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1.
Protein J ; 39(3): 198-216, 2020 06.
Article in English | MEDLINE | ID: covidwho-1718840

ABSTRACT

The devastating effects of the recent global pandemic (termed COVID-19 for "coronavirus disease 2019") caused by the severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) are paramount with new cases and deaths growing at an exponential rate. In order to provide a better understanding of SARS CoV-2, this article will review the proteins found in the SARS CoV-2 that caused this global pandemic.


Subject(s)
Betacoronavirus/chemistry , Betacoronavirus/physiology , Coronavirus Infections/virology , Pneumonia, Viral/virology , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Sequence , Betacoronavirus/genetics , COVID-19 , Coronavirus Envelope Proteins , Coronavirus Infections/drug therapy , Coronavirus Infections/metabolism , Coronavirus Nucleocapsid Proteins , Drug Discovery/methods , Genome, Viral , Host-Pathogen Interactions/drug effects , Humans , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Pandemics , Phosphoproteins , Pneumonia, Viral/drug therapy , Pneumonia, Viral/metabolism , Polyproteins , Protein Interaction Maps/drug effects , SARS-CoV-2 , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Proteins/genetics , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism , Viroporin Proteins
2.
Proteins ; 90(5): 1102-1114, 2022 May.
Article in English | MEDLINE | ID: covidwho-1704604

ABSTRACT

Coronaviruses, especially severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), present an ongoing threat to human wellbeing. Consequently, elucidation of molecular determinants of their function and interaction with the host is an important task. Whereas some of the coronaviral proteins are extensively characterized, others remain understudied. Here, we use molecular dynamics simulations to analyze the structure and dynamics of the SARS-CoV-2 envelope (E) protein (a viroporin) in the monomeric form. The protein consists of the hydrophobic α-helical transmembrane domain (TMD) and amphiphilic α-helices H2 and H3, connected by flexible linkers. We show that TMD has a preferable orientation in the membrane, while H2 and H3 reside at the membrane surface. Orientation of H2 is strongly influenced by palmitoylation of cysteines Cys40, Cys43, and Cys44. Glycosylation of Asn66 affects the orientation of H3. We also observe that the monomeric E protein both generates and senses the membrane curvature, preferably localizing with the C-terminus at the convex regions of the membrane; the protein in the pentameric form displays these properties as well. Localization to curved regions may be favorable for assembly of the E protein oligomers, whereas induction of curvature may facilitate the budding of the viral particles. The presented results may be helpful for a better understanding of the function of the coronaviral E protein and viroporins in general, and for overcoming the ongoing SARS-CoV-2 pandemic.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry , Viral Envelope Proteins/chemistry
3.
Microbiol Spectr ; 10(1): e0061821, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1622002

ABSTRACT

The host transmembrane protein MARCH8 is a RING finger E3 ubiquitin ligase that downregulates various host transmembrane proteins, such as MHC-II. We have recently reported that MARCH8 expression in virus-producing cells impairs viral infectivity by reducing virion incorporation of not only HIV-1 envelope glycoprotein but also vesicular stomatitis virus G-glycoprotein through two different pathways. However, the MARCH8 inhibition spectrum remains largely unknown. Here, we show the antiviral spectrum of MARCH8 using viruses pseudotyped with a variety of viral envelope glycoproteins. Infection experiments revealed that viral envelope glycoproteins derived from the rhabdovirus, arenavirus, coronavirus, and togavirus (alphavirus) families were sensitive to MARCH8-mediated inhibition. Lysine mutations at the cytoplasmic tails of rabies virus-G, lymphocytic choriomeningitis virus glycoproteins, SARS-CoV and SARS-CoV-2 spike proteins, and Chikungunya virus and Ross River virus E2 proteins conferred resistance to MARCH8. Immunofluorescence showed impaired downregulation of the mutants of these viral envelope glycoproteins by MARCH8, followed by lysosomal degradation, suggesting that MARCH8-mediated ubiquitination leads to intracellular degradation of these envelopes. Indeed, rabies virus-G and Chikungunya virus E2 proteins proved to be clearly ubiquitinated. We conclude that MARCH8 has inhibitory activity on a variety of viral envelope glycoproteins whose cytoplasmic lysine residues are targeted by this antiviral factor. IMPORTANCE A member of the MARCH E3 ubiquitin ligase family, MARCH8, downregulates many different kinds of host transmembrane proteins, resulting in the regulation of cellular homeostasis. On the other hands, MARCH8 acts as an antiviral factor when it binds to and downregulates HIV-1 envelope glycoprotein and vesicular stomatitis virus G-glycoprotein that are viral transmembrane proteins. This study reveals that, as in the case of cellular membrane proteins, MARCH8 shows broad-spectrum inhibition against various viral envelope glycoproteins by recognizing their cytoplasmic lysine residues, resulting in lysosomal degradation.


Subject(s)
Antiviral Agents/pharmacology , Lysine/drug effects , Ubiquitin-Protein Ligases/pharmacology , Viral Envelope Proteins/chemistry , Blotting, Western , Down-Regulation , HEK293 Cells , HeLa Cells , Humans , Immunoprecipitation , Lysine/metabolism , Ubiquitination/physiology , Viral Envelope Proteins/drug effects
4.
PLoS Comput Biol ; 17(12): e1009664, 2021 12.
Article in English | MEDLINE | ID: covidwho-1571973

ABSTRACT

The evolution of circulating viruses is shaped by their need to evade antibody response, which mainly targets the viral spike. Because of the high density of spikes on the viral surface, not all antigenic sites are targeted equally by antibodies. We offer here a geometry-based approach to predict and rank the probability of surface residues of SARS spike (S protein) and influenza H1N1 spike (hemagglutinin) to acquire antibody-escaping mutations utilizing in-silico models of viral structure. We used coarse-grained MD simulations to estimate the on-rate (targeting) of an antibody model to surface residues of the spike protein. Analyzing publicly available sequences, we found that spike surface sequence diversity of the pre-pandemic seasonal influenza H1N1 and the sarbecovirus subgenus highly correlates with our model prediction of antibody targeting. In particular, we identified an antibody-targeting gradient, which matches a mutability gradient along the main axis of the spike. This identifies the role of viral surface geometry in shaping the evolution of circulating viruses. For the 2009 H1N1 and SARS-CoV-2 pandemics, a mutability gradient along the main axis of the spike was not observed. Our model further allowed us to identify key residues of the SARS-CoV-2 spike at which antibody escape mutations have now occurred. Therefore, it can inform of the likely functional role of observed mutations and predict at which residues antibody-escaping mutation might arise.


Subject(s)
Evolution, Molecular , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Animals , Antibodies, Viral/biosynthesis , Antigens, Viral/chemistry , Antigens, Viral/genetics , COVID-19/epidemiology , COVID-19/immunology , COVID-19/virology , Computational Biology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/genetics , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Host Microbial Interactions/genetics , Host Microbial Interactions/immunology , Humans , Immune Evasion/genetics , Influenza, Human/immunology , Influenza, Human/virology , Models, Immunological , Molecular Dynamics Simulation , Mutation , Pandemics , Spike Glycoprotein, Coronavirus/chemistry , Viral Envelope Proteins/chemistry
5.
Viruses ; 13(7)2021 07 08.
Article in English | MEDLINE | ID: covidwho-1300294

ABSTRACT

The emergence of novel viral infections of zoonotic origin and mutations of existing human pathogenic viruses represent a serious concern for public health. It warrants the establishment of better interventions and protective therapies to combat the virus and prevent its spread. Surface glycoproteins catalyzing the fusion of viral particles and host cells have proven to be an excellent target for antivirals as well as vaccines. This review focuses on recent advances for computational structure-based design of antivirals and vaccines targeting viral fusion machinery to control seasonal and emerging respiratory viruses.


Subject(s)
Computer Simulation , Viral Envelope Proteins/analysis , Viral Envelope Proteins/chemistry , Viral Matrix Proteins/analysis , Viral Matrix Proteins/chemistry , Animals , Antiviral Agents , Clinical Trials as Topic , Humans , Mice , Respiratory Tract Infections/virology , Vaccinology/methods , Viral Vaccines/analysis , Viruses/chemistry , Viruses/classification
6.
Viruses ; 13(6)2021 05 29.
Article in English | MEDLINE | ID: covidwho-1282636

ABSTRACT

An effective vaccine for the hepatitis C virus (HCV) is a major unmet medical and public health need, and it requires an antigen that elicits immune responses to multiple key conserved epitopes. Decades of research have generated a number of vaccine candidates; based on these data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice. One bottleneck in the development of an E1E2-based vaccine is that the antigen is challenging to produce in large quantities and at high levels of purity and antigenic/functional integrity. This review describes the production and characterization of E1E2-based vaccine antigens, both membrane-associated and a novel secreted form of E1E2, with a particular emphasis on the major challenges facing the field and how those challenges can be addressed.


Subject(s)
Hepacivirus/chemistry , Hepatitis C/prevention & control , Viral Envelope Proteins/chemistry , Viral Hepatitis Vaccines/chemistry , Animals , Epitopes/immunology , HEK293 Cells , Hepacivirus/genetics , Hepacivirus/immunology , Hepatitis C/virology , Humans , Mice , Models, Molecular , Protein Conformation , Protein Multimerization , Viral Envelope Proteins/immunology , Viral Envelope Proteins/metabolism
7.
Int J Mol Sci ; 22(12)2021 Jun 18.
Article in English | MEDLINE | ID: covidwho-1273462

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 coronavirus deeply affected the world community. It gave a strong impetus to the development of not only approaches to diagnostics and therapy, but also fundamental research of the molecular biology of this virus. Fluorescence microscopy is a powerful technology enabling detailed investigation of virus-cell interactions in fixed and live samples with high specificity. While spatial resolution of conventional fluorescence microscopy is not sufficient to resolve all virus-related structures, super-resolution fluorescence microscopy can solve this problem. In this paper, we review the use of fluorescence microscopy to study SARS-CoV-2 and related viruses. The prospects for the application of the recently developed advanced methods of fluorescence labeling and microscopy-which in our opinion can provide important information about the molecular biology of SARS-CoV-2-are discussed.


Subject(s)
Microscopy, Fluorescence , SARS-CoV-2/physiology , COVID-19/pathology , COVID-19/virology , Endocytosis , Fluorescent Dyes/chemistry , Genes, Reporter , Humans , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , Virus Internalization
8.
Int J Mol Sci ; 22(11)2021 Jun 01.
Article in English | MEDLINE | ID: covidwho-1259507

ABSTRACT

The COVID-19 pandemic is caused by the 2019-nCoV/SARS-CoV-2 virus. This severe acute respiratory syndrome is currently a global health emergency and needs much effort to generate an urgent practical treatment to reduce COVID-19 complications and mortality in humans. Viral infection activates various cellular responses in infected cells, including cellular stress responses such as unfolded protein response (UPR) and autophagy, following the inhibition of mTOR. Both UPR and autophagy mechanisms are involved in cellular and tissue homeostasis, apoptosis, innate immunity modulation, and clearance of pathogens such as viral particles. However, during an evolutionary arms race, viruses gain the ability to subvert autophagy and UPR for their benefit. SARS-CoV-2 can enter host cells through binding to cell surface receptors, including angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP1). ACE2 blockage increases autophagy through mTOR inhibition, leading to gastrointestinal complications during SARS-CoV-2 virus infection. NRP1 is also regulated by the mTOR pathway. An increased NRP1 can enhance the susceptibility of immune system dendritic cells (DCs) to SARS-CoV-2 and induce cytokine storm, which is related to high COVID-19 mortality. Therefore, signaling pathways such as mTOR, UPR, and autophagy may be potential therapeutic targets for COVID-19. Hence, extensive investigations are required to confirm these potentials. Since there is currently no specific treatment for COVID-19 infection, we sought to review and discuss the important roles of autophagy, UPR, and mTOR mechanisms in the regulation of cellular responses to coronavirus infection to help identify new antiviral modalities against SARS-CoV-2 virus.


Subject(s)
Autophagy , COVID-19/pathology , Neuropilin-1/metabolism , Unfolded Protein Response , Antiviral Agents/pharmacology , Autophagy/drug effects , COVID-19/virology , Humans , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Signal Transduction/drug effects , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism
9.
Autoimmunity ; 54(4): 213-224, 2021 06.
Article in English | MEDLINE | ID: covidwho-1201340

ABSTRACT

Currently, the novel coronavirus pneumonia has been widespread globally, and there is no specific medicine. In response to the emergency, we employed bioinformatics methods to investigate the virus's pathogenic mechanism, finding possible control methods. We speculated in previous studies that E protein was associated with viral infectivity. The present study adopted the domain search techniques to analyse the E protein. According to the results, the E protein could bind iron or haem. The iron and haem bound by the E protein came from the attacked haemoglobin and phagocytes. When E protein was attached to haem, it synthesised oxygen and water into superoxide anions, hydrogen peroxide and hydroxyl radicals. When the iron-bound E protein and the haem-bound E protein worked together, they converted superoxide anions and hydrogen peroxide into oxygen and water. These were the "ROS attack" and "ROS escape" of the virus. "ROS attack" damaged the tissues or cells exposed on the surface of the virus, and "ROS escape" decomposed the superoxide anion and hydrogen peroxide that attacked the virus. When NK cells were exposed to infected cells, viruses that had not shed from the infected cells' surface damaged them through "ROS attack". In addition, lymphocytes such as T cells and B cells, which could be close to the antigen of the virus surface, were also easily damaged or killed by the "ROS attack", generating a decrease in lymphocytes. When memory B cells were exposed to the virus's surface antigen, they were also damaged by "ROS attack", resulting in the patient's re-infection. The virus applied the "ROS escape" to decompose hydrogen peroxide released by phagocytes into oxygen and water. The surrounding cells were replenished with oxygen, and the patient was in a "happy hypoxia" state. When the phagocytes swallowed the virus, the E protein converted superoxide anions into oxygen and water. In this way, the virus parasitized in the vesicles of the phagocyte. While virus was in the lysosome, the E protein generated ROS to damage nearby hydrolases. In this way, the virus parasitized the lysosome. Excessive hydroxyl free radicals destroyed the membrane structure of the lysosome, causing the hydrolase release from lysosome, autophagy of phagocytic cells and subsequent cell death. As a result, the colonizing phagocytes of the virus was associated with asymptomatic infection or retest-positive. Briefly, the virus inhibited the immune system through "ROS escape", and damaged the immune system by "ROS attack". The destruction instigated a strong cytokine storm, leading to organ failure and complications.


Subject(s)
COVID-19/etiology , COVID-19/metabolism , Disease Susceptibility , Host-Pathogen Interactions , Immune System/immunology , Immune System/metabolism , Iron/metabolism , Reactive Oxygen Species/metabolism , SARS-CoV-2/physiology , Amino Acid Sequence , Catalysis , Computational Biology/methods , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Immune System/pathology , Models, Molecular , Protein Conformation , Structure-Activity Relationship , Superoxide Dismutase/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism
10.
mBio ; 12(2)2021 03 16.
Article in English | MEDLINE | ID: covidwho-1138303

ABSTRACT

An emerging class of cellular inhibitory proteins has been identified that targets viral glycoproteins. These include the membrane-associated RING-CH (MARCH) family of E3 ubiquitin ligases that, among other functions, downregulate cell surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to function by catalyzing the ubiquitination of the cytoplasmic tails (CTs) of target proteins, leading to their degradation. MARCH proteins have recently been reported to target retroviral envelope glycoproteins (Env) and vesicular stomatitis virus G glycoprotein (VSV-G). However, the mechanism of antiviral activity remains poorly defined. Here we show that MARCH8 antagonizes the full-length forms of HIV-1 Env, VSV-G, Ebola virus glycoprotein (EboV-GP), and the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), thereby impairing the infectivity of virions pseudotyped with these viral glycoproteins. This MARCH8-mediated targeting of viral glycoproteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that MARCH8 protein antagonism of VSV-G is CT dependent. In contrast, MARCH8-mediated targeting of HIV-1 Env, EboV-GP, and SARS-CoV-2 S protein by MARCH8 does not require the CT, suggesting a novel mechanism of MARCH-mediated antagonism of these viral glycoproteins. Confocal microscopy data demonstrate that MARCH8 traps the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in several relevant human cell types is rapidly inducible by type I interferon. These results help to inform the mechanism by which MARCH proteins exert their antiviral activity and provide insights into the role of cellular inhibitory factors in antagonizing the biogenesis, trafficking, and virion incorporation of viral glycoproteins.IMPORTANCE Viral envelope glycoproteins are an important structural component on the surfaces of enveloped viruses that direct virus binding and entry and also serve as targets for the host adaptive immune response. In this study, we investigate the mechanism of action of the MARCH family of cellular proteins that disrupt the trafficking and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into how host cell factors antagonize viral replication, perhaps opening new avenues for therapeutic intervention in the replication of a diverse group of highly pathogenic enveloped viruses.


Subject(s)
Membrane Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Viral Envelope Proteins/metabolism , Amino Acid Sequence , Cells, Cultured , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Interferons/pharmacology , Intracellular Space/metabolism , Membrane Proteins/genetics , Mutation , RNA Viruses/classification , RNA Viruses/metabolism , Species Specificity , Ubiquitin-Protein Ligases/genetics , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Virion/metabolism , Virus Replication
11.
J Struct Biol ; 213(2): 107713, 2021 06.
Article in English | MEDLINE | ID: covidwho-1117175

ABSTRACT

The high SARS-CoV-2 reproductive number driving the COVID-19 pandemic has been a mystery. Our recent in vitro, and in vivo coronaviral pathogenesis studies involving Mouse Hepatitis Virus (MHV-A59) suggest a crucial role for a small host membrane-virus contact initiator region of the Spike protein, called the fusion peptide that enhances the virus fusogenicity and infectivity. Here I study the Spike from five human ß-coronaviruses (HCoV) including the SARS-CoV-2, and MHV-A59 for comparison. The structural and dynamics analyses of the Spike show that its fusion loop spatially organizes three fusion peptides contiguous to each other to synergistically trigger the virus-host membrane fusion process. I propose a Contact Initiation Model based on the architecture of the Spike quaternary structure that explains the obligatory participation of the fusion loop in the initiation of the host membrane contact for the virus fusion process. Among all the HCoV Spikes in this study, SARS-CoV-2 has the most hydrophobic surface and the extent of hydrophobicity correlates with the reproductive number and infectivity of the other HCoV. Comparison between results from standard and replica exchange molecular dynamics reveal the unique physicochemical properties of the SARS-CoV-2 fusion peptides, accrued in part from the presence of consecutive prolines that impart backbone rigidity which aids the virus fusogenicity. The priming of the Spike by its cleavage and subsequent fusogenic conformational transition steered by the fusion loop may be critical for the SARS-CoV-2 spread. The importance of the fusion loop makes it an apt target for anti-virals and vaccine candidates.


Subject(s)
COVID-19/prevention & control , Peptides/chemistry , Protein Domains , Protein Structure, Secondary , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Sequence , COVID-19/epidemiology , COVID-19/virology , Humans , Models, Molecular , Pandemics , Peptides/genetics , Peptides/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Sequence Homology, Amino Acid , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Static Electricity , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Virus Internalization
12.
Int J Biol Macromol ; 177: 1-9, 2021 Apr 30.
Article in English | MEDLINE | ID: covidwho-1071367

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from China has become a global threat due to the continuous rise in cases of Coronavirus disease 2019 (COVID-19). The problem with COVID-19 therapeutics is due to complexity of the mechanism of the pathogenesis of this virus. In this review, an extensive analysis of genome architecture and mode of pathogenesis of SARS-CoV-2 with an emphasis on therapeutic approaches is performed. SARS-CoV-2 genome consists of a single, ~29.9 kb long RNA having significant sequence similarity to BAT-CoV, SARS-CoV and MERS-CoV genome. Two-third part of SARS-Cov-2 genome comprises of ORF (ORF1ab) resulting in the formation of 2 polyproteins, pp1a and pp1ab, later processed into 16 smaller non-structural proteins (NSPs). The four major structural proteins of SARS-CoV-2 are the spike surface glycoprotein (S), a small envelope (E), membrane (M), and nucleocapsid (N) proteins. S protein helps in receptor binding and membrane fusion and hence plays the most important role in the transmission of CoVs. Priming of S protein is done by serine 2 transmembrane protease and thus plays a key role in virus and host cell fusion. This review highlights the possible mechanism of action of SARS-CoV-2 to search for possible therapeutic options.


Subject(s)
COVID-19/drug therapy , Genome, Viral , SARS-CoV-2/genetics , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Humans , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/metabolism , Peptide Hydrolases/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/physiology , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism
13.
Methods Enzymol ; 653: 207-235, 2021.
Article in English | MEDLINE | ID: covidwho-1051391

ABSTRACT

The SARS-CoV-2 3a protein is a putative ion channel implicated in virus life cycle and pathogenesis. We recently expressed, purified, and reconstituted 3a into lipid nanodiscs to solve its structure by cryo-EM to 2.1Å resolution. In this chapter, we describe methods we developed in order to facilitate the study of this protein in other laboratories. We emphasize factors that enabled rapid progression from gene sequence to reconstituted protein (3 weeks in the case of 3a) and provide general observations and tips for adapting these protocols to other membrane proteins of interest.


Subject(s)
Ion Channels/chemistry , Nanostructures , SARS-CoV-2/chemistry , Viral Envelope Proteins/chemistry , Viroporin Proteins/chemistry , Lipid Bilayers/chemistry
14.
Viruses ; 12(9)2020 09 22.
Article in English | MEDLINE | ID: covidwho-973229

ABSTRACT

Coronaviruses (CoVs) are enveloped, positive sense, single strand RNA viruses that cause respiratory, intestinal and neurological diseases in mammals and birds. Following replication, CoVs assemble on intracellular membranes including the endoplasmic reticulum Golgi intermediate compartment (ERGIC) where the envelope protein (E) functions in virus assembly and release. In consequence, E potentially contains membrane-modifying peptides. To search for such peptides, the E coding sequence of Mouse Hepatitis Virus (MHV) was inspected for its amino acid conservation, proximity to the membrane and/or predicted amphipathic helices. Peptides identified in silico were synthesized and tested for membrane-modifying activity in the presence of giant unilamellar vesicles (GUVs) consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), sphingomyelin and cholesterol. To confirm the presence of membrane binding peptides identified in the context of a full-length E protein, the wild type and a number of mutants in the putative membrane binding peptide were expressed in Lenti-X-293T mammalian and insect cells, and the distribution of E antigen within the expressing cell was assessed. Our data identify a role for the post-transmembrane region of MHV E in membrane binding.


Subject(s)
Murine hepatitis virus/chemistry , Peptides/chemistry , Viral Envelope Proteins/chemistry , Amino Acid Sequence , Animals , Cell Line , Coronavirus Infections , Humans , Intracellular Membranes/metabolism , Mice , Murine hepatitis virus/genetics , Murine hepatitis virus/metabolism , Mutation , Peptides/chemical synthesis , Peptides/metabolism , Sf9 Cells , Spodoptera , Unilamellar Liposomes/metabolism , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism
15.
J Gen Virol ; 102(1)2021 01.
Article in English | MEDLINE | ID: covidwho-910292

ABSTRACT

Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293TCD81KO). Clones made in 293TCD81KO cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T cells and Freestyle 293-F (293-F) cells exhibit important differences. We found that 293-F-produced sE2 harbours mostly complex-type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex-type glycans and high-mannose or hybrid-type glycans. Moreover, sE2 produced in 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293-F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.


Subject(s)
Hepacivirus/physiology , Viral Envelope Proteins/metabolism , Antibody Affinity , Gene Knockdown Techniques , HEK293 Cells , Hepacivirus/immunology , Hepatitis C/virology , Hepatitis C Antibodies/immunology , Hepatitis C Antigens/immunology , Hepatitis C Antigens/metabolism , Humans , Mannose/chemistry , Polysaccharides/chemistry , Protein Binding , Receptors, Virus/genetics , Receptors, Virus/metabolism , Tetraspanin 28/genetics , Tetraspanin 28/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/immunology
16.
Virol J ; 17(1): 165, 2020 10 29.
Article in English | MEDLINE | ID: covidwho-895011

ABSTRACT

BACKGROUND: In order to obtain antibodies that recognize natural proteins, it is possible to predict the antigenic determinants of natural proteins, which are eventually embodied as polypeptides. The polypeptides can be coupled with corresponding vectors to stimulate the immune system to produce corresponding antibodies, which is also a simple and effective vaccine development method. The discovery of epitopes is helpful to the development of SARS-CoV-2 vaccine. METHODS: The analyses were related to epitopes on 3 proteins, including spike (S), envelope (E) and membrane (M) proteins, which are located on the lipid envelope of the SARS-CoV-2. Based on the NCBI Reference Sequence: NC_045512.2, the conformational and linear B cell epitopes of the surface protein were predicted separately by various prediction methods. Furthermore, the conservation of the epitopes, the adaptability and other evolutionary characteristics were also analyzed, the sequences of the whole genome of SARS-CoV-2 were obtained from the GISAID. RESULTS: 7 epitopes were predicted, including 6 linear epitopes and 1 conformational epitope. One of the linear and one of the conformational consist of identical sequence, but represent different forms of epitopes. It is worth mentioning that all 6 identified epitopes were conserved in nearly 3500 SARS-CoV-2 genomes, showing that it is helpful to obtain stable and long-acting epitopes under the condition of high frequency of amino acid mutation, which deserved further study at the experiment level. CONCLUSION: The findings would facilitate the vaccine development, had the potential to be directly applied on the prevention in this disease, but also have the potential to prevent the possible threats caused by other types of coronavirus.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/virology , Epitopes, B-Lymphocyte/immunology , Pneumonia, Viral/virology , Viral Envelope Proteins/immunology , Viral Matrix Proteins/immunology , COVID-19 , COVID-19 Vaccines , Computational Biology , Coronavirus Envelope Proteins , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Humans , Immunogenicity, Vaccine/immunology , Models, Molecular , Pandemics , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology , Viral Envelope Proteins/chemistry , Viral Vaccines/immunology
17.
PLoS One ; 15(8): e0237300, 2020.
Article in English | MEDLINE | ID: covidwho-842269

ABSTRACT

The outbreak of COVID-19 across the world has posed unprecedented and global challenges on multiple fronts. Most of the vaccine and drug development has focused on the spike proteins and viral RNA-polymerases and main protease for viral replication. Using the bioinformatics and structural modelling approach, we modelled the structure of the envelope (E)-protein of novel SARS-CoV-2. The E-protein of this virus shares sequence similarity with that of SARS- CoV-1, and is highly conserved in the N-terminus regions. Incidentally, compared to spike proteins, E proteins demonstrate lower disparity and mutability among the isolated sequences. Using homology modelling, we found that the most favorable structure could function as a gated ion channel conducting H+ ions. Combining pocket estimation and docking with water, we determined that GLU 8 and ASN 15 in the N-terminal region were in close proximity to form H-bonds which was further validated by insertion of the E protein in an ERGIC-mimic membrane. Additionally, two distinct "core" structures were visible, the hydrophobic core and the central core, which may regulate the opening/closing of the channel. We propose this as a mechanism of viral ion channeling activity which plays a critical role in viral infection and pathogenesis. In addition, it provides a structural basis and additional avenues for vaccine development and generating therapeutic interventions against the virus.


Subject(s)
Betacoronavirus/chemistry , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Vaccines , Computer Simulation , Coronavirus Envelope Proteins , Coronavirus Infections/virology , Humans , Hydrogen , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Models, Molecular , Pneumonia, Viral/virology , Point Mutation , Protein Conformation , SARS-CoV-2 , Structural Homology, Protein , Vaccines, Attenuated , Vaccines, Inactivated , Viral Envelope Proteins/immunology , Viral Vaccines , Water/chemistry
18.
ACS Sens ; 5(10): 3043-3048, 2020 10 23.
Article in English | MEDLINE | ID: covidwho-801107

ABSTRACT

Mass testing is fundamental to face the pandemic caused by the coronavirus SARS-CoV-2 discovered at the end of 2019. To this aim, it is necessary to establish reliable, fast, and cheap tools to detect viral particles in biological material so to identify the people capable of spreading the infection. We demonstrate that a colorimetric biosensor based on gold nanoparticle (AuNP) interaction induced by SARS-CoV-2 lends itself as an outstanding tool for detecting viral particles in nasal and throat swabs. The extinction spectrum of a colloidal solution of multiple viral-target gold nanoparticles-AuNPs functionalized with antibodies targeting three surface proteins of SARS-CoV-2 (spike, envelope, and membrane)-is red-shifted in few minutes when mixed with a solution containing the viral particle. The optical density of the mixed solution measured at 560 nm was compared to the threshold cycle (Ct) of a real-time PCR (gold standard for detecting the presence of viruses) finding that the colorimetric method is able to detect very low viral load with a detection limit approaching that of the real-time PCR. Since the method is sensitive to the infecting viral particle rather than to its RNA, the achievements reported here open a new perspective not only in the context of the current and possible future pandemics, but also in microbiology, as the biosensor proves itself to be a powerful though simple tool for measuring the viral particle concentration.


Subject(s)
Betacoronavirus/chemistry , Colorimetry/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Nasal Mucosa/virology , Pharynx/virology , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Biosensing Techniques , COVID-19 , Gold , Humans , Membrane Proteins/chemistry , Metal Nanoparticles , Pandemics , Photochemistry , Polymerase Chain Reaction , SARS-CoV-2 , Specimen Handling , Spike Glycoprotein, Coronavirus/chemistry , Threshold Limit Values , Viral Envelope Proteins/chemistry
19.
Open Biol ; 10(9): 200209, 2020 09.
Article in English | MEDLINE | ID: covidwho-751934

ABSTRACT

Coronavirus E protein is a small membrane protein found in the virus envelope. Different coronavirus E proteins share striking biochemical and functional similarities, but sequence conservation is limited. In this report, we studied the E protein topology from the new SARS-CoV-2 virus both in microsomal membranes and in mammalian cells. Experimental data reveal that E protein is a single-spanning membrane protein with the N-terminus being translocated across the membrane, while the C-terminus is exposed to the cytoplasmic side (Ntlum/Ctcyt). The defined membrane protein topology of SARS-CoV-2 E protein may provide a useful framework to understand its interaction with other viral and host components and contribute to establish the basis to tackle the pathogenesis of SARS-CoV-2.


Subject(s)
Betacoronavirus/metabolism , Eukaryota/metabolism , Viral Envelope Proteins/metabolism , Amino Acid Sequence , Betacoronavirus/isolation & purification , COVID-19 , Cell Membrane/metabolism , Coronavirus Envelope Proteins , Coronavirus Infections/pathology , Coronavirus Infections/virology , Eukaryota/cytology , Humans , Microsomes/metabolism , Mutation , Pandemics , Phylogeny , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SARS-CoV-2 , Sequence Alignment , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/classification , Viral Envelope Proteins/genetics
20.
Microbes Infect ; 22(10): 592-597, 2020.
Article in English | MEDLINE | ID: covidwho-744191

ABSTRACT

The Envelope (E) protein of SARS-CoV-2 is the most enigmatic protein among the four structural ones. Most of its current knowledge is based on the direct comparison to the SARS E protein, initially mistakenly undervalued and subsequently proved to be a key factor in the ER-Golgi localization and in tight junction disruption. We compared the genomic sequences of E protein of SARS-CoV-2, SARS-CoV and the closely related genomes of bats and pangolins obtained from the GISAID and GenBank databases. When compared to the known SARS E protein, we observed a significant difference in amino acid sequence in the C-terminal end of SARS-CoV-2 E protein. Subsequently, in silico modelling analyses of E proteins conformation and docking provide evidences of a strengthened binding of SARS-CoV-2 E protein with the tight junction-associated PALS1 protein. Based on our computational evidences and on data related to SARS-CoV, we believe that SARS-CoV-2 E protein interferes more stably with PALS1 leading to an enhanced epithelial barrier disruption, amplifying the inflammatory processes, and promoting tissue remodelling. These findings raise a warning on the underestimated role of the E protein in the pathogenic mechanism and open the route to detailed experimental investigations.


Subject(s)
COVID-19/metabolism , Membrane Proteins/chemistry , Nucleoside-Phosphate Kinase/chemistry , SARS-CoV-2/chemistry , Tight Junctions/chemistry , Viral Envelope Proteins/chemistry , Amino Acid Sequence , Animals , COVID-19/genetics , Chiroptera/virology , Databases, Genetic , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Dynamics Simulation , Nucleoside-Phosphate Kinase/genetics , Nucleoside-Phosphate Kinase/metabolism , Pangolins/virology , SARS Virus/chemistry , SARS Virus/genetics , SARS Virus/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Tight Junctions/metabolism , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism
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