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1.
Viruses ; 14(2)2022 02 17.
Article in English | MEDLINE | ID: covidwho-1786043

ABSTRACT

Various adenoviruses are being used as viral vectors for the generation of vaccines against chronic and emerging diseases (e.g., AIDS, COVID-19). Here, we report the improved capsid structure for one of these vectors, human adenovirus D26 (HAdV-D26), at 3.4 Å resolution, by reprocessing the previous cryo-electron microscopy dataset and obtaining a refined model. In addition to overall improvements in the model, the highlights of the structure include (1) locating a segment of the processed peptide of VIII that was previously believed to be released from the mature virions, (2) reorientation of the helical appendage domain (APD) of IIIa situated underneath the vertex region relative to its counterpart observed in the cleavage defective (ts1) mutant of HAdV-C5 that resulted in the loss of interactions between the APD and hexon bases, and (3) the revised conformation of the cleaved N-terminal segments of pre-protein VI (pVIn), located in the hexon cavities, is highly conserved, with notable stacking interactions between the conserved His13 and Phe18 residues. Taken together, the improved model of HAdV-D26 capsid provides a better understanding of protein-protein interactions in HAdV capsids and facilitates the efforts to modify and/or design adenoviral vectors with altered properties. Last but not least, we provide some insights into clotting factors (e.g., FX and PF4) binding to AdV vectors.


Subject(s)
Adenoviruses, Human/chemistry , Capsid/chemistry , Capsid/ultrastructure , Cryoelectron Microscopy/methods , Adenoviruses, Human/genetics , Capsid Proteins/genetics , Humans , Models, Molecular , Protein Conformation , Protein Interaction Domains and Motifs , Virus Assembly , Virus Internalization
2.
Virus Genes ; 58(2): 143-145, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1777771

ABSTRACT

Virus like particles (VLPs) are used as a tool to study the mutations in the structural genes that influence the virus assembly and entry process. We observed that Chikungunya VLP with the E1:V291I mutation produced more fluorescence-positive cells in Vero cells than the other mutant VLPs (E1:A226V, D284E, and E2:V264A) and wild-type VLP tested in this study. According to the findings, the V291I mutation may aid the virus's ability to enter the cells more efficiently than wild-type VLPs. The study concludes that VLP is a useful model for studying the virus entry process in cells.


Subject(s)
Chikungunya Fever , Animals , Chlorocebus aethiops , Mutation , Vero Cells , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Virus Assembly
3.
Viruses ; 14(2)2022 01 25.
Article in English | MEDLINE | ID: covidwho-1715763

ABSTRACT

Epithelial cells are apico-basolateral polarized cells that line all tubular organs and are often targets for infectious agents. This review focuses on the release of human RNA virus particles from both sides of polarized human cells grown on transwells. Most viruses that infect the mucosa leave their host cells mainly via the apical side while basolateral release is linked to virus propagation within the host. Viruses do this by hijacking the cellular factors involved in polarization and trafficking. Thus, understanding epithelial polarization is essential for a clear understanding of virus pathophysiology.


Subject(s)
Epithelial Cells/virology , RNA Viruses/physiology , Virus Release , Cell Polarity , Humans , Virion/physiology , Virus Assembly , Virus Replication
4.
Viruses ; 14(2)2022 01 23.
Article in English | MEDLINE | ID: covidwho-1651072

ABSTRACT

The COVID-19 pandemic is driven by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) that emerged in 2019 and quickly spread worldwide. Genomic surveillance has become the gold standard methodology used to monitor and study this fast-spreading virus and its constantly emerging lineages. The current deluge of SARS-CoV-2 genomic data generated worldwide has put additional pressure on the urgent need for streamlined bioinformatics workflows. Here, we describe a workflow developed by our group to process and analyze large-scale SARS-CoV-2 Illumina amplicon sequencing data. This workflow automates all steps of SARS-CoV-2 reference-based genomic analysis: data processing, genome assembly, PANGO lineage assignment, mutation analysis and the screening of intrahost variants. The pipeline is capable of processing a batch of around 100 samples in less than half an hour on a personal laptop or in less than five minutes on a server with 50 threads. The workflow presented here is available through Docker or Singularity images, allowing for implementation on laptops for small-scale analyses or on high processing capacity servers or clusters. Moreover, the low requirements for memory and CPU cores and the standardized results provided by ViralFlow highlight it as a versatile tool for SARS-CoV-2 genomic analysis.


Subject(s)
Automation, Laboratory/methods , Genome, Viral , Mutation , SARS-CoV-2/classification , SARS-CoV-2/genetics , Workflow , Computational Biology/instrumentation , Computational Biology/methods , Genomics/instrumentation , Genomics/methods , Humans , Phylogeny , Spike Glycoprotein, Coronavirus/genetics , Virus Assembly/genetics
5.
Int J Biol Macromol ; 200: 487-497, 2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1634879

ABSTRACT

Virus-like particles (VLPs) are nano-scale particles that are morphologically similar to a live virus but which lack a genetic component. Since the pandemic spread of COVID-19, much focus has been placed on coronavirus (CoV)-related VLPs. CoVs contain four structural proteins, though the minimum requirement for VLP formation differs among virus species. CoV VLPs are commonly produced in mammalian and insect cell systems, sometimes in the form of chimeric VLPs that enable surface display of CoV epitopes. VLPs are an ideal model for virological research and have been applied as vaccines and diagnostic reagents to aid in clinical disease control. This review summarizes and updates the research progress on the characteristics of VLPs from different known CoVs, mainly focusing on assembly, in vitro expression systems for VLP generation, VLP chimerism, protein-based nanoparticles and their applications in basic research and clinical settings, which may aid in development of novel VLP vaccines against emerging coronavirus diseases such as SARS-CoV-2.


Subject(s)
Coronavirus/genetics , Coronavirus/immunology , Vaccines, Virus-Like Particle/biosynthesis , Vaccines, Virus-Like Particle/genetics , Animals , Chimerism , Epitopes , Humans , SARS-CoV-2/immunology , Vaccines, Virus-Like Particle/therapeutic use , Viral Proteins , Virus Assembly
6.
J Virol ; 94(13)2020 06 16.
Article in English | MEDLINE | ID: covidwho-1583223

ABSTRACT

Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homology, and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approximately 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging infectious disease with zoonotic potential and has reservoirs in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans, with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data have found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This information can guide therapeutic solutions to block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.


Subject(s)
Calcium/metabolism , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Host-Pathogen Interactions , Ions/metabolism , Membrane Fusion , Middle East Respiratory Syndrome Coronavirus/physiology , Virus Internalization , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Line , Chlorocebus aethiops , Humans , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Models, Molecular , Mutation , Protein Binding , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship , Vero Cells , Virulence , Virus Assembly
7.
Viruses ; 13(11)2021 11 19.
Article in English | MEDLINE | ID: covidwho-1538549

ABSTRACT

A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular 'sponges', stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ∼2400 Gag and ∼120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak-strong-moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules.


Subject(s)
HIV Infections/virology , HIV-1 , Nucleocapsid Proteins/immunology , Viral Proteases/immunology , HIV-1/immunology , HIV-1/physiology , Humans , Virus Assembly
8.
Viruses ; 13(10)2021 09 26.
Article in English | MEDLINE | ID: covidwho-1485180

ABSTRACT

Nascent HIV-1 particles incorporate the viral envelope glycoprotein and multiple host transmembrane proteins during assembly at the plasma membrane. At least some of these host transmembrane proteins on the surface of virions are reported as pro-viral factors that enhance virus attachment to target cells or facilitate trans-infection of CD4+ T cells via interactions with non-T cells. In addition to the pro-viral factors, anti-viral transmembrane proteins are incorporated into progeny virions. These virion-incorporated transmembrane proteins inhibit HIV-1 entry at the point of attachment and fusion. In infected polarized CD4+ T cells, HIV-1 Gag localizes to a rear-end protrusion known as the uropod. Regardless of cell polarization, Gag colocalizes with and promotes the virion incorporation of a subset of uropod-directed host transmembrane proteins, including CD162, CD43, and CD44. Until recently, the functions of these virion-incorporated proteins had not been clear. Here, we review the recent findings about the roles played by virion-incorporated CD162, CD43, and CD44 in HIV-1 spread to CD4+ T cells.


Subject(s)
HIV Infections/metabolism , Hyaluronan Receptors/metabolism , Leukosialin/metabolism , Membrane Glycoproteins/metabolism , Cell Membrane/metabolism , HIV Infections/genetics , HIV-1/genetics , HIV-1/metabolism , HIV-1/pathogenicity , Host-Pathogen Interactions , Humans , Hyaluronan Receptors/genetics , Leukosialin/genetics , Membrane Glycoproteins/genetics , Membrane Proteins/metabolism , T-Lymphocytes/metabolism , T-Lymphocytes/virology , Virion/metabolism , Virus Assembly , Virus Attachment , gag Gene Products, Human Immunodeficiency Virus/metabolism
9.
Dev Cell ; 56(20): 2790-2807.e8, 2021 10 25.
Article in English | MEDLINE | ID: covidwho-1446559

ABSTRACT

SARS-CoV-2 virions are surrounded by a lipid bilayer that contains membrane proteins such as spike, responsible for target-cell binding and virus fusion. We found that during SARS-CoV-2 infection, spike becomes lipid modified, through the sequential action of the S-acyltransferases ZDHHC20 and 9. Particularly striking is the rapid acylation of spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics, and biochemical approaches, we show that this massive lipidation controls spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid-rich lipid nanodomains in the early Golgi, where viral budding occurs. Finally, S-acylation of spike allows the formation of viruses with enhanced fusion capacity. Our study points toward S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets.


Subject(s)
Acylation/physiology , COVID-19/drug therapy , Membrane Lipids/metabolism , SARS-CoV-2/pathogenicity , Acyltransferases/metabolism , Golgi Apparatus/metabolism , Golgi Apparatus/virology , Humans , Virus Assembly/physiology
10.
mBio ; 12(5): e0237121, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1440804

ABSTRACT

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.


Subject(s)
Betacoronavirus/metabolism , SARS-CoV-2/metabolism , Membrane Glycoproteins/metabolism , Nucleocapsid Proteins/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Virus Assembly/physiology
11.
Int J Biol Sci ; 17(14): 3889-3897, 2021.
Article in English | MEDLINE | ID: covidwho-1438863

ABSTRACT

Intraviral protein-protein interactions (PPIs) of SARS-CoV-2 in host cells may provide useful information for deep understanding of virology of SARS-CoV-2. In this study, 22 of 55 interactions of the structural and accessory proteins of SARS-CoV-2 were identified by biomolecular fluorescence complementation (BiFC) assay. The nucleocapsid (N) protein was found to have the most interactions among the structural and accessory proteins of SARS-CoV-2, and also specifically interacted with the putative packaging signal (PS) of SARS-CoV-2. We also demonstrated that the PS core containing PS576 RNA bears a functional PS, important for the assembly of the viral RNA into virus like particles (VLPs), and the packaging of SARS-CoV-2 RNA was N dependent.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , SARS-CoV-2/metabolism , Virus Assembly , HEK293 Cells , Humans , Phosphoproteins/metabolism , Protein Interaction Maps
12.
Nat Commun ; 12(1): 5333, 2021 09 09.
Article in English | MEDLINE | ID: covidwho-1402067

ABSTRACT

The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain, and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. Here we report a proteomic screen for cellular factors that interact with the cytoplasmic tail of S. We confirm interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding site promotes exit from the endoplasmic reticulum, and although binding to COPI should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle.


Subject(s)
COVID-19/metabolism , Cell Membrane/metabolism , Giant Cells/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , COP-Coated Vesicles/metabolism , Chlorocebus aethiops , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , HEK293 Cells , Humans , Protein Binding , Protein Domains , Proteomics , Vero Cells , Virus Assembly/genetics
13.
FASEB J ; 34(8): 9832-9842, 2020 08.
Article in English | MEDLINE | ID: covidwho-1388029

ABSTRACT

To date, the recently discovered SARS-CoV-2 virus has afflicted >6.9 million people worldwide and disrupted the global economy. Development of effective vaccines or treatments for SARS-CoV-2 infection will be aided by a molecular-level understanding of SARS-CoV-2 proteins and their interactions with host cell proteins. The SARS-CoV-2 nucleocapsid (N) protein is highly homologous to the N protein of SARS-CoV, which is essential for viral RNA replication and packaging into new virions. Emerging models indicate that nucleocapsid proteins of other viruses can form biomolecular condensates to spatiotemporally regulate N protein localization and function. Our bioinformatic analyses, in combination with pre-existing experimental evidence, suggest that the SARS-CoV-2 N protein is capable of forming or regulating biomolecular condensates in vivo by interaction with RNA and key host cell proteins. We discuss multiple models, whereby the N protein of SARS-CoV-2 may harness this activity to regulate viral life cycle and host cell response to viral infection.


Subject(s)
Coronavirus Nucleocapsid Proteins/chemistry , SARS-CoV-2/chemistry , Binding Sites , Computational Biology , Cytoplasmic Granules/chemistry , Humans , Phosphoproteins/chemistry , Protein Binding , Protein Domains , Protein Kinases/chemistry , SARS-CoV-2/physiology , Virus Assembly , Virus Replication
14.
Curr Opin Virol ; 50: 159-170, 2021 10.
Article in English | MEDLINE | ID: covidwho-1363948

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiologic agent that causes Coronavirus Disease 2019 (COVID-19) pandemic, is a newly emerging respiratory RNA virus with exceptional transmissibility and pathogenicity. Numerous COVID-19 related studies have been fast-tracked, with the ultimate goal to end the pandemic. Here we review the major stages of SARS-CoV-2 infection cycle in cells, with specific emphasis on essential host factors. Insights into the cell biology of SARS-CoV-2 infection have accelerated the development of host-directed therapeutics, as shown by dozens of clinical trials evaluating COVID-19 treatments using host-targeting compounds.


Subject(s)
COVID-19/etiology , SARS-CoV-2/physiology , COVID-19/drug therapy , Cathepsin L/physiology , Humans , RNA, Viral/biosynthesis , SARS-CoV-2/genetics , Virus Assembly , Virus Internalization
15.
Sci China Life Sci ; 65(2): 280-294, 2022 02.
Article in English | MEDLINE | ID: covidwho-1356045

ABSTRACT

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) has spread rapidly throughout the world. SARS-CoV-2 is an enveloped, plus-stranded RNA virus with a single-stranded RNA genome of approximately 30,000 nucleotides. The SARS-CoV-2 genome encodes 29 proteins, including 16 nonstructural, 4 structural and 9 accessory proteins. To date, over 1,228 experimental structures of SARS-CoV-2 proteins have been deposited in the Protein Data Bank (PDB), including 16 protein structures, two functional domain structures of nucleocapsid (N) protein, and scores of complexes. Overall, they exhibit high similarity to SARS-CoV proteins. Here, we summarize the progress of structural and functional research on SARS-CoV-2 proteins. These studies provide structural and functional insights into proteins of SARS-CoV-2, and further elucidate the daedal relationship between different components at the atomic level in the viral life cycle, including attachment to the host cell, viral genome replication and transcription, genome packaging and assembly, and virus release. It is important to understand the structural and functional properties of SARS-CoV-2 proteins as it will facilitate the development of anti-CoV drugs and vaccines to prevent and control the current SARS-CoV-2 pandemic.


Subject(s)
COVID-19/virology , Genome, Viral , SARS-CoV-2/genetics , Viral Proteins/genetics , Humans , Protein Conformation , SARS-CoV-2/physiology , Transcription, Genetic , Viral Proteins/chemistry , Virus Assembly , Virus Replication
16.
Nat Commun ; 12(1): 4629, 2021 07 30.
Article in English | MEDLINE | ID: covidwho-1333939

ABSTRACT

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events - e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.


Subject(s)
COVID-19/immunology , SARS-CoV-2/immunology , Virus Assembly/immunology , Virus Release/immunology , Virus Replication/immunology , Animals , COVID-19/epidemiology , COVID-19/virology , Chlorocebus aethiops , Cryoelectron Microscopy , Electron Microscope Tomography , Humans , Pandemics/prevention & control , SARS-CoV-2/physiology , SARS-CoV-2/ultrastructure , Vero Cells , Virus Assembly/physiology , Virus Release/physiology , Virus Replication/physiology
17.
PLoS Pathog ; 17(5): e1009519, 2021 05.
Article in English | MEDLINE | ID: covidwho-1232468

ABSTRACT

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.


Subject(s)
Amiloride/pharmacology , COVID-19/drug therapy , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Amiloride/pharmacokinetics , Animals , Antiviral Agents/pharmacology , Binding Sites/drug effects , COVID-19/virology , Chlorocebus aethiops , Coronavirus Envelope Proteins/chemistry , Humans , Ion Channels/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Binding/drug effects , Protein Conformation/drug effects , Protein Domains , Vero Cells , Virus Assembly/drug effects
18.
Cells ; 10(4)2021 04 09.
Article in English | MEDLINE | ID: covidwho-1178118

ABSTRACT

Research on infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is currently restricted to BSL-3 laboratories. SARS-CoV2 virus-like particles (VLPs) offer a BSL-1, replication-incompetent system that can be used to evaluate virus assembly and virus-cell entry processes in tractable cell culture conditions. Here, we describe a SARS-CoV2 VLP system that utilizes nanoluciferase (Nluc) fragment complementation to track assembly and entry. We utilized the system in two ways. Firstly, we investigated the requirements for VLP assembly. VLPs were produced by concomitant synthesis of three viral membrane proteins, spike (S), envelope (E), and matrix (M), along with the cytoplasmic nucleocapsid (N). We discovered that VLP production and secretion were highly dependent on N proteins. N proteins from related betacoronaviruses variably substituted for the homologous SARS-CoV2 N, and chimeric betacoronavirus N proteins effectively supported VLP production if they contained SARS-CoV2 N carboxy-terminal domains (CTD). This established the CTDs as critical features of virus particle assembly. Secondly, we utilized the system by investigating virus-cell entry. VLPs were produced with Nluc peptide fragments appended to E, M, or N proteins, with each subsequently inoculated into target cells expressing complementary Nluc fragments. Complementation into functional Nluc was used to assess virus-cell entry. We discovered that each of the VLPs were effective at monitoring virus-cell entry, to various extents, in ways that depended on host cell susceptibility factors. Overall, we have developed and utilized a VLP system that has proven useful in identifying SARS-CoV2 assembly and entry features.


Subject(s)
COVID-19/metabolism , SARS-CoV-2/physiology , Virion/physiology , Virus Assembly , Virus Internalization , Coronavirus Envelope Proteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Nucleocapsid Proteins/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Viral Matrix Proteins/metabolism
19.
Virus Res ; 297: 198382, 2021 05.
Article in English | MEDLINE | ID: covidwho-1118716

ABSTRACT

Coronaviruses are a large group of RNA viruses that infect a wide range of animal species. The replication strategy of coronaviruses involves recombination and mutation events that lead to the possibility of cross-species transmission. The high plasticity of the viral receptor due to a continuous modification of the host species habitat may be the cause of cross-species transmission that can turn into a threat to other species including the human population. The successive emergence of highly pathogenic coronaviruses such as the Severe Acute Respiratory Syndrome (SARS) in 2003, the Middle East Respiratory Syndrome Coronavirus in 2012, and the recent SARS-CoV-2 has incentivized a number of studies on the molecular basis of the coronavirus and its pathogenesis. The high degree of interrelatedness between humans and wild and domestic animals and the modification of animal habitats by human urbanization, has favored new viral spreads. Hence, knowledge on the main clinical signs of coronavirus infection in the different hosts and the distinctive molecular characteristics of each coronavirus is essential to prevent the emergence of new coronavirus diseases. The coronavirus infections routinely studied in veterinary medicine must be properly recognized and diagnosed not only to prevent animal disease but also to promote public health.


Subject(s)
Coronavirus Infections , Coronavirus , Host Specificity , Viral Zoonoses , Animals , Coronavirus/chemistry , Coronavirus/genetics , Coronavirus/physiology , Coronavirus Infections/transmission , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Genome, Viral , Humans , Open Reading Frames , RNA, Viral , Viral Proteins , Viral Structures , Viral Transcription , Viral Zoonoses/transmission , Viral Zoonoses/virology , Virus Assembly , Virus Replication
20.
Viruses ; 13(3)2021 02 26.
Article in English | MEDLINE | ID: covidwho-1115433

ABSTRACT

Ubiquitination of proteins is a post-translational modification process with many different cellular functions, including protein stability, immune signaling, antiviral functions and virus replication. While ubiquitination of viral proteins can be used by the host as a defense mechanism by destroying the incoming pathogen, viruses have adapted to take advantage of this cellular process. The ubiquitin system can be hijacked by viruses to enhance various steps of the replication cycle and increase pathogenesis. Emerging viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), flaviviruses like Zika and dengue, as well as highly pathogenic viruses like Ebola and Nipah, have the ability to directly use the ubiquitination process to enhance their viral-replication cycle, and evade immune responses. Some of these mechanisms are conserved among different virus families, especially early during virus entry, providing an opportunity to develop broad-spectrum antivirals. Here, we discuss the mechanisms used by emergent viruses to exploit the host ubiquitin system, with the main focus on the role of ubiquitin in enhancing virus replication.


Subject(s)
Ubiquitin/metabolism , Virus Diseases/metabolism , Virus Replication , Viruses/metabolism , Immune Evasion , Ubiquitination , Viral Proteins/metabolism , Virus Assembly , Virus Diseases/immunology , Virus Diseases/virology , Virus Internalization , Virus Release , Viruses/classification , Viruses/immunology , Viruses/pathogenicity
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