Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Using cryoEM and cryoET to visualize membrane penetration of a non-enveloped virus.

ABSTRACT: Key to cell entry by non-enveloped viruses is virus-cell interactions at the cell or endosomal membrane. Here, we detail our protocols to capture such interactions between non-enveloped virus bluetongue virus (BTV) and vesicular membrane by cryogenic electron microscopy (cryoEM) and tomography (cryoET). Key steps include virus isolation, liposome preparation, virus-liposome incubation and vitrification, cryoEM and cryoET imaging, data processing for 3D reconstruction, and subtomogram averaging. The protocols can be generally applicable to studies of cell entry by other non-enveloped viruses. For complete details on the use and execution of this protocol, please refer to Xia et al. (2021).

SUBMITTER: Xia X

PROVIDER: S-EPMC9667302 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Using cryoEM and cryoET to visualize membrane penetration of a non-enveloped virus.

Xia Xian X Zhou Z Hong ZH

STAR protocols 20221111 4

Key to cell entry by non-enveloped viruses is virus-cell interactions at the cell or endosomal membrane. Here, we detail our protocols to capture such interactions between non-enveloped virus bluetongue virus (BTV) and vesicular membrane by cryogenic electron microscopy (cryoEM) and tomography (cryoET). Key steps include virus isolation, liposome preparation, virus-liposome incubation and vitrification, cryoEM and cryoET imaging, data processing for 3D reconstruction, and subtomogram averaging. ...[more]

PMID: 36595958

Similar Datasets

Non-enveloped virus membrane penetration: New advances leading to new insights.

Project description:Host cell membranes pose a particular challenge for non-enveloped viruses. Whereas enveloped viruses enter cells by fusing their lipid envelopes with the cellular membrane, non-enveloped viruses generally must (1) enter cells via endocytosis, then (2) penetrate the cellular endomembrane to reach the cytosol. Only then can the viruses begin to replicate (or transit to the nucleus to replicate). Although membrane penetration of non-enveloped viruses is a crucial entry step, many of the precise molecular details of this process remain unclear. Recent findings have begun to untangle the various mechanisms by which non-enveloped viral proteins disrupt and penetrate cellular endomembranes. Specifically, high-resolution microscopy studies have revealed precise conformational changes in viral proteins that enable penetration, while biochemical studies have identified key host proteins that promote viral penetration and transport. This brief article summarizes new discoveries in the membrane penetration process for three of the most intensely studied families of non-enveloped viruses: reoviruses, papillomaviruses, and polyomaviruses.

| S-EPMC9731489 | biostudies-literature

Structural rearrangements in the membrane penetration protein of a non-enveloped virus.

Project description:Non-enveloped virus particles (those that lack a lipid-bilayer membrane) must breach the membrane of a target host cell to gain access to its cytoplasm. So far, the molecular mechanism of this membrane penetration step has resisted structural analysis. The spike protein VP4 is a principal component in the entry apparatus of rotavirus, a non-enveloped virus that causes gastroenteritis and kills 440,000 children each year. Trypsin cleavage of VP4 primes the virus for entry by triggering a rearrangement that rigidifies the VP4 spikes. We have determined the crystal structure, at 3.2 A resolution, of the main part of VP4 that projects from the virion. The crystal structure reveals a coiled-coil stabilized trimer. Comparison of this structure with the two-fold clustered VP4 spikes in a approximately 12 A resolution image reconstruction from electron cryomicroscopy of trypsin-primed virions shows that VP4 also undergoes a second rearrangement, in which the oligomer reorganizes and each subunit folds back on itself, translocating a potential membrane-interaction peptide from one end of the spike to the other. This rearrangement resembles the conformational transitions of membrane fusion proteins of enveloped viruses.

| S-EPMC1780043 | biostudies-literature

EMC1-dependent stabilization drives membrane penetration of a partially destabilized non-enveloped virus.

Project description:Destabilization of a non-enveloped virus generates a membrane transport-competent viral particle. Here we probe polyomavirus SV40 endoplasmic reticulum (ER)-to-cytosol membrane transport, a decisive infection step where destabilization initiates this non-enveloped virus for membrane penetration. We find that a member of the ER membrane protein complex (EMC) called EMC1 promotes SV40 ER membrane transport and infection. Surprisingly, EMC1 does so by using its predicted transmembrane residue D961 to bind to and stabilize the membrane-embedded partially destabilized SV40, thereby preventing premature viral disassembly. EMC1-dependent stabilization enables SV40 to engage a cytosolic extraction complex that ejects the virus into the cytosol. Thus EMC1 acts as a molecular chaperone, bracing the destabilized SV40 in a transport-competent state. Our findings reveal the novel principle that coordinated destabilization-stabilization drives membrane transport of a non-enveloped virus.

| S-EPMC5224922 | biostudies-literature

Functional refolding of the penetration protein on a non-enveloped virus.

Project description:A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell1. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP42,3. Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an 'upright' to a 'reversed' conformation. The reversed structure projects a previously buried 'foot' domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses.

| S-EPMC8297411 | biostudies-literature

Regulated Erlin-dependent release of the B12 transmembrane J-protein promotes ER membrane penetration of a non-enveloped virus.

Project description:The molecular mechanism by which non-enveloped viruses penetrate biological membranes remains enigmatic. The non-enveloped polyomavirus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol and cause infection. We previously demonstrated that SV40 creates its own membrane penetration structure by mobilizing select transmembrane proteins to distinct puncta in the ER membrane called foci that likely function as the cytosol entry sites. How these ER membrane proteins reorganize into the foci is unknown. B12 is a transmembrane J-protein that mobilizes into the foci to promote cytosol entry of SV40. Here we identify two closely related ER membrane proteins Erlin1 and Erlin2 (Erlin1/2) as B12-interaction partners. Strikingly, SV40 recruits B12 to the foci by inducing release of this J-protein from Erlin1/2. Our data thus reveal how a non-enveloped virus promotes its own membrane translocation by triggering the release and recruitment of a critical transport factor to the membrane penetration site.

| S-EPMC5484543 | biostudies-literature

Precision requirements and data compression in CryoEM/CryoET.

Project description:With larger, higher speed detectors and improved automation, individual CryoEM instruments are capable of producing a prodigious amount of data each day, which must then be stored, processed and archived. While it has become routine to use lossless compression on raw counting-mode movies, the averages which result after correcting these movies no longer compress well. These averages could be considered sufficient for long term archival, yet they are conventionally stored with 32 bits of precision, despite high noise levels. Derived images are similarly stored with excess precision, providing an opportunity to decrease project sizes and improve processing speed. We present a simple argument based on propagation of uncertainty for safe bit truncation of flat-fielded images combined with lossless compression. The same method can be used for most derived images throughout the processing pipeline. We test the proposed strategy on two standard, data-limited CryoEM data sets, demonstrating that these limits are safe for real-world use. We find that 5 bits of precision is sufficient for virtually any raw CryoEM data and that 8-12 bits is sufficient for intermediate averages or final 3-D structures. Additionally, we detail and recommend specific rules for discretization of data as well as a practical compressed data representation that is tuned to the specific needs of CryoEM.

| S-EPMC9645247 | biostudies-literature

Topaz-Denoise: general deep denoising models for cryoEM and cryoET.

Project description:Cryo-electron microscopy (cryoEM) is becoming the preferred method for resolving protein structures. Low signal-to-noise ratio (SNR) in cryoEM images reduces the confidence and throughput of structure determination during several steps of data processing, resulting in impediments such as missing particle orientations. Denoising cryoEM images can not only improve downstream analysis but also accelerate the time-consuming data collection process by allowing lower electron dose micrographs to be used for analysis. Here, we present Topaz-Denoise, a deep learning method for reliably and rapidly increasing the SNR of cryoEM images and cryoET tomograms. By training on a dataset composed of thousands of micrographs collected across a wide range of imaging conditions, we are able to learn models capturing the complexity of the cryoEM image formation process. The general model we present is able to denoise new datasets without additional training. Denoising with this model improves micrograph interpretability and allows us to solve 3D single particle structures of clustered protocadherin, an elongated particle with previously elusive views. We then show that low dose collection, enabled by Topaz-Denoise, improves downstream analysis in addition to reducing data collection time. We also present a general 3D denoising model for cryoET. Topaz-Denoise and pre-trained general models are now included in Topaz. We expect that Topaz-Denoise will be of broad utility to the cryoEM community for improving micrograph and tomogram interpretability and accelerating analysis.

| S-EPMC7567117 | biostudies-literature

Enveloped and non-enveloped virus survival on microfiber towels.

Project description:BackgroundHandwashing is an important intervention which can reduce indirect disease transmission, however soap and water for handwashing purposes is not available in some low-resource regions. When handwashing with soap and water is not possible, individuals may use alternatives such as the Supertowel (a microfiber towel with an antimicrobial coating). Testing of viral inactivation as a result of antimicrobial treatment on the Supertowel, however, has been limited. The goal of this study is to provide information about the performance of the Supertowel's antimicrobial treatment against viruses, which will help inform the use of the towels as handwashing alternatives.MethodsWe seeded the Supertowel and a regular microfiber towel with two bacteriophages (enveloped Phi6 and non-enveloped MS2) and monitored viral inactivation over time. Additionally, we assessed if temperature, humidity, whether the towel was initially wet or dry, or virus type had an effect on viral decay rate constants. Virus concentrations were measured repeatedly over 24 h.ResultsWe found that neither towel type (whether the towel was a Supertowel or a regular microfiber towel) nor humidity were significant variables in our model of decay rate constants (P = 0.06 and P = 0.22, respectively). We found that the variables of temperature, whether towels were initially wet versus dry, and virus type were significantly different from 0, suggesting that these variables explained variance in the decay rate constant (P = 6.55 × 10-13, P = 0.001, and P < 2 × 10-16, respectively). Higher temperatures, dry towels, and enveloped viruses all resulted in increases in the decay rate constant.ConclusionsViruses seeded onto a Supertowel decay similar to viruses seeded onto a regular towel indicating that the virucidal potential of the Supertowel is minimal.

| S-EPMC10106082 | biostudies-literature

A Non-enveloped Virus Hijacks Host Disaggregation Machinery to Translocate across the Endoplasmic Reticulum Membrane.

Project description:Mammalian cytosolic Hsp110 family, in concert with the Hsc70:J-protein complex, functions as a disaggregation machinery to rectify protein misfolding problems. Here we uncover a novel role of this machinery in driving membrane translocation during viral entry. The non-enveloped virus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a critical infection step. Combining biochemical, cell-based, and imaging approaches, we find that the Hsp110 family member Hsp105 associates with the ER membrane J-protein B14. Here Hsp105 cooperates with Hsc70 and extracts the membrane-penetrating SV40 into the cytosol, potentially by disassembling the membrane-embedded virus. Hence the energy provided by the Hsc70-dependent Hsp105 disaggregation machinery can be harnessed to catalyze a membrane translocation event.

| S-EPMC4526233 | biostudies-literature

Influence of membrane composition on the binding and folding of a membrane lytic peptide from the non-enveloped flock house virus.

Project description:Using a combination of coarse-grained and atomistic molecular dynamics simulations we have investigated the membrane binding and folding properties of the membrane lytic peptide of Flock House virus (FHV). FHV is an animal virus and an excellent model system for studying cell entry mechanisms in non-enveloped viruses. FHV undergoes a maturation event where the 44 C-terminal amino acids are cleaved from the major capsid protein, forming the membrane lytic (γ) peptides. Under acidic conditions, γ is released from the capsid interior allowing the peptides to bind and disrupt membranes. The first 21 N-terminal residues of γ, termed γ1, have been resolved in the FHV capsid structure and γ1 has been the subject of in vitro studies. γ1 is structurally dynamic as it adopts helical secondary structure inside the capsid and on membranes, but it is disordered in solution. In vitro studies have shown the binding free energies to POPC or POPG membranes are nearly equivalent, but binding to POPC is enthalpically driven, while POPG binding is entropically driven. Through coarse-grained and multiple microsecond all-atom simulations the membrane binding and folding properties of γ1 are investigated against homogeneous and heterogeneous bilayers to elucidate the dependence of the microenvironment on the structural properties of γ1. Our studies provide a rationale for the thermodynamic data and suggest binding of γ1 to POPG bilayers occurs in a disordered state, but γ1 must adopt a helical conformation when binding POPC bilayers.

| S-EPMC5482360 | 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