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MBio Oct 2021In 2019, a new pandemic virus belonging to the betacoronavirus family emerged, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This new coronavirus...
In 2019, a new pandemic virus belonging to the betacoronavirus family emerged, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This new coronavirus appeared in Wuhan, China, and is responsible for severe respiratory pneumonia in humans, namely, coronavirus disease 2019 (COVID-19). Having infected almost 200 million people worldwide and caused more than 4.1 million deaths as of today, this new disease has raised a significant number of questions about its molecular mechanism of replication and, in particular, how infectious viral particles are produced. Although viral entry is well characterized, the full assembly steps of SARS-CoV-2 have still not been fully described. Coronaviruses, including SARS-CoV-2, have four main structural proteins, namely, the spike glycoprotein (S), the membrane glycoprotein (M), the envelope protein (E), and the nucleocapsid protein (N). All these proteins have key roles in the process of coronavirus assembly and budding. In this review, we gathered the current knowledge about betacoronavirus structural proteins involved in viral particle assembly, membrane curvature and scission, and then egress in order to suggest and question a coherent model for SARS-CoV-2 particle production and release.
Topics: Betacoronavirus; Membrane Glycoproteins; Nucleocapsid Proteins; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Virus Assembly
PubMed: 34579570
DOI: 10.1128/mBio.02371-21 -
Nano Letters Apr 2021Designer virus-inspired proteins drive the manufacturing of more effective, safer gene-delivery systems and simpler models to study viral assembly. However,...
Designer virus-inspired proteins drive the manufacturing of more effective, safer gene-delivery systems and simpler models to study viral assembly. However, self-assembly of engineered viromimetic proteins on specific nucleic acid templates, a distinctive viral property, has proved difficult. Inspired by viral packaging signals, we harness the programmability of CRISPR-Cas12a to direct the nucleation and growth of a self-assembling synthetic polypeptide into virus-like particles (VLP) on specific DNA molecules. Positioning up to ten nuclease-dead Cas12a (dCas12a) proteins along a 48.5 kbp DNA template triggers particle growth and full DNA encapsidation at limiting polypeptide concentrations. Particle growth rate is further increased when dCas12a is dimerized with a polymerization silk-like domain. Such improved self-assembly efficiency allows for discrimination between cognate versus noncognate DNA templates by the synthetic polypeptide. CRISPR-guided VLPs will help to develop programmable bioinspired nanomaterials with applications in biotechnology as well as viromimetic scaffolds to improve our understanding of viral self-assembly.
Topics: Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Nucleocapsid; Virion; Virus Assembly
PubMed: 33729813
DOI: 10.1021/acs.nanolett.0c04640 -
Viruses Jan 2022The hepatitis C virus (HCV) co-opts numerous cellular elements, including proteins, lipids, and microRNAs, to complete its viral life cycle. The cellular RNA-binding...
The hepatitis C virus (HCV) co-opts numerous cellular elements, including proteins, lipids, and microRNAs, to complete its viral life cycle. The cellular RNA-binding protein, poly(rC)-binding protein 1 (PCBP1), was previously reported to bind to the 5' untranslated region (UTR) of the HCV genome; however, its importance in the viral life cycle has remained unclear. Herein, we sought to clarify the role of PCBP1 in the HCV life cycle. Using the HCV cell culture (HCVcc) system, we found that knockdown of endogenous PCBP1 resulted in an overall decrease in viral RNA accumulation, yet resulted in an increase in extracellular viral titers. To dissect PCBP1's specific role in the HCV life cycle, we carried out assays for viral entry, translation, genome stability, RNA replication, as well as virion assembly and secretion. We found that PCBP1 knockdown did not directly affect viral entry, translation, RNA stability, or RNA replication, but resulted in an overall increase in infectious particle secretion. This increase in virion secretion was evident even when viral RNA synthesis was inhibited, and blocking virus secretion could partially restore the viral RNA accumulation decreased by PCBP1 knockdown. We therefore propose a model where endogenous PCBP1 normally limits virion assembly and secretion, which increases viral RNA accumulation in infected cells by preventing the departure of viral genomes packaged into virions. Overall, our findings improve our understanding of how cellular RNA-binding proteins influence viral genomic RNA utilization during the HCV life cycle.
Topics: Cell Line; DNA-Binding Proteins; Hepacivirus; Humans; RNA, Viral; RNA-Binding Proteins; Viral Genome Packaging; Virion; Virus Assembly
PubMed: 35215884
DOI: 10.3390/v14020291 -
Nucleic Acids Research Oct 2022Liquid-liquid phase separation (LLPS) has assumed a prominent role in biological cell systems, where it underpins the formation of subcellular compartments necessary for...
Liquid-liquid phase separation (LLPS) has assumed a prominent role in biological cell systems, where it underpins the formation of subcellular compartments necessary for cell function. We investigated the underlying mechanism of LLPS in virus infected cells, where virus inclusion bodies are formed by an RNA-binding phosphoprotein (NS2) of Bluetongue virus to serve as sites for subviral particle assembly and virus maturation. We show that NS2 undergoes LLPS that is dependent on protein phosphorylation and RNA-binding and that LLPS occurrence is accompanied by a change in protein secondary structure. Site-directed mutagenesis identified two critical arginine residues in NS2 responsible for specific RNA binding and thus for NS2-RNA complex driven LLPS. Reverse genetics identified the same residues as essential for VIB assembly in infected cells and virus viability. Our findings suggest that a specific arginine-RNA interaction in the context of a phosphorylated state drives LLPS in this, and possibly other, virus infections.
Topics: Animals; Phosphorylation; Virus Assembly; Bluetongue virus; RNA; Arginine
PubMed: 36259663
DOI: 10.1093/nar/gkac904 -
Wiley Interdisciplinary Reviews.... Jul 2020Viruses are highly ordered supramolecular complexes that have evolved to propagate by hijacking the host cell's machinery. Although viruses are very diverse, spreading... (Review)
Review
Viruses are highly ordered supramolecular complexes that have evolved to propagate by hijacking the host cell's machinery. Although viruses are very diverse, spreading through cells of all kingdoms of life, they share common functions and properties. Next to the general interest in virology, fundamental viral mechanisms are of growing importance in other disciplines such as biomedicine and (bio)nanotechnology. However, in order to optimally make use of viruses and virus-like particles, for instance as vehicle for targeted drug delivery or as building blocks in electronics, it is essential to understand their basic chemical and physical properties and characteristics. In this context, the number of studies addressing the mechanisms governing viral properties and processes has recently grown drastically. This review summarizes a specific part of these scientific achievements, particularly addressing physical virology approaches aimed to understand the self-assembly of viruses and the mechanical properties of viral particles. Using a physicochemical perspective, we have focused on fundamental studies providing an overview of the molecular basis governing these key aspects of viral systems. This article is categorized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.
Topics: Biomechanical Phenomena; Genome, Viral; Humans; Virion; Virus Assembly; Viruses
PubMed: 31960585
DOI: 10.1002/wnan.1613 -
Cell Host & Microbe Nov 2019Dengue virus assembly requires cleavage of viral C-prM-E polyprotein into three structural proteins (capsid, premembrane, and envelope), packaging of viral RNA with C...
Dengue virus assembly requires cleavage of viral C-prM-E polyprotein into three structural proteins (capsid, premembrane, and envelope), packaging of viral RNA with C protein into nucleocapsid, and budding of prM and E proteins into virions. The molecular mechanisms underlying these assembly events are unclear. Here, we show that dengue nonstructural protein 2A (NS2A protein) recruits viral RNA, structural proteins, and protease to the site of virion assembly and coordinates nucleocapsid and virus formation. The last 285 nucleotides of viral 3' UTR serve as a "recruiting signal for packaging" that binds to a cytosolic loop of NS2A. This interaction allows NS2A to recruit nascent RNA from the replication complex to the virion assembly site. NS2A also recruits the C-prM-E polyprotein and NS2B-NS3 protease to the virion assembly site by interacting with prM, E, and NS3, leading to coordinated C-prM-E cleavage. Mature C protein assembles onto genomic RNA to form nucleocapsid, followed by prM and E envelopment and virion formation.
Topics: Aedes; Animals; Cell Line; Chlorocebus aethiops; Cricetinae; Dengue Virus; HEK293 Cells; Humans; Nucleocapsid; RNA Helicases; RNA, Viral; Serine Endopeptidases; Vero Cells; Viral Envelope Proteins; Viral Nonstructural Proteins; Viral Proteins; Virus Assembly
PubMed: 31631053
DOI: 10.1016/j.chom.2019.09.015 -
Journal of Virology Oct 2023The influenza A virus genome consists of eight distinct viral RNAs (vRNAs) that are typically packaged into a single virion as an octameric complex. How this genome...
The influenza A virus genome consists of eight distinct viral RNAs (vRNAs) that are typically packaged into a single virion as an octameric complex. How this genome complex is assembled and incorporated into the virion is poorly understood, but previous research suggests a coordinative role for packaging signals present in all vRNAs. Here, we show that disruption of two packaging signals in a model H7N7 influenza A virus results in a mixture of virions with unusual vRNA content, including empty virions, virions with one to four vRNAs, and virions with octameric complexes composed of vRNA duplicates. Our results suggest that (i) the assembly of error-free octameric complexes proceeds through a series of defined vRNA sub-complexes and (ii) virions can bud without incorporating complete octameric complexes.
Topics: Genome, Viral; Influenza A virus; Influenza A Virus, H7N7 Subtype; RNA, Viral; Viral Genome Packaging; Virion; Virus Assembly
PubMed: 37811996
DOI: 10.1128/jvi.01076-23 -
Molecular & Cellular Proteomics : MCP Aug 2010Viral capsid assembly, in which viral proteins self-assemble into complexes of well defined architecture, is a fascinating biological process. Although viral structure...
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.
Topics: Capsid; Microscopy, Atomic Force; Norwalk virus; Osmolar Concentration; Particle Size; Spectrometry, Mass, Electrospray Ionization; Virus Assembly
PubMed: 20418222
DOI: 10.1074/mcp.M900620-MCP200 -
Journal of the American Chemical Society Jan 2019Disruption of virus capsid assembly has compelling antiviral potential that has been applied to hepatitis B virus (HBV). HBV core protein assembly can be modulated by...
Disruption of virus capsid assembly has compelling antiviral potential that has been applied to hepatitis B virus (HBV). HBV core protein assembly can be modulated by heteroaryldihydropyrimidines (HAPs), and such molecules are collectively termed core protein allosteric modulators (CpAMs). Although the antiviral effects of CpAMs are acknowledged, the mechanism of action remains an open question. Challenging aspects of characterizing misdirected assembly are the large size and nonuniform nature of the final particles. In this study of HBV assembly, we observed a competition between normative and CpAM-induced aberrant assembly with electron microscopy and resistive-pulse sensing on nanofluidic devices. This competition was a function of the strength of the association energy between individual core proteins, which is proportional to ionic strength. At strong association energy, assembly reactions primarily yielded morphologically normal HBV capsids, despite the presence of HAP-TAMRA. At weak association energy, HAP-TAMRA led to increased assembly product size and disrupted morphology. The smallest particles were T = 4 icosahedra, whereas the larger particles were defective spheres, ellipsoids, and bacilliform cylinders, with regions of T = 4 geometry interspersed with flat regions. Deviation from spherical geometry progressively increased with particle size, which is consistent with the interpretation of a competition between two alternative assembly pathways.
Topics: Antiviral Agents; Capsid; Hepatitis B virus; Osmolar Concentration; Particle Size; Pyrimidines; Rhodamines; Sodium Chloride; Virus Assembly
PubMed: 30537810
DOI: 10.1021/jacs.8b10131 -
Trends in Microbiology Jan 2024The recent revolution in imaging techniques and results from RNA footprinting in situ reveal how the bacteriophage MS2 genome regulates both particle assembly and genome... (Review)
Review
The recent revolution in imaging techniques and results from RNA footprinting in situ reveal how the bacteriophage MS2 genome regulates both particle assembly and genome release. We have proposed a model in which multiple packaging signal (PS) RNA-coat protein (CP) contacts orchestrate different stages of a viral life cycle. Programmed formation and release of specific PS contacts with CP regulates viral particle assembly and genome uncoating during cell entry. We hypothesize that molecular frustration, a concept introduced to understand protein folding, can be used to better rationalize how PSs function in both particle assembly and genome release. More broadly this concept may explain the directionality of viral life cycles, for example, the roles of host cofactors in HIV infection. We propose that this is a universal principle in virology that explains mechanisms of host-virus interaction and suggests diverse therapeutic interventions.
Topics: Humans; Capsid Proteins; RNA, Viral; HIV Infections; Genome, Viral; Virus Assembly
PubMed: 37507296
DOI: 10.1016/j.tim.2023.07.003