Insights into virus capsid assembly from non-covalent mass spectrometry.
Ontology highlight
ABSTRACT:
SUBMITTER: Morton VL
PROVIDER: S-EPMC7168407 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The assembly of hundreds of identical proteins into an icosahedral virus capsid is a remarkable feat of molecular engineering. How this occurs is poorly understood. Key intermediates have been anticipated at the end of the assembly reaction, but it has not been possible to detect them. In this work we have used charge detection mass spectrometry to identify trapped intermediates from late in the assembly of the hepatitis B virus T = 4 capsid, a complex of 120 protein dimers. Prominent intermediates are found with 104/105, 110/111, and 117/118 dimers. Cryo-EM observations indicate the intermediates are incomplete capsids and, hence, on the assembly pathway. On the basis of their stability and kinetic accessibility we have proposed plausible structures. The prominent trapped intermediate with 104 dimers is attributed to an icosahedron missing two neighboring facets, the 111-dimer species is assigned to an icosahedron missing a single facet, and the intermediate with 117 dimers is assigned to a capsid missing a ring of three dimers in the center of a facet.
| S-EPMC3985884 | biostudies-literature
Project description:Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane-integrated enzymes assembling up to hexameric complexes. We further indicate a lipid-dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent-free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provide a versatile platform for the analysis of membrane proteins in a native environment.
| S-EPMC5291076 | biostudies-literature
Project description:Viral capsid assembly, in which viral proteins self-assemble into complexes of well defined architecture, is a fascinating biological process. Although viral structure and assembly processes have been the subject of many excellent structural biology studies in the past, questions still remain regarding the intricate mechanisms that underlie viral structure, stability, and assembly. Here we used native mass spectrometry-based techniques to study the structure, stability, and assembly of Norwalk virus-like particles. Although detailed structural information on the fully assembled capsid exists, less information is available on potential capsid (dis)assembly intermediates, largely because of the inherent heterogeneity and complexity of the disassembly pathways. We used native mass spectrometry and atomic force microscopy to investigate the (dis)assembly of the Norwalk virus-like particles as a function of solution pH, ionic strength, and VP1 protein concentration. Native MS analysis at physiological pH revealed the presence of the complete capsid (T = 3) consisting of 180 copies of VP1. The mass of these capsid particles extends over 10 million Da, ranking them among the largest protein complexes ever analyzed by native MS. Although very stable under acidic conditions, the capsid was found to be sensitive to alkaline treatment. At elevated pH, intermediate structures consisting of 2, 4, 6, 18, 40, 60, and 80 copies of VP1 were observed with the VP1(60) (3.36-MDa) and VP1(80) (4.48-MDa) species being most abundant. Atomic force microscopy imaging and ion mobility mass spectrometry confirmed the formation of these latter midsize spherical particles at elevated pH. All these VP1 oligomers could be reversely assembled into the original capsid (VP1(180)). From the MS data collected over a range of experimental conditions, we suggest a disassembly model in which the T = 3 VP1(180) particles dissociate into smaller oligomers, predominantly dimers, upon alkaline treatment prior to reassembly into VP1(60) and VP1(80) species.
| S-EPMC2938053 | biostudies-literature
Project description:The structural and functional analysis of the core protein of hepatitis B virus is important for a full understanding of the viral life cycle and the development of novel therapeutic agents. The majority of the core protein (CP149) comprises the capsid assembly domain, and the C-terminal region (residues 150-183) is responsible for nucleic acid binding. Protein monomers associate to form dimeric structural subunits, and helices 3 and 4 (residues 50-111 of the assembly domain) have been shown to be important for this as they constitute the interdimer interface. Here, using mass spectrometry coupled with ion mobility spectrometry, we demonstrate the conformational flexibility of the CP149 dimer. Limited proteolysis was used to locate involvement in this feature to the C-terminal region. A genetically fused CP dimer was found to show decreased disorder, consistent with a more restricted C-terminus at the fusion junction. Incubation of CP149 dimer with heteroaryldihydropyrimidine-1, a small molecule known to interfere with the assembly process, was shown to result in oligomers different in shape to the capsid assembly-competent oligomers of the fused CP dimer. We suggest that heteroaryldihydropyrimidine-1 affects the dynamics of CP149 dimer in solution, likely affecting the ratio between assembly active and inactive states. Therefore, assembly of the less dynamic fused dimer is less readily misdirected by heteroaryldihydropyrimidine-1. These studies of the flexibility and oligomerization properties of hepatitis B virus core protein illustrate both the importance of C-terminal dynamics in function and the utility of gas-phase techniques for structural and dynamical biomolecular analysis.
| S-EPMC3762345 | biostudies-literature
Project description:We have combined ion mobility spectrometry-mass spectrometry with tandem mass spectrometry to characterise large, non-covalently bound macromolecular complexes in terms of mass, shape (cross-sectional area) and stability (dissociation) in a single experiment. The results indicate that the quaternary architecture of a complex influences its residual shape following removal of a single subunit by collision-induced dissociation tandem mass spectrometry. Complexes whose subunits are bound to several neighbouring subunits to create a ring-like three-dimensional (3D) architecture undergo significant collapse upon dissociation. In contrast, subunits which have only a single neighbouring subunit within a complex retain much of their original shape upon complex dissociation. Specifically, we have determined the architecture of two transient, on-pathway intermediates observed during in vitro viral capsid assembly. Knowledge of the mass, stoichiometry and cross-sectional area of each viral assembly intermediate allowed us to model a range of potential structures based on the known X-ray structure of the coat protein building blocks. Comparing the cross-sectional areas of these potential architectures before and after dissociation provided tangible evidence for the assignment of the topologies of the complexes, which have been found to encompass both the 3-fold and the 5-fold symmetry axes of the final icosahedral viral shell. Such insights provide unique information about virus assembly pathways that could allow the design of anti-viral therapeutics directed at the assembly step. This methodology can be readily applied to the structural characterisation of many other non-covalently bound macromolecular complexes and their assembly pathways.
| S-EPMC4789508 | biostudies-literature
Project description:African swine fever virus (ASFV), the cause of a highly contagious hemorrhagic and fatal disease of domestic pigs, has a complex multilayer structure. The inner capsid of ASFV located underneath the inner membrane enwraps the genome-containing nucleoid and is likely the assembly of proteolytic products from the virally encoded polyproteins pp220 and pp62. Here, we report the crystal structure of ASFV p150△NC, a major middle fragment of the pp220 proteolytic product p150. The structure of ASFV p150△NC contains mainly helices and has a triangular plate-like shape. The triangular plate is approximately 38 Å in thickness, and the edge of the triangular plate is approximately 90 Å long. The structure of ASFV p150△NC is not homologous to any of the known viral capsid proteins. Further analysis of the cryo-electron microscopy maps of the ASFV and the homologous faustovirus inner capsids revealed that p150 or the p150-like protein of faustovirus assembles to form screwed propeller-shaped hexametric and pentametric capsomeres of the icosahedral inner capsids. Complexes of the C terminus of p150 and other proteolytic products of pp220 likely mediate interactions between the capsomeres. Together, these findings provide new insights into the assembling of ASFV inner capsid and provide a reference for understanding the assembly of the inner capsids of nucleocytoplasmic large DNA viruses (NCLDV). IMPORTANCE African swine fever virus has caused catastrophic destruction to the pork industry worldwide since it was first discovered in Kenya in 1921. The architecture of ASFV is complicated, with two protein shells and two membrane envelopes. Currently, mechanisms involved in the assembly of the ASFV inner core shell are less understood. The structural studies of the ASFV inner capsid protein p150 performed in this research enable the building of a partial model of the icosahedral ASFV inner capsid, which provides a structural basis for understanding the structure and assembly of this complex virion. Furthermore, the structure of ASFV p150△NC represents a new type of fold for viral capsid assembly, which could be a common fold for the inner capsid assembly of nucleocytoplasmic large DNA viruses (NCLDV) and would facilitate the development of vaccine and antivirus drugs against these complex viruses.
| S-EPMC10308890 | biostudies-literature
Project description:Using mass spectrometry (MS) to obtain information about a higher order structure of protein requires that a protein's structural properties are encoded into the mass of that protein. Covalent labeling (CL) with reagents that can irreversibly modify solvent accessible amino acid side chains is an effective way to encode structural information into the mass of a protein, as this information can be read-out in a straightforward manner using standard MS-based proteomics techniques. The differential reactivity of proteins under two or more conditions can be used to distinguish protein topologies, conformations, and/or binding sites. CL-MS methods have been effectively used for the structural analysis of proteins and protein complexes, particularly for systems that are difficult to study by other more traditional biochemical techniques. This review provides an overview of the non-specific CL approaches that have been combined with MS with a particular emphasis on the reagents that are commonly used, including hydroxyl radicals, carbenes, and diethylpyrocarbonate. We describe the reagent and protein factors that affect the reactivity of amino acid side chains. We also include details about experimental design and workflow, data analysis, recent applications, and some future prospects of CL-MS methods.
| S-EPMC6051898 | biostudies-literature
Project description:The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host-derived molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass-spectrometry (dMS) followed by targeted analysis to facilitate this discovery. To trace the non-human dMS identified peptides back to its genetic origin by next generation sequencing (NGS) cDNA libraries were generated using degenerate oligos corresponding to the identified peptides. In a pilot study DPS identified both viral and human biomarkers. Based on the identified peptides that are differentially expressed in the virus positive tumor sample, degenerate oligos were designed. Degenerate oligos or LNA modified degenerate oligos or a modified oligo(dT) SMART primer were used to facilitate reverse transcription from viral or viral and host RNA. NGS analysis revealed seven times more MCV reads in degenerate oligo primed RNAseq samples compared to polyA-based sequencing reads.
2020-10-20 | GSE157610 | GEO
Project description:In all herpesviruses, the capsid is icosahedral in shape, composed of 162 capsomers, and assembled in the infected cell nucleus. Once a closed capsid has formed, it is packaged with the virus DNA and transported to the cytoplasm where further morphogenetic events take place. Herpesvirus capsid populations are highly uniform in shape, and this property has made them attractive for structural analysis particularly by cryo electron microscopy followed by three-dimensional image reconstruction. Here we describe what is known about herpesvirus capsid structure and assembly with emphasis on herpes simplex virus and on the contribution of structural studies. The overall analysis has demonstrated that herpesvirus capsids are formed by a pathway resembling that established for dsDNA bacteriophage such as P22 and HK97. For example herpes capsid assembly is found to: (1) involve a scaffolding protein not present in the mature virus; (2) proceed through a fragile, spherical procapsid intermediate; and (3) result in incorporation of a portal complex at a unique capsid vertex.
| S-EPMC3171831 | biostudies-literature
Project description:Antigen-antibody epitope mapping is essential for understanding binding mechanisms and developing new protein therapeutics. In this study, we investigate diethylpyrocarbonate (DEPC) covalent labeling-mass spectrometry as a means of analyzing antigen-antibody interactions using the well-characterized model system of TNFα in complex with three different antibodies. Results show that residues buried in the epitope undergo substantial decreases in labeling, as expected. Interestingly, serine, threonine, and tyrosine residues at the edges of the epitope undergo unexpected increases in labeling. The increased labeling of these weakly nucleophilic residues is caused by the formation of hydrophobic pockets upon antibody binding that presumably increase local DEPC concentrations. Residues that are distant from the epitope generally do not undergo changes in labeling extent; however, some that do change experience variations in their local microenvironment due to side-chain reorganization or stabilization of the TNFα trimer that occurs upon binding. Overall, DEPC labeling of antigen-antibody complexes is found to depend on both changes in solvent exposure and changes to the residue microenvironment.
| S-EPMC8785103 | biostudies-literature