11821 ◽  
Author(s):  
Julia O. Jackson ◽  
Richard Longnecker
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Fusion Pore ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Viral Fusion Proteins ◽  
Simplex Virus

ABSTRACT Membrane fusion induced by enveloped viruses proceeds through the actions of viral fusion proteins. Once activated, viral fusion proteins undergo large protein conformational changes to execute membrane fusion. Fusion is thought to proceed through a “hemifusion” intermediate in which the outer membrane leaflets of target and viral membranes mix (lipid mixing) prior to fusion pore formation, enlargement, and completion of fusion. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins—glycoprotein D (gD), glycoprotein B (gB), and a heterodimer of glycoprotein H and L (gH/gL)—to accomplish fusion. gD is primarily thought of as a receptor-binding protein and gB as a fusion protein. The role of gH/gL in fusion has remained enigmatic. Despite experimental evidence that gH/gL may be a fusion protein capable of inducing hemifusion in the absence of gB, the recently solved crystal structure of HSV-2 gH/gL has no structural homology to any known viral fusion protein. We found that in our hands, all HSV entry proteins—gD, gB, and gH/gL—were required to observe lipid mixing in both cell-cell- and virus-cell-based hemifusion assays. To verify that our hemifusion assay was capable of detecting hemifusion, we used glycosylphosphatidylinositol (GPI)-linked hemagglutinin (HA), a variant of the influenza virus fusion protein, HA, known to stall the fusion process before productive fusion pores are formed. Additionally, we found that a mutant carrying an insertion within the short gH cytoplasmic tail, 824L gH, is incapable of executing hemifusion despite normal cell surface expression. Collectively, our findings suggest that HSV gH/gL may not function as a fusion protein and that all HSV entry glycoproteins are required for both hemifusion and fusion. The previously described gH 824L mutation blocks gH/gL function prior to HSV-induced lipid mixing.

scholarly journals Critical Residues and Contacts within Domain IV of Autographa californica Multiple Nucleopolyhedrovirus GP64 Contribute to Its Refolding during Membrane Fusion

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
Qianlong Yu ◽  
Lisha Bai ◽  
Ning Ji ◽  
Xiaorong Yue ◽  
Yuanyuan Jiang ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Fusion Pore ◽  
Viral Fusion ◽  
Class Iii ◽  
Viral Fusion Protein ◽  
Domain I ◽  
Autographa Californica Multiple Nucleopolyhedrovirus ◽  
Pore Expansion

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 is a class III viral fusion protein that mediates low-pH-triggered membrane fusion during virus entry. Although the structure of GP64 in a postfusion conformation has been solved, its prefusion structure and the mechanism of how the protein refolds to execute fusion are unknown. In its postfusion structure, GP64 is composed of five domains (domains I to V). Domain IV (amino acids [aa] 374 to 407) contains two loops (loop 1 and loop 2) that form a hydrophobic pocket at the membrane-distal end of the molecule. To determine the roles of domain IV, we used alanine-scanning mutagenesis to replace each of the individual residues and the contact-forming residues within domain IV and evaluate their contributions to GP64-mediated membrane fusion and virus infection. In many cases, replacement of a single amino acid had no significant impact on GP64. However, replacement of R392 or disruption of the N381-N385, N384-Y388, N385-W393, or K389-W393 contact resulted in poor cell surface expression and fusion loss of the modified GP64, whereas replacement of E390 or G391 or disruption of the N381-K389, N381-Q401, or N381-I403 contact reduced the cell surface expression level of the constructs and the ability of GP64 to mediate fusion pore expansion. In contrast, replacement of N407 or disruption of contact D404-S406 appeared to restrict fusion pore expansion without affecting expression. Combined with the finding that these constructs remain in the prefusion conformation or have a dramatically less efficient transition from the prefusion to the postfusion state under acidic conditions, we proposed that domain IV is necessary for refolding of GP64 during membrane fusion. IMPORTANCE Baculovirus GP64 is grouped with rhabdovirus G, herpesvirus gB, and thogotovirus glycoproteins as a class III viral fusion protein. In their postfusion structures, these proteins contain five domains (domains I to V). Distinct from domain IV of rhabdovirus G and herpesvirus gB proteins, which is composed of β-sheets, domain IV of GP64 is a loop region; the same domain in thogotovirus glycoproteins has not been solved. In addition, domain IV is proximal to domain I (fusion domain) in prefusion structures of vesicular stomatitis virus (VSV) G and human cytomegalovirus (HCMV) gB but resides at the domain I-distal end of the molecule in a postfusion conformation. In this study, we identified that highly conserved residues and contacts within domain IV of AcMNPV GP64 are necessary for low-pH-triggered conformational change and fusion pore expansion. Our results highlight the roles of domain IV of class III viral fusion proteins in refolding during membrane fusion.

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

2017 ◽  
Vol 91 (19) ◽  
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.

scholarly journals Role of Metastability and Acidic pH in Membrane Fusion by Tick-Borne Encephalitis Virus

2001 ◽  
Vol 75 (16) ◽  
pp. 7392-7398 ◽  
Author(s):  
Karin Stiasny ◽  
Steven L. Allison ◽  
Christian W. Mandl ◽  
Franz X. Heinz
Keyword(s):  
Influenza Virus ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Low Ph ◽  
Class I ◽  
Acidic Ph ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Tick Borne Encephalitis ◽  
Tbe Virus

ABSTRACT The envelope protein E of the flavivirus tick-borne encephalitis (TBE) virus is, like the alphavirus E1 protein, a class II viral fusion protein that differs structurally and probably mechanistically from class I viral fusion proteins. The surface of the native TBE virion is covered by an icosahedrally symmetrical network of E homodimers, which mediate low-pH-induced fusion in endosomes. At the pH of fusion, the E homodimers are irreversibly converted to a homotrimeric form, which we have found by intrinsic fluorescence measurements to be more stable than the native dimers. Thus, the TBE virus E protein is analogous to the prototypical class I fusion protein, the influenza virus hemagglutinin (HA), in that it is initially synthesized in a metastable state that is energetically poised to be converted to the fusogenic state by exposure to low pH. However, in contrast to what has been observed with influenza virus HA, this transition could not be triggered by input of heat energy alone and membrane fusion could be induced only when the virus was exposed to an acidic pH. In a previous study we showed that the dimer-to-trimer transition appears to be a two-step process involving a reversible dissociation of the dimer followed by an irreversible trimerization of the dissociated monomeric subunits. Because the dimer-monomer equilibrium in the first step apparently depends on the protonation state of E, the lack of availability of monomers for the trimerization step at neutral pH could explain why low pH is essential for fusion in spite of the metastability of the native E dimer.

scholarly journals Unusual Topological Arrangement of Structural Motifs in the Baboon Reovirus Fusion-Associated Small Transmembrane Protein

2005 ◽  
Vol 79 (10) ◽  
pp. 6216-6226 ◽  
Author(s):  
Sandra Dawe ◽  
Jennifer A. Corcoran ◽  
Eileen K. Clancy ◽  
Jayme Salsman ◽  
Roy Duncan
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Transmembrane Protein ◽  
Topological Analysis ◽  
Viral Fusion ◽  
Structural Motifs ◽  
Membrane Fusion Protein ◽  
Viral Fusion Protein ◽  
Fast Proteins ◽  
Tm Domain

ABSTRACT Select members of the Reoviridae are the only nonenveloped viruses known to induce syncytium formation. The fusogenic orthoreoviruses accomplish cell-cell fusion through a distinct class of membrane fusion-inducing proteins referred to as the fusion-associated small transmembrane (FAST) proteins. The p15 membrane fusion protein of baboon reovirus is unique among the FAST proteins in that it contains two hydrophobic regions (H1 and H2) recognized as potential transmembrane (TM) domains, suggesting a polytopic topology. However, detailed topological analysis of p15 indicated only the H1 domain is membrane spanning. In the absence of an N-terminal signal peptide, the H1 TM domain serves as a reverse signal-anchor to direct p15 membrane insertion and a bitopic Nexoplasmic/Ccytoplasmic topology. This topology results in the translocation of the smallest ectodomain (∼20 residues) of any known viral fusion protein, with the majority of p15 positioned on the cytosolic side of the membrane. Mutagenic analysis indicated the unusual presence of an N-terminal myristic acid on the small p15 ectodomain is essential to the fusion process. Furthermore, the only other hydrophobic region (H2) present in p15, aside from the TM domain, is located within the endodomain. Consequently, the p15 ectodomain is devoid of a fusion peptide motif, a hallmark feature of membrane fusion proteins. The exceedingly small, myristoylated ectodomain and the unusual topological distribution of structural motifs in this nonenveloped virus membrane fusion protein necessitate alternate models of protein-mediated membrane fusion.

scholarly journals Outer nuclear membrane fusion of adjacent nuclei in varicella-zoster virus-induced syncytia

Virology ◽  
2017 ◽  
Vol 512 ◽  
pp. 34-38 ◽  
Author(s):  
Wei Wang ◽  
Lianwei Yang ◽  
Xiumin Huang ◽  
Wenkun Fu ◽  
Dequan Pan ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Membrane Fusion ◽  
Nuclear Membrane ◽  
Outer Nuclear Membrane ◽  
Varicella Zoster

scholarly journals Inner/Outer Nuclear Membrane Fusion in Nuclear Pore Assembly

2010 ◽  
Vol 21 (23) ◽  
pp. 4197-4211 ◽  
Author(s):  
Boris Fichtman ◽  
Corinne Ramos ◽  
Beth Rasala ◽  
Amnon Harel ◽  
Douglass J. Forbes
Keyword(s):  
Membrane Fusion ◽  
Nuclear Membrane ◽  
Nuclear Pore ◽  
Channel Formation ◽  
Fusion Event ◽  
Diffusion Channel ◽  
Unique Challenge ◽  
Outer Nuclear Membrane ◽  
Nuclear Membranes ◽  
Nuclear Pore Assembly

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.

scholarly journals The Pre-Transmembrane Domain of the Autographa californica Multicapsid Nucleopolyhedrovirus GP64 Protein Is Critical for Membrane Fusion and Virus Infectivity

2009 ◽  
Vol 83 (21) ◽  
pp. 10993-11004 ◽  
Author(s):  
Zhaofei Li ◽  
Gary W. Blissard
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Aromatic Amino Acids ◽  
Autographa Californica ◽  
Fusion Pore ◽  
Virus Infectivity ◽  
Viral Fusion ◽  
Fusion Activity

ABSTRACT The envelope glycoprotein, GP64, of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is a class III viral fusion protein that mediates pH-triggered membrane fusion during virus entry. Viral fusion glycoproteins from many viruses contain a short region in the ectodomain and near the transmembrane domain, referred to as the pre-transmembrane (PTM) domain. In some cases, the PTM domain is rich in aromatic amino acids and plays an important role in membrane fusion. Although the 23-amino-acid (aa) PTM domain of AcMNPV GP64 lacks aromatic amino acids, we asked whether this region might also play a significant role in membrane fusion. We generated alanine scanning and single and multiple amino acid substitutions in the GP64 PTM domain. We specifically focused on amino acid positions conserved between baculovirus GP64 and thogotovirus GP75 proteins, as well as hydrophobic and charged amino acids. For each PTM-modified construct, we examined trimerization, cell surface localization, and membrane fusion activity. Membrane merger and pore formation were also examined. We identified eight aa positions that are important for membrane fusion activity. Critical positions were not clustered in the linear sequence but were distributed throughout the PTM domain. While charged residues were not critical or essential, three hydrophobic amino acids (L465, L476, and L480) played an important role in membrane fusion activity and appear to be involved in formation of the fusion pore. We also asked whether selected GP64 constructs were capable of rescuing a gp64null AcMNPV virus. These studies suggested that several conserved residues (T463, G460, G462, and G474) were not required for membrane fusion but were important for budding and viral infectivity.

scholarly journals Biochemical Analysis of Coronavirus Spike Glycoprotein Conformational Intermediates during Membrane Fusion

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Miyuki Kawase ◽  
Michiyo Kataoka ◽  
Kazuya Shirato ◽  
Shutoku Matsuyama
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Biochemical Analysis ◽  
Spike Protein ◽  
Viral Fusion ◽  
Enveloped Viruses ◽  
Viral Fusion Protein ◽  
Protease Digestion

ABSTRACT A fusion protein expressed on the surface of enveloped viruses mediates fusion of the viral and cellular membranes to facilitate virus infection. Pre- and postfusion structures of viral fusion proteins have been characterized, but conformational changes between them remain poorly understood. Here, we examined the intermediate conformation of the murine coronavirus fusion protein, called the spike protein, which must be cleaved by a cellular protease following receptor binding. Western blot analysis of protease digestion products revealed that two subunits (67 and 69 kDa) are produced from a single spike protein (180 kDa). These two subunits were considered to be by-products derived from conformational changes and were useful for probing the intermediate conformation of the spike protein. Interaction with a heptad repeat (HR) peptide revealed that these subunits adopt packed and unpacked conformations, respectively, and two-dimensional electrophoresis revealed a trimeric assembly. Based on biochemical observations, we propose an asymmetric trimer model for the intermediate structure of the spike protein. Receptor binding induces the membrane-binding potential of the trimer, in which at least one HR motif forms a packed-hairpin structure, while membrane fusion subunits are covered by the receptor-binding subunit, thereby preventing the spike protein from forming the typical homotrimeric prehairpin structure predicted by the current model of class I viral fusion protein. Subsequent proteolysis induces simultaneous packing of the remaining unpacked HRs upon assembly of three HRs at the central axis to generate a six-helix bundle. Our model proposes a key mechanism for membrane fusion of enveloped viruses. IMPORTANCE Recent studies using single-particle cryo-electron microscopy (cryoEM) revealed the mechanism underlying activation of viral fusion protein at the priming stage. However, characterizing the subsequent triggering stage underpinning transition from pre- to postfusion structures is difficult because single-particle cryoEM excludes unstable structures that appear as heterogeneous shapes. Therefore, population-based biochemical analysis is needed to capture features of unstable proteins. Here, we analyzed protease digestion products of a coronavirus fusion protein during activation; their sizes appear to be affected directly by the conformational state. We propose a model for the viral fusion protein in the intermediate state, which involves a compact structure and conformational changes that overcome steric hindrance within the three fusion protein subunits.

scholarly journals Highly basic clusters in the HSV-1 nuclear egress complex drive membrane budding by inducing lipid ordering

2021 ◽  
Author(s):  
Michael K Thorsen ◽  
Alex Lai ◽  
Michelle W. Lee ◽  
David P. Hoogerheide ◽  
Gerard C.L. Wong ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Electrostatic Interactions ◽  
Membrane Curvature ◽  
Membrane Deformation ◽  
Nuclear Egress ◽  
Membrane Bound ◽  
Membrane Budding ◽  
Protein Scaffolding

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and also promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC/membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in-vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an "off" switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.

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