Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Expression, Purification and Crystallization of the Herpesvirus Nuclear Egress Complex (NEC).

ABSTRACT: The protocol describes the production and crystallization of the soluble form of the nuclear egress complex (NEC) from Herpes simplex virus 1 and Pseudorabies virus. The NEC is a heterodimer that consists of conserved proteins UL31 and UL34. NEC oligomerization deforms the inner nuclear membrane around the capsid in infected cells, thereby mediating capsid budding into the perinuclear space during nuclear egress. We have successfully developed a protocol for large-scale preparation of highly pure NEC from two different viruses in a prokaryotic expression system, which enabled us to crystallize these viral protein complexes and determine their structures. This procedure may be adapted to purify and crystallize other soluble protein complexes.

SUBMITTER: Bigalke JM

PROVIDER: S-EPMC5199029 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Expression, Purification and Crystallization of the Herpesvirus Nuclear Egress Complex (NEC).

Bigalke Janna M JM Heldwein Ekaterina E EE

Bio-protocol 20160701 14

The protocol describes the production and crystallization of the soluble form of the nuclear egress complex (NEC) from Herpes simplex virus 1 and Pseudorabies virus. The NEC is a heterodimer that consists of conserved proteins UL31 and UL34. NEC oligomerization deforms the inner nuclear membrane around the capsid in infected cells, thereby mediating capsid budding into the perinuclear space during nuclear egress. We have successfully developed a protocol for large-scale preparation of highly pur ...[more]

PMID: 28042595

Similar Datasets

Properties of Oligomeric Interaction of the Cytomegalovirus Core Nuclear Egress Complex (NEC) and Its Sensitivity to an NEC Inhibitory Small Molecule.

Project description:Herpesviral nuclear egress is a regulated process shared by all family members, ensuring the efficient cytoplasmic release of viral capsids. In the case of human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50-pUL53 heterodimer that builds hexameric lattices for capsid binding and multicomponent interaction, including NEC-associated host factors. A characteristic feature of NEC interaction is the N-terminal hook structure of pUL53 that binds to an alpha-helical groove of pUL50, thus termed as hook-into-groove interaction. This central regulatory element is essential for viral replication and shows structural-functional conservation, which has been postulated as a next-generation target of antiviral strategies. However, a solid validation of this concept has been missing. In the present study, we focused on the properties of oligomeric HCMV core NEC interaction and the antiviral activity of specifically targeted prototype inhibitors. Our data suggest the following: (i) transiently expressed, variably tagged versions of HCMV NEC proteins exert hook-into-groove complexes, putatively in oligomeric assemblies that are distinguishable from heterodimers, as shown by in vitro assembly and coimmunoprecipitation approaches; (ii) this postulated oligomeric binding pattern was further supported by the use of a pUL50::pUL53 fusion construct also showing a pronounced multi-interaction potency; (iii) using confocal imaging cellular NEC-associated proteins were found partly colocalized with the tagged core NECs; (iv) a small inhibitory molecule, recently identified by an in vitro binding inhibition assay, was likewise active in blocking pUL50-pUL53 oligomeric assembly and in exerting antiviral activity in HCMV-infected fibroblasts. In summary, the findings refine the previous concept of HCMV core NEC formation and nominate this drug-accessible complex as a validated antiviral drug target.

| S-EPMC8002134 | biostudies-literature

Crystal Structure of the Herpesvirus Nuclear Egress Complex Provides Insights into Inner Nuclear Membrane Remodeling.

Project description:Although nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Together, the conserved herpesvirus proteins pUL31 and pUL34 form the heterodimeric nuclear egress complex (NEC), which, in turn, mediates the formation of tight-fitting membrane vesicles around capsids at the inner nuclear membrane. Here, we present the crystal structure of the pseudorabies virus NEC. The structure revealed that a zinc finger motif in pUL31 and an extensive interaction network between the two proteins stabilize the complex. Comprehensive mutational analyses, characterized both in situ and in vitro, indicated that the interaction network is not redundant but rather complementary. Fitting of the NEC crystal structure into the recently determined cryoEM-derived hexagonal lattice, formed in situ by pUL31 and pUL34, provided details on the molecular basis of NEC coat formation and inner nuclear membrane remodeling.

| S-EPMC4700048 | biostudies-literature

Unexpected features and mechanism of heterodimer formation of a herpesvirus nuclear egress complex.

Project description:Herpesvirus nucleocapsids escape from the nucleus in a process orchestrated by a highly conserved, viral nuclear egress complex. In human cytomegalovirus, the complex consists of two proteins, UL50 and UL53. We solved structures of versions of UL53 and the complex by X-ray crystallography. The UL53 structures, determined at 1.93 and 3.0 Å resolution, contained unexpected features including a Bergerat fold resembling that found in certain nucleotide-binding proteins, and a Cys3His zinc finger. Substitutions of zinc-coordinating residues decreased UL50-UL53 co-localization in transfected cells, and, when incorporated into the HCMV genome, ablated viral replication. The structure of the complex, determined at 2.47 Å resolution, revealed a mechanism of heterodimerization in which UL50 clamps onto helices of UL53 like a vise. Substitutions of particular residues on the interaction interface disrupted UL50-UL53 co-localization in transfected cells and abolished virus production. The structures and the identification of contacts can be harnessed toward the rational design of novel and highly specific antiviral drugs and will aid in the detailed understanding of nuclear egress.

| S-EPMC4687685 | biostudies-literature

Patterns of Autologous and Nonautologous Interactions Between Core Nuclear Egress Complex (NEC) Proteins of α-, β- and γ-Herpesviruses.

Project description:Nuclear egress is a regulated process shared by α-, β- and γ-herpesviruses. The core nuclear egress complex (NEC) is composed of the membrane-anchored protein homologs of human cytomegalovirus (HCMV) pUL50, murine cytomegalovirus (MCMV) pM50, Epstein-Barr virus (EBV) BFRF1 or varicella zoster virus (VZV) Orf24, which interact with the autologous NEC partners pUL53, pM53, BFLF2 or Orf27, respectively. Their recruitment of additional proteins leads to the assembly of a multicomponent NEC, coordinately regulating viral nucleocytoplasmic capsid egress. Here, the functionality of VZV, HCMV, MCMV and EBV core NECs was investigated by coimmunoprecipitation and confocal imaging analyses. Furthermore, a recombinant MCMV, harboring a replacement of ORF M50 by UL50, was analyzed both in vitro and in vivo. In essence, core NEC interactions were strictly limited to autologous NEC pairs and only included one measurable nonautologous interaction between the homologs of HCMV and MCMV. A comparative analysis of MCMV-WT versus MCMV-UL50-infected murine fibroblasts revealed almost identical phenotypes on the levels of protein and genomic replication kinetics. In infected BALB/c mice, virus spread to lung and other organs was found comparable between these viruses, thus stating functional complementarity. In conclusion, our study underlines that herpesviral core NEC proteins are functionally conserved regarding complementarity of core NEC interactions, which were found either virus-specific or restricted within subfamilies.

| S-EPMC7150769 | biostudies-literature

Getting to and through the inner nuclear membrane during herpesvirus nuclear egress.

Project description:Herpesviruses, like most DNA viruses, replicate and package their genomes into capsids in the host cell nucleus. Capsids then transit to the cytoplasm in a fascinating process called nuclear egress, which includes several unusual steps: Movement of capsids from the nuclear interior to the periphery, disruption of the nuclear lamina, capsid budding through the inner nuclear membrane, and fusion of enveloped particles with the outer nuclear membrane. Here, we review recent advances and emerging questions relating to herpesvirus nuclear egress, emphasizing controversies regarding mechanisms for capsid trafficking to the nuclear periphery, and implications of recent structures of the two-subunit, viral nuclear egress complex for the process, particularly at the step of budding through the inner nuclear membrane.

| S-EPMC5503802 | biostudies-literature

Have NEC Coat, Will Travel: Structural Basis of Membrane Budding During Nuclear Egress in Herpesviruses.

Project description:Herpesviruses are unusual among enveloped viruses because they bud twice yet acquire a single envelope. Furthermore, unlike other DNA viruses that replicate in the nucleus, herpesviruses do not exit it by passing through the nuclear pores or by rupturing the nuclear envelope. Instead, herpesviruses have a complex mechanism of nuclear escape whereby nascent capsids bud at the inner nuclear membrane to form perinuclear virions that subsequently fuse with the outer nuclear membrane, releasing capsids into the cytosol. This makes them some of the very few known viruses that bud into the nuclear envelope. The envelope acquired during nuclear budding does not end up in the mature viral particle but instead allows the capsid to translocate from the nucleus into the cytosol. The viral nuclear egress complex (NEC) is a critical player in the nuclear egress, yet its function and mechanism have remained enigmatic. Recent studies have demonstrated that the NEC buds membranes without the help of other proteins by forming a honeycomb coat, which established the NEC as the first virally encoded budding machine that operates at the nuclear, as opposed to cytoplasmic, membrane. This review discusses our current understanding of the NEC budding mechanism, with the emphasis on studies that illuminated the structure of the NEC coat and its role in capsid budding during herpesvirus nuclear escape.

| S-EPMC5367388 | biostudies-literature

Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication.

Project description:Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1-168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50-UL53 binding in vitro, eliminated UL50-UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein-protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target.

| S-EPMC4517201 | biostudies-literature

HCMV-NEC analysis - Proteomic analysis of the multimeric nuclear egress complex of human cytomegalovirus

Project description:Herpesviral capsids are assembled in the host cell nucleus before being translocated to the cytoplasm where they undergo further maturation. The crossing of nuclear envelope represents a major event that requires formation of the nuclear egress complex (NEC). We investigated the protein composition of specific HCMV-NEC complex in infected infected human primary fibroblasts.

2014-06-30 | PXD000536 | Pride

The Herpesvirus Nuclear Egress Complex Component, UL31, Can Be Recruited to Sites of DNA Damage Through Poly-ADP Ribose Binding.

Project description:The herpes simplex virus (HSV) UL31 gene encodes a conserved member of the herpesvirus nuclear egress complex that not only functions in the egress of DNA containing capsids from the nucleus, but is also required for optimal replication of viral DNA and its packaging into capsids. Here we report that the UL31 protein from HSV-2 can be recruited to sites of DNA damage by sequences found in its N-terminus. The N-terminus of UL31 contains sequences resembling a poly (ADP-ribose) (PAR) binding motif suggesting that PAR interactions might mediate UL31 recruitment to damaged DNA. Whereas PAR polymerase inhibition prevented UL31 recruitment to damaged DNA, inhibition of signaling through the ataxia telangiectasia mutated DNA damage response pathway had no effect. These findings were further supported by experiments demonstrating direct and specific interaction between HSV-2 UL31 and PAR using purified components. This study reveals a previously unrecognized function for UL31 and may suggest that the recognition of PAR by UL31 is coupled to the nuclear egress of herpesvirus capsids, influences viral DNA replication and packaging, or possibly modulates the DNA damage response mounted by virally infected cells.

| S-EPMC5432524 | biostudies-literature

Expression, purification, crystallization and preliminary X-ray analysis of the HER3-9E12 Fab complex.

Project description:9E12 is a fully human immunoglobulin G1/? monoclonal antibody that is specific for the epidermal growth factor receptor 3 (HER3), the overexpression of which has been detected in many tumour types and is associated with poor survival outcomes. To date, knowledge of the molecular mechanism for targeted antibodies that directly inhibit HER3 signalling is limited. Because knowledge of such therapeutic antibodies would help basic immunological therapeutics, structural insights into the HER3-9E12 Fab complex are important. Recombinant human HER3 and Fab fragments of the 9E12 antibody were cloned, expressed and crystallized, and crystallographic data sets were collected. The crystals belonged to space group P1, with unit-cell parameters a=74.4, b=98.6, c=99.6 Å, ?=106.0, ?=95.0, ?=102.5° and diffracted to a resolution of 2.1?Å.

| S-EPMC4051538 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data