ABSTRACT
This article is aimed at analyzing the structure and function of the spike (S) proteins of porcine enteric coronaviruses, including transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) by applying bioinformatics methods. The physical and chemical properties, hydrophilicity and hydrophobicity, transmembrane region, signal peptide, phosphorylation and glycosylation sites, epitope, functional domains, and motifs of S proteins of porcine enteric coronaviruses were predicted and analyzed through online software. The results showed that S proteins of TGEV, PEDV, SADS-CoV, and PDCoV all contained transmembrane regions and signal peptide. TGEV S protein contained 139 phosphorylation sites, 24 glycosylation sites, and 53 epitopes. PEDV S protein had 143 phosphorylation sites, 22 glycosylation sites, and 51 epitopes. SADS-CoV S protein had 109 phosphorylation sites, 20 glycosylation sites, and 43 epitopes. PDCoV S protein had 124 phosphorylation sites, 18 glycosylation sites, and 52 epitopes. Moreover, TGEV, PEDV, and PDCoV S proteins all contained two functional domains and two motifs, spike_rec_binding and corona_S2. The corona_S2 consisted of S2 subunit heptad repeat 1 (HR1) and S2 subunit heptad repeat 2 (HR2) region profiles. Additionally, SADS-CoV S protein was predicted to contain only one functional domain, the corona_S2. This analysis of the biological functions of porcine enteric coronavirus spike proteins can provide a theoretical basis for the design of antiviral drugs.
Subject(s)
Coronavirus Infections/epidemiology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/ultrastructure , Alphacoronavirus/metabolism , Alphacoronavirus/pathogenicity , Animals , Computational Biology/methods , Coronavirus/immunology , Coronavirus/ultrastructure , Databases, Genetic , Deltacoronavirus/metabolism , Deltacoronavirus/pathogenicity , Epitopes/immunology , Porcine epidemic diarrhea virus/metabolism , Porcine epidemic diarrhea virus/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Swine/virology , Swine Diseases/virology , Transmissible gastroenteritis virus/metabolism , Transmissible gastroenteritis virus/pathogenicityABSTRACT
In the current pandemic times, medical physicists may not be aware that there is an interesting story on two significant discoveries related to the coronavirus. One is the invention of the polymerase chain reaction (PCR) and the other is the first electron microscopic observation and identification of the coronavirus. Both of them were disregarded by the reviewers and major journals declined to publish these discoveries. These days, PCR, for example, is a widespread method for analyzing DNA, having a profound effect on healthcare, especially now during the Covid-19 pandemic. Prejudice or perhaps ignorance prevail in every aspect of our society, and there is no exception in scientific research. We need to, however, learn from these two stories and be open-minded about novel discoveries and findings - as they may be just disruptive in the "right" way to lead to an unexpected breakthrough.
Subject(s)
Coronavirus , Periodicals as Topic/history , Prejudice/history , Scholarly Communication/history , COVID-19 , Coronavirus/classification , Coronavirus/ultrastructure , Coronavirus Infections , History, 20th Century , Humans , Microscopy, Immunoelectron/history , Pandemics , Pneumonia, Viral , Polymerase Chain Reaction/historyABSTRACT
PURPOSE OF REVIEW: COronaVirus Disease 2019 (COVID-19) has spread at unprecedented speed and scale into a global pandemic with cardiovascular risk factors and complications emerging as important disease modifiers. We aim to review available clinical and biomedical literature on cardiovascular risks of COVID-19. RECENT FINDINGS: SARS-CoV2, the virus responsible for COVID-19, enters the cell via ACE2 expressed in select organs. Emerging epidemiological evidence suggest cardiovascular risk factors are associated with increased disease severity and mortality in COVID-19 patients. Patients with a more severe form of COVID-19 are also more likely to develop cardiac complications such as myocardial injury and arrhythmia. The true incidence of and mechanism underlying these events remain elusive. Cardiovascular diseases appear intricately linked with COVID-19, with cardiac complications contributing to the elevated morbidity/mortality of COVID-19. Robust epidemiologic and biologic studies are urgently needed to better understand the mechanism underlying these associations to develop better therapies.
Subject(s)
Cardiovascular Diseases/mortality , Cardiovascular Diseases/physiopathology , Coronavirus Infections/mortality , Coronavirus Infections/physiopathology , Pneumonia, Viral/mortality , Pneumonia, Viral/physiopathology , Angiotensin-Converting Enzyme 2 , Betacoronavirus/metabolism , Betacoronavirus/pathogenicity , Betacoronavirus/ultrastructure , COVID-19 , Cardiovascular Diseases/epidemiology , Cardiovascular Diseases/virology , Comorbidity , Coronavirus/metabolism , Coronavirus/pathogenicity , Coronavirus/ultrastructure , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Humans , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , Risk Factors , SARS-CoV-2 , Treatment OutcomeABSTRACT
Coronaviruses (CoVs) are by far the largest group of known positive-sense RNA viruses having an extensive range of natural hosts. In the past few decades, newly evolved Coronaviruses have posed a global threat to public health. The immune response is essential to control and eliminate CoV infections, however, maladjusted immune responses may result in immunopathology and impaired pulmonary gas exchange. Gaining a deeper understanding of the interaction between Coronaviruses and the innate immune systems of the hosts may shed light on the development and persistence of inflammation in the lungs and hopefully can reduce the risk of lung inflammation caused by CoVs. In this review, we provide an update on CoV infections and relevant diseases, particularly the host defense against CoV-induced inflammation of lung tissue, as well as the role of the innate immune system in the pathogenesis and clinical treatment.
Subject(s)
Coronavirus Infections/immunology , Coronavirus/immunology , Adaptive Immunity , Animals , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , B-Lymphocytes/immunology , Coronavirus/classification , Coronavirus/physiology , Coronavirus/ultrastructure , Coronavirus Infections/pathology , Dendritic Cells/immunology , Humans , Immunity, Innate , Inflammation , Lung/immunology , Lung/pathology , Pneumonia, Viral/immunology , Pneumonia, Viral/pathology , Receptors, Pattern Recognition/immunology , Receptors, Pattern Recognition/metabolism , T-Lymphocytes/immunologyABSTRACT
A new coronavirus outbreak emerged on the 31st of December 2019 in Wuhan, China, causing commotion among the medical community and the rest of the world. This new species of coronavirus has been termed 2019-nCoV and has caused a considerable number of cases of infection and deaths in China and, to a growing degree, beyond China, becoming a worldwide public health emergency.2019-nCoV has high homology to other pathogenic coronaviruses, such as those originating from bat-related zoonosis (SARS-CoV), which caused approximately 646 deaths in China at the start of the decade. The mortality rate for 2019-nCoV is not as high (approximately 2-3%), but its rapid propagation has resulted in the activation of protocols to stop its spread. This pathogen has the potential to become a pandemic. It is therefore vital to follow the personal care recommendations issued by the World Health Organization
Un nuevo brote de coronavirus surgió el pasado 31 de diciembre de 2019 en Wuhan, China, causando conmoción entre la comunidad médica y el resto del mundo. Esta nueva especie de coronavirus fue denominada como 2019-nCoV, causante de un gran número de casos y fallecimientos en China y en cantidad creciente fuera de ella, convirtiéndose en una emergencia de salud pública a nivel mundial.2019-nCoV es un virus con alta homología con otros coronavirus patogénicos, como los originados por zoonosis con murciélagos (SARS-CoV) causantes de aproximadamente 646 muertes en China a principios de la década. Su tasa de mortalidad no es tan elevada (aproximadamente del 2-3%), pero su rápida propagación ha propiciado la activación de protocolos para detener su diseminación. Este patógeno tiene el potencial para convertirse en pandemia, por lo que es vital seguir las recomendaciones de cuidado personal dictadas por la Organización Mundial de la Salud
Subject(s)
Humans , Coronavirus Infections/epidemiology , Coronavirus/pathogenicity , Pneumonia, Viral/epidemiology , Disease Transmission, Infectious/prevention & control , RNA, Viral/genetics , Pandemics , Disease Outbreaks , Coronavirus Infections/prevention & control , Communicable Disease Control/methods , Coronavirus/ultrastructureABSTRACT
Porcine deltacoronavirus (PDCoV) is a newly emerging enteric pathogen in swine that causes diarrhea in neonatal piglets and creates an additional economic burden on porcine industries in Asia and North America. In this study, a PDCoV isolate, CHN-SC2015, was isolated from Sichuan Province in southwest China. The isolate was characterized by a cytopathic effect, immunofluorescence, and electron microscopy. CHN-SC2015 titers in LLC-PK cells ranged from 104.31 to 108.22 TCID50/mL during the first 30 passages. During serial passage, 11 nucleotide mutations occurred in the S gene, resulting in nine amino acid changes. A whole genome sequencing analysis demonstrated that CHN-SC2015 shares 97.5%-99.1% identity with 59 reference strains in GenBank. Furthermore, CHN-SC2015 contained 6-nt deletion and 9-nt insertion in the ORF1ab gene, 3-nt deletion in the S gene and 11-nt deletion in its 3'UTR compared with other reference strains available in GenBank. A phylogenetic analysis showed that CHN-SC2015 is more closely related to other PDCoV strains in China than to the strains from Southeast Asia, USA, Japan, and South Korea, indicating the diversity of genetic relationships and regional and epidemic characteristics among these strains. A recombination analysis indicated that CHN-SC2015 experienced recombination events between SHJS/SL/2016 and TT-1115. In vivo infection demonstrated that CHN-SC2015 is highly pathogenic to sucking piglets, causing diarrhea, vomiting, dehydration, and death. Virus was shed daily in the feces of infected piglets and upon necropsy, was found distributed in the gastrointestinal tract and in multiple organs. CHN-SC2015 is the first systematically characterized strain from southwest China hitherto reported. Our results enrich the body of information on the epidemiology, pathogenicity and molecular evolution associated with PDCoV.
Subject(s)
Coronavirus Infections/veterinary , Coronavirus/physiology , Disease Susceptibility , Swine Diseases/epidemiology , Swine Diseases/virology , Animals , China/epidemiology , Coronavirus/classification , Coronavirus/isolation & purification , Coronavirus/ultrastructure , Evolution, Molecular , Genome, Viral , Genomics/methods , Mutation , Phylogeny , Swine , Swine Diseases/pathology , Virus ReplicationABSTRACT
Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe diarrhea. The virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to address questions regarding virus-host cell interactions for this genera of coronavirus. Here, we presented a detailed study of PDCoV-induced replication organelles. All positive-strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the Alpha-, Beta-, and Gammacoronavirus genera have been characterized. All coronavirus genera induced the formation of double-membrane vesicles (DMVs). In addition, Alpha- and Betacoronaviruses induce the formation of convoluted membranes, while Gammacoronaviruses induce the formation of zippered endoplasmic reticulum (ER) with tethered double-membrane spherules. However, the structures induced by Deltacoronaviruses, particularly the presence of convoluted membranes or double-membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis, and progeny particle release determined. Subsequently, virus-induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double-membrane vesicles. Significantly, however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum, small associated tethered vesicles, and double-membrane spherules. These structures strongly resemble the replication organelle induced by avian Gammacoronavirus infectious bronchitis virus.
Subject(s)
Coronavirus , Endoplasmic Reticulum/ultrastructure , Intracellular Membranes/ultrastructure , Organelles/ultrastructure , Virus Replication , Animals , Cell Line , Coronavirus/physiology , Coronavirus/ultrastructure , Coronavirus Infections/virology , Endoplasmic Reticulum/virology , Host-Pathogen Interactions , Intracellular Membranes/virology , Kinetics , Organelles/virology , RNA, Viral/biosynthesis , SwineABSTRACT
Transmissible gastroenteritis (TGE) is a contagious and infectious disease that is characterized by severe vomiting and diarrhea of swine , especially piglet, and caused by transmissible gastroenteritis coronavirus (TGEV) . TGEV infection provokes mitochondrial damage of porcine intestinal epthelial cell (IPEC), which is responsible for inflammation and cell death. In our previous study, we have demonstrated that circular RNA circEZH2 was down-regulated during TGEV infection and promoted the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) via targeting miR-22 in porcine intestinal epithelial cell line (IPEC-J2). Activation of NF-κB is an important factor for mitochondrial damage. Mitochondrial permeability transition pore (mPTP) opening is a key reason for mitochondrial damage. So, we speculate that circEZH2 may regulate TGEV-induced mPTP opening via NF-kB pathway. In the present study, we found that mPTP opening of IPEC-J2 was occured during TGEV infection and suppressed by circEZH2 via attaching miR-22. Hexokinase 2 (HK2) and interleukin 6 (IL-6) were identified as the targets of miR-22. Silencing HK2 enhanced TGEV-induced mPTP opening, while no effect on NF-κB pathway. Silencing IL-6 promoted TGEV-induced mPTP opening and inhibited NF-κB pathway. Inhibitor of NF-κB increased TGEV-induced mPTP opening. The data revealed that TGEV-induced mPTP opening was regulated via two pathways: circEZH2/miR-22/HK2 axis and circEZH2/miR-22/IL-6/NF-κB axis.
Subject(s)
Coronavirus/pathogenicity , MicroRNAs/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , RNA, Circular/metabolism , Transmissible gastroenteritis virus/pathogenicity , Animals , Cell Line , Coronavirus/ultrastructure , MicroRNAs/genetics , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Mitochondrial Permeability Transition Pore , RNA, Circular/genetics , SwineABSTRACT
Coronaviruses (CoVs) have caused outbreaks of deadly pneumonia in humans since the beginning of the 21st century. The severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 and was responsible for an epidemic that spread to five continents with a fatality rate of 10% before being contained in 2003 (with additional cases reported in 2004). The Middle-East respiratory syndrome coronavirus (MERS-CoV) emerged in the Arabian Peninsula in 2012 and has caused recurrent outbreaks in humans with a fatality rate of 35%. SARS-CoV and MERS-CoV are zoonotic viruses that crossed the species barrier using bats/palm civets and dromedary camels, respectively. No specific treatments or vaccines have been approved against any of the six human coronaviruses, highlighting the need to investigate the principles governing viral entry and cross-species transmission as well as to prepare for zoonotic outbreaks which are likely to occur due to the large reservoir of CoVs found in mammals and birds. Here, we review our understanding of the infection mechanism used by coronaviruses derived from recent structural and biochemical studies.
Subject(s)
Coronavirus/physiology , Coronavirus/ultrastructure , Virion/ultrastructure , Virus Internalization , Animals , Host-Pathogen Interactions , HumansABSTRACT
Porcine deltacoronavirus (PDCoV) causes severe diarrhea and vomiting in affected piglets. The aim of this study was to establish the basic, in vitro characteristics of the life cycle such as replication kinetics, cellular ultrastructure, virion morphology, and induction of autophagy of PDCoV. Time-course analysis of viral subgenomic and genomic RNA loads and infectious titers indicated that one replication cycle of PDCoV takes 5 to 6 h. Electron microscopy showed that PDCoV infection induced the membrane rearrangements with double-membrane vesicles and large virion-containing vacuoles. The convoluted membranes structures described in alpha- and beta-coronavirus were not observed. PDCoV infection also increased the number of autophagosome-like vesicles in the cytoplasm of cells, and the autophagy response was detected by LC3 I/II and p62 Western blot analysis. For the first time, this study presents the picture of the PDCoV infection cycle, which is crucial to help elucidate the molecular mechanism of deltacoronavirus replication.
Subject(s)
Coronavirus/physiology , Virus Replication , Animals , Autophagy , Coronavirus/classification , Coronavirus/ultrastructure , Coronavirus Infections/veterinary , Life Cycle Stages , Swine , Swine Diseases/virology , Virion/ultrastructureABSTRACT
Porcine deltacoronavirus (PDCoV) is a newly identified virus that causes watery diarrhea in newborn piglets and results in significant economic losses to the pig industry. Since first reported in Hong Kong in 2012, PDCoV has been subsequently detected in USA, South Korea, Thailand, and mainland China. Here we isolated a strain of PDCoV, named CHN-GD-2016, from the intestinal content of a diseased newborn piglet with severe diarrhea in a pig farm in Guangdong, China. PDCoV CHN-GD-2016 could be identified by immunofluorescence with PDCoV specific rabbit antisera, and typical crown-shaped particles with spiky surface projections of this PDCoV were observed with electron microscopy. Genomic analysis showed that the PDCoV CHN-GD-2016 was closely related to other Chinese PDCoV strains, with the highest sequence similarity with the strain CHN/Tianjin/2016. Importantly, inoculation of newborn piglets with 1 × 105 TCID50 of CHN-GD-2016 by oral feeding successfully reproduced clear clinical symptoms, including vomiting, dehydration, and severe diarrhea in piglets. In addition, the virus RNA in rectal swabs from 1 to 7 days post inoculation was detected, macroscopic and microscopic lesions in small intestine were observed, and viral antigen was also detected in the small intestines with immunohistochemical staining. Collectively, the data show in this study confirms that PDCoV is present in Guangdong, China and is highly pathogenic in newborn piglets.
Subject(s)
Coronavirus Infections/veterinary , Coronavirus/isolation & purification , Diarrhea/veterinary , Swine Diseases/pathology , Swine Diseases/virology , Swine , Animals , Animals, Newborn , China , Coronavirus/classification , Coronavirus/genetics , Coronavirus/ultrastructure , Coronavirus Infections/pathology , Coronavirus Infections/virology , Diarrhea/pathology , Diarrhea/virology , Genome, Viral , Microscopy, Electron , Microscopy, Fluorescence , Phylogeny , Sequence Analysis, DNA , Sequence HomologyABSTRACT
Coronavirus spike proteins from different genera are divergent, although they all mediate coronavirus entry into cells by binding to host receptors and fusing viral and cell membranes. Here, we determined the cryo-electron microscopy structure of porcine deltacoronavirus (PdCoV) spike protein at 3.3-Å resolution. The trimeric protein contains three receptor-binding S1 subunits that tightly pack into a crown-like structure and three membrane fusion S2 subunits that form a stalk. Each S1 subunit contains two domains, an N-terminal domain (S1-NTD) and C-terminal domain (S1-CTD). PdCoV S1-NTD has the same structural fold as alpha- and betacoronavirus S1-NTDs as well as host galectins, and it recognizes sugar as its potential receptor. PdCoV S1-CTD has the same structural fold as alphacoronavirus S1-CTDs, but its structure differs from that of betacoronavirus S1-CTDs. PdCoV S1-CTD binds to an unidentified receptor on host cell surfaces. PdCoV S2 is locked in the prefusion conformation by structural restraint of S1 from a different monomeric subunit. PdCoV spike possesses several structural features that may facilitate immune evasion by the virus, such as its compact structure, concealed receptor-binding sites, and shielded critical epitopes. Overall, this study reveals that deltacoronavirus spikes are structurally and evolutionally more closely related to alphacoronavirus spikes than to betacoronavirus spikes; it also has implications for the receptor recognition, membrane fusion, and immune evasion by deltacoronaviruses as well as coronaviruses in general. IMPORTANCE In this study, we determined the cryo-electron microscopy structure of porcine deltacoronavirus (PdCoV) spike protein at a 3.3-Å resolution. This is the first atomic structure of a spike protein from the deltacoronavirus genus, which is divergent in amino acid sequences from the well-studied alpha- and betacoronavirus spike proteins. Here, we described the overall structure of the PdCoV spike and the detailed structure of each of its structural elements. Moreover, we analyzed the functions of each of the structural elements. Based on the structures and functions of these structural elements, we discussed the evolution of PdCoV spike protein in relation to the spike proteins from other coronavirus genera. This study combines the structure, function, and evolution of PdCoV spike protein and provides many insights into its receptor recognition, membrane fusion, and immune evasion.
Subject(s)
Coronavirus/ultrastructure , Cryoelectron Microscopy , Spike Glycoprotein, Coronavirus/ultrastructure , Animals , Humans , Sf9 Cells , Spodoptera , SwineABSTRACT
Coronavirus particles serve three fundamentally important functions in infection. The virion provides the means to deliver the viral genome across the plasma membrane of a host cell. The virion is also a means of escape for newly synthesized genomes. Lastly, the virion is a durable vessel that protects the genome on its journey between cells. This review summarizes the available X-ray crystallography, NMR, and cryoelectron microscopy structural data for coronavirus structural proteins, and looks at the role of each of the major structural proteins in virus entry and assembly. The potential wider conservation of the nucleoprotein fold identified in the Arteriviridae and Coronaviridae families and a speculative model for the evolution of corona-like virus architecture are discussed.
Subject(s)
Coronavirus/ultrastructure , Genome, Viral , Nucleoproteins/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Viral Envelope Proteins/chemistry , Virion/ultrastructure , Animals , Biological Evolution , Coronavirus/chemistry , Coronavirus/genetics , Coronavirus Infections/virology , Epithelial Cells/virology , Gene Expression , Host-Pathogen Interactions , Humans , Nucleoproteins/genetics , Nucleoproteins/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Virion/chemistry , Virion/genetics , Virus Assembly/genetics , Virus InternalizationSubject(s)
Coronavirus/ultrastructure , Genome, Viral , Phylogeny , RNA, Viral/genetics , Virion/ultrastructure , Animals , Coronavirus/classification , Coronavirus/genetics , Coronavirus/pathogenicity , Coronavirus Infections/pathology , Coronavirus Infections/virology , Humans , Terminology as Topic , Virion/genetics , Virion/pathogenicityABSTRACT
All eukaryotic positive-stranded RNA (+RNA) viruses appropriate host cell membranes and transform them into replication organelles, specialized micro-environments that are thought to support viral RNA synthesis. Arteriviruses (order Nidovirales) belong to the subset of +RNA viruses that induce double-membrane vesicles (DMVs), similar to the structures induced by e.g. coronaviruses, picornaviruses and hepatitis C virus. In the last years, electron tomography has revealed substantial differences between the structures induced by these different virus groups. Arterivirus-induced DMVs appear to be closed compartments that are continuous with endoplasmic reticulum membranes, thus forming an extensive reticulovesicular network (RVN) of intriguing complexity. This RVN is remarkably similar to that described for the distantly related coronaviruses (also order Nidovirales) and sets them apart from other DMV-inducing viruses analysed to date. We review here the current knowledge and open questions on arterivirus replication organelles and discuss them in the light of the latest studies on other DMV-inducing viruses, particularly coronaviruses. Using the equine arteritis virus (EAV) model system and electron tomography, we present new data regarding the biogenesis of arterivirus-induced DMVs and uncover numerous putative intermediates in DMV formation. We generated cell lines that can be induced to express specific EAV replicase proteins and showed that DMVs induced by the transmembrane proteins nsp2 and nsp3 form an RVN and are comparable in topology and architecture to those formed during viral infection. Co-expression of the third EAV transmembrane protein (nsp5), expressed as part of a self-cleaving polypeptide that mimics viral polyprotein processing in infected cells, led to the formation of DMVs whose size was more homogenous and closer to what is observed upon EAV infection, suggesting a regulatory role for nsp5 in modulating membrane curvature and DMV formation.
Subject(s)
Arterivirus/ultrastructure , Cell Membrane/ultrastructure , Endoplasmic Reticulum/ultrastructure , Organelles/ultrastructure , Organelles/virology , Viral Nonstructural Proteins/genetics , Animals , Arterivirus/genetics , Arterivirus/metabolism , Arterivirus Infections/veterinary , Arterivirus Infections/virology , Cell Line , Cell Membrane/virology , Coronavirus/genetics , Coronavirus/metabolism , Coronavirus/ultrastructure , Electron Microscope Tomography , Endoplasmic Reticulum/virology , Gene Expression , Host-Pathogen Interactions , Viral Nonstructural Proteins/metabolismABSTRACT
HKU1 is a human betacoronavirus that causes mild yet prevalent respiratory disease, and is related to the zoonotic SARS and MERS betacoronaviruses, which have high fatality rates and pandemic potential. Cell tropism and host range is determined in part by the coronavirus spike (S) protein, which binds cellular receptors and mediates membrane fusion. As the largest known class I fusion protein, its size and extensive glycosylation have hindered structural studies of the full ectodomain, thus preventing a molecular understanding of its function and limiting development of effective interventions. Here we present the 4.0 Å resolution structure of the trimeric HKU1 S protein determined using single-particle cryo-electron microscopy. In the pre-fusion conformation, the receptor-binding subunits, S1, rest above the fusion-mediating subunits, S2, preventing their conformational rearrangement. Surprisingly, the S1 C-terminal domains are interdigitated and form extensive quaternary interactions that occlude surfaces known in other coronaviruses to bind protein receptors. These features, along with the location of the two protease sites known to be important for coronavirus entry, provide a structural basis to support a model of membrane fusion mediated by progressive S protein destabilization through receptor binding and proteolytic cleavage. These studies should also serve as a foundation for the structure-based design of betacoronavirus vaccine immunogens.
Subject(s)
Coronavirus/chemistry , Coronavirus/ultrastructure , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/ultrastructure , Cell Line , Cryoelectron Microscopy , Humans , Membrane Fusion , Models, Molecular , Protein Binding , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/metabolism , Proteolysis , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Viral Vaccines/chemistry , Viral Vaccines/immunology , Virus InternalizationABSTRACT
Acute gastroenteritis accounts for a significant burden of medically attended illness in children under the age of five. For this study, four multiplex reverse transcription PCR assays were used to determine the incidence of adenovirus, astrovirus, coronavirus, norovirus GI and GII, rotavirus, and sapovirus in stool samples submitted for viral electron microscopy (EM) to the Children's Hospital Colorado. Of 1105 stool samples available, viral RNA/DNA was detected in 247 (26.2%) of 941 pediatric samples (median age = 2.97 years, 54% male) with 28 (3.0%) positive for more than one virus. Adenovirus, astrovirus, norovirus GI, norovirus GII, rotavirus, and sapovirus were detected in 95 (10.0%), 33 (3.5%), 8 (0.9%), 90 (9.6%), 49 (5.2%), and 2 (0.2%) of the pediatric samples, respectively. No coronaviruses were identified. Sequencing of norovirus positive samples indicated an outbreak of norovirus strain GII.4 in 2006 with evidence of numerous circulating strains. Multiple samples from the same immunocompromised patients demonstrated symptomatic shedding of norovirus for up to 32 weeks and astrovirus for 12 weeks. RT-PCR detected 99 of 111 (89%) adenovirus-positive samples versus 12 (11%) by EM, and 186 of 192 (97%) sapovirus/astrovirus/norovirus-positive samples versus 21 (11%) by EM. Noroviruses and adenoviruses are common causes of gastroenteritis in children. Immunocompromised patients can be infected with multiple viruses and shed viruses in their stools for prolonged periods. This data support the superiority of RT-PCR compared to EM for diagnosis of viral gastroenteritis.
Subject(s)
Adenoviridae Infections/epidemiology , Adenoviridae/isolation & purification , Adenovirus Infections, Human/epidemiology , Caliciviridae Infections/epidemiology , Enterovirus Infections/epidemiology , Gastroenteritis/epidemiology , Gastroenteritis/virology , Rotavirus Infections/epidemiology , Adenoviridae/genetics , Adenoviridae/ultrastructure , Child , Child, Preschool , Colorado/epidemiology , Coronavirus/isolation & purification , Coronavirus/ultrastructure , Disease Outbreaks , Feces/virology , Female , Gastroenteritis/etiology , Humans , Infant , Male , Microscopy, Electron , Multiplex Polymerase Chain Reaction , Norovirus/isolation & purification , Norovirus/ultrastructure , RNA Viruses/genetics , RNA Viruses/isolation & purification , RNA Viruses/ultrastructure , RNA, Viral/genetics , RNA, Viral/isolation & purification , Rotavirus/genetics , Rotavirus/isolation & purification , Sapovirus/isolation & purification , Sapovirus/ultrastructure , Time Factors , Virus SheddingABSTRACT
Purification of intact enveloped virus particles can be useful as a first step in understanding the structure and function of both viral and host proteins that are incorporated into the virion. Purified preparations of virions can be used to address these questions using techniques such as mass spectrometry proteomics. Recent studies on the proteome of coronavirus virions have shown that in addition to the structural proteins, accessory and non-structural virus proteins and a wide variety of host cell proteins associate with virus particles. To further study the presence of virion proteins, high-quality sample preparation is crucial to ensure reproducible analysis by the wide variety of methods available for proteomic analysis.
Subject(s)
Coronavirus/isolation & purification , Proteome/isolation & purification , Viral Proteins/isolation & purification , Virion/isolation & purification , Animals , Chlorocebus aethiops , Coronavirus/metabolism , Coronavirus/ultrastructure , Cryoelectron Microscopy , Proteome/metabolism , Proteomics , Vero Cells , Viral Proteins/metabolism , Virion/metabolism , Virion/ultrastructureABSTRACT
Coronaviruses (CoVs) generate specialized membrane compartments, which consist of double membrane vesicles connected to convoluted membranes, the so-called replicative structures, where viral RNA synthesis takes place. These sites harbor the CoV replication-transcription complexes (RTCs): multi-protein complexes consisting of 16 nonstructural proteins (nsps), the CoV nucleocapsid protein (N) and presumably host proteins. To successfully establish functional membrane-bound RTCs all of the viral and host constituents need to be correctly spatiotemporally organized during viral infection. Few studies, however, have investigated the dynamic processes involved in the formation and functioning of the (subunits of) CoV RTCs and the replicative structures in living cells. In this chapter we describe several protocols to perform time-lapse imaging of CoV-infected cells and to study the kinetics of (subunits of) the CoV replicative structures. The approaches described are not limited to CoV-infected cells; they can also be applied to other virus-infected or non-infected cells.