scholarly journals Cryo-EM analysis of a feline coronavirus spike protein reveals a unique structure and camouflaging glycans

2020 ◽  
Vol 117 (3) ◽  
pp. 1438-1446 ◽  
Author(s):  
Tzu-Jing Yang ◽  
Yen-Chen Chang ◽  
Tzu-Ping Ko ◽  
Piotr Draczkowski ◽  
Yu-Chun Chien ◽  
...  
Keyword(s):  
Viral Entry ◽  
Protein Structures ◽  
Structural Features ◽  
Host Recognition ◽  
Complex Type ◽  
Site Specific ◽  
S Protein ◽  
Feline Infectious Peritonitis ◽  
Structural Insights ◽  
Feline Coronavirus

Feline infectious peritonitis virus (FIPV) is an alphacoronavirus that causes a nearly 100% mortality rate without effective treatment. Here we report a 3.3-Å cryoelectron microscopy (cryo-EM) structure of the serotype I FIPV spike (S) protein, which is responsible for host recognition and viral entry. Mass spectrometry provided site-specific compositions of densely distributed high-mannose and complex-type N-glycans that account for 1/4 of the total molecular mass; most of the N-glycans could be visualized by cryo-EM. Specifically, the N-glycans that wedge between 2 galectin-like domains within the S1 subunit of FIPV S protein result in a unique propeller-like conformation, underscoring the importance of glycosylation in maintaining protein structures. The cleavage site within the S2 subunit responsible for activation also showed distinct structural features and glycosylation. These structural insights provide a blueprint for a better molecular understanding of the pathogenesis of FIP.

scholarly journals Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins

2020 ◽  
pp. mcp.RA120.002295 ◽  
Author(s):  
Yong Zhang ◽  
Wanjun Zhao ◽  
Yonghong Mao ◽  
Yaohui Chen ◽  
Shisheng Wang ◽  
...  
Keyword(s):  
Insect Cells ◽  
Host Cells ◽  
Protein Subunit ◽  
Genome Sequences ◽  
Complex Type ◽  
Site Specific ◽  
Differential Processing ◽  
S Protein ◽  
Shed Light

The glycoprotein spike (S) on the surface of SARS-CoV-2 is a determinant for viral invasion and host immune response. Herein, we characterized the site-specific N-glycosylation of S protein at the level of intact glycopeptides. All 22 potential N-glycosites were identified in the S-protein protomer and were found to be preserved among the 753 SARS-CoV-2 genome sequences. The glycosites exhibited glycoform heterogeneity as expected for a human cell-expressed protein subunit. We identified masses that correspond to 157 N-glycans, primarily of the complex type. In contrast, the insect cell-expressed S protein contained 38 N-glycans, completely of the high-mannose type. Our results revealed that the glycan types were highly determined by the differential processing of N-glycans among human and insect cells, regardless of the glycosites’ location. Moreover, the N-glycan compositions were conserved among different sizes of subunits. Our study indicate that the S protein N-glycosylation occurs regularly at each site, albeit the occupied N-glycans were diverse and heterogenous. This N-glycosylation landscape and the differential N-glycan patterns among distinct host cells are expected to shed light on the infection mechanism and present a positive view for the development of vaccines and targeted drugs.

An Updated Review on Betacoronavirus Viral Entry Inhibitors: Learning from Past Discoveries to Advance COVID-19 Drug Discovery

2021 ◽  
Vol 21 ◽  
Author(s):  
Dima A. Sabbah ◽  
Rima Hajjo ◽  
Sanaa K. Bardaweel ◽  
Haizhen A. Zhong
Keyword(s):  
Viral Entry ◽  
Clinical Importance ◽  
Structural Features ◽  
Biomedical Literature ◽  
Host Cells ◽  
Virus Binding ◽  
Structural Determinants ◽  
S Protein ◽  
Host Tropism ◽  
Entry Inhibitors

: One year after its first outbreak reported in China, coronavirus disease 2019 (COVID-19) pandemic is still sweeping the World causing serious infections and claiming more fatalities. COVID-19 is caused by the novel corona virus SARS-CoV-2, which belongs to the genus Betacoronavirus (β-CoVs) which is of greatest clinical importance since it contains many other viruses that cause respiratory disease in humans including OC43, HKU1, SARS-CoV and MERS. The spike (S) glycoprotein of β-CoVs is a key virulence factor determining disease pathogenesis and host tropism, and it also mediates virus binding to host’s receptors to allow viral entry into host cells, i.e., the first step in virus lifecycle. This, viral entry inhibitors are considered promising putative drugs for COVID-19. Herein, we mined the biomedical literature for viral entry inhibitors of other corona viruses, with special emphasis on β-CoVs entry inhibitors. We also outlined the structural features of SARS-CoV-2 S protein and how it differs from other β-CoVs to better understand the structural determinants of S protein binding to its human receptor (ACE2). This review highlighted several promising viral entry inhibitors as potential treatments for COVID-19.

scholarly journals Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins

2020 ◽  
Author(s):  
Yong Zhang ◽  
Wanjun Zhao ◽  
Yonghong Mao ◽  
Yaohui Chen ◽  
Shisheng Wang ◽  
...  
Keyword(s):  
Insect Cells ◽  
Host Cells ◽  
Genome Sequences ◽  
Complex Type ◽  
Site Specific ◽  
Differential Processing ◽  
S Protein ◽  
Protein Subunits ◽  
Shed Light

SummaryThe glycoprotein spike (S) on the surface of SARS-CoV-2 is a determinant for viral invasion and host immune response. Herein, we characterized the site-specific N-glycosylation of S protein at the level of intact glycopeptides. All 22 potential N-glycosites were identified in the S-protein protomer and were found to be preserved among the 753 SARS-CoV-2 genome sequences. The glycosites exhibited glycoform heterogeneity as expected for a human cell-expressed protein subunits. We identified masses that correspond to 157 N-glycans, primarily of the complex type. In contrast, the insect cell-expressed S protein contained 38 N-glycans, primarily of the high-mannose type. Our results revealed that the glycan types were highly determined by the differential processing of N-glycans among human and insect cells. This N-glycosylation landscape and the differential N-glycan patterns among distinct host cells are expected to shed light on the infection mechanism and present a positive view for the development of vaccines and targeted drugs.

scholarly journals A Tale of Two Viruses: The Distinct Spike Glycoproteins of Feline Coronaviruses

Viruses ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 83 ◽  
Author(s):  
Javier A. Jaimes ◽  
Jean K. Millet ◽  
Alison E. Stout ◽  
Nicole M. André ◽  
Gary R. Whittaker
Keyword(s):  
Clinical Manifestations ◽  
Clinical Settings ◽  
Viral Agent ◽  
Functional Characteristics ◽  
S Protein ◽  
Feline Infectious Peritonitis ◽  
Taxonomical Classification ◽  
Biological Outcomes ◽  
Feline Coronavirus ◽  
S Proteins

Feline coronavirus (FCoV) is a complex viral agent that causes a variety of clinical manifestations in cats, commonly known as feline infectious peritonitis (FIP). It is recognized that FCoV can occur in two different serotypes. However, differences in the S protein are much more than serological or antigenic variants, resulting in the effective presence of two distinct viruses. Here, we review the distinct differences in the S proteins of these viruses, which are likely to translate into distinct biological outcomes. We introduce a new concept related to the non-taxonomical classification and differentiation among FCoVs by analyzing and comparing the genetic, structural, and functional characteristics of FCoV and the FCoV S protein among the two serotypes and FCoV biotypes. Based on our analysis, we suggest that our understanding of FIP needs to consider whether the presence of these two distinct viruses has implications in clinical settings.

scholarly journals Computational Modulation of the V3 Region of Glycoprotein gp125 of HIV-2

2021 ◽  
Vol 22 (4) ◽  
pp. 1948
Author(s):  
Patrícia A. Serra ◽  
Nuno Taveira ◽  
Rita C. Guedes
Keyword(s):  
Viral Entry ◽  
Network Formation ◽  
Protein Structures ◽  
3D Structure ◽  
3D Models ◽  
Structural Features ◽  
Host Cells ◽  
Computer Assisted ◽  
Function Relationship ◽  
Dynamics Simulations

HIV-2 infection is frequently neglected in HIV/AIDS campaigns. However, a special emphasis must be given to HIV-2 as an untreated infection that also leads to AIDS and death, and for which the efficacy of most available drugs is limited against HIV-2. HIV envelope glycoproteins mediate binding to the receptor CD4 and co-receptors at the surface of the target cell, enabling fusion with the cell membrane and viral entry. Here, we developed and optimized a computer-assisted drug design approach of an important HIV-2 glycoprotein that allows us to explore and gain further insights at the molecular level into protein structures and interactions crucial for the inhibition of HIV-2 cell entry. The 3D structure of a key HIV-2ROD gp125 region was generated by a homology modeling campaign. To disclose the importance of the main structural features and compare them with experimental results, 3D-models of six mutants were also generated. These mutations revealed the selective impact on the behavior of the protein. Furthermore, molecular dynamics simulations were performed to optimize the models, and the dynamic behavior was tackled to account for structure flexibility and interactions network formation. Structurally, the mutations studied lead to a loss of aromatic features, which is very important for the establishment of π-π interactions and could induce a structural preference by a specific coreceptor. These new insights into the structure-function relationship of HIV-2 gp125 V3 and surrounding regions will help in the design of better models and the design of new small molecules capable to inhibit the attachment and binding of HIV with host cells.

scholarly journals Structural Insights of SARS-CoV-2 Spike Protein from Delta and Omicron Variants

2021 ◽  
Author(s):  
Ali Sadek ◽  
David Zaha ◽  
Mahmoud Salama Ahmed
Keyword(s):  
Viral Entry ◽  
Structural Changes ◽  
Structural Basis ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
X Ray ◽  
X Ray Crystallography ◽  
Binding Domains ◽  
Structural Insights ◽  
Sheer Number

Given the continuing heavy toll of the COVID-19 pandemic and the emergence of the Delta (B.1.617.2) and Omicron (B.1.1.529) variants, the WHO declared both as variants of concern (VOC). There are valid concerns that the latest Omicron variant might have increased infectivity and pathogenicity. In addition, the sheer number of S protein mutations in the Omicron variant raise concerns of potential immune evasion and resistance to therapeutics such as monoclonal antibodies. However, structural insights that underpin the potential increased pathogenicity are unknown. Here we adopted an artificial intelligence (AI)-based approach to predict the structural changes induced by mutations of the Delta and Omicron variants in the spike (S) protein using Alphafold. This was followed by docking the human angiotensin-converting enzyme 2 (ACE2) with the predicted S proteins for Wuhan-Hu-1, Delta, and Omicron variants. Our in-silico structural analysis indicates that S protein for Omicron variant has a higher binding affinity to ACE-2 receptor, compared to Wuhan-Hu-1 and Delta variants. In addition, the recognition sites of the receptor binding domains for Delta and Omicron variants showed lower electronegativity compared to Wuhan-Hu-1. Importantly, further molecular insights revealed significant changes induced at fusion protein (FP) site, which may mediate enhanced viral entry. These results represent the first computational analysis of structural changes associated with Omicron variant using Alphafold, Collectively, our results highlight potential structural basis for enhanced pathogenicity of the Omicron variant, however further validation using X-ray crystallography and cryo-EM are warranted.

scholarly journals Subarachnoid diverticulum associated with feline infectious peritonitis in a Siberian cat

2020 ◽  
Vol 6 (2) ◽  
pp. 205511692094147
Author(s):  
Christopher Hoey ◽  
George Nye ◽  
Angela Fadda ◽  
Janet Bradshaw ◽  
Emi N Barker
Keyword(s):  
Cervical Spinal Cord ◽  
Serum Biochemistry ◽  
Acute Onset ◽  
Neurological Deficits ◽  
Rt Pcr ◽  
Feline Infectious Peritonitis ◽  
Case Summary ◽  
Choroid Plexitis ◽  
The Brain ◽  
Feline Coronavirus

Case summary A 7-month-old Siberian cat was presented for investigation of acute onset multifocal neurological deficits. Neurological examination documented dull mental status and an ambulatory left hemiparesis. Serum biochemistry documented marked hyperglobulinaemia. MRI of the brain identified marked leptomeningeal contrast enhancement extending along the brainstem caudally to involve the cranial cervical spinal cord. MRI of the cervical spine further identified a subarachnoid diverticulum that extended from the level of the obex to the C2–C3 vertebrae. Cerebrospinal fluid quantitative RT-PCR was positive for the presence of feline coronavirus. Histopathology revealed pyogranulomatous meningitis and choroid plexitis, uveitis and nephritis. Relevance and novel information This article describes the first reported case of a subarachnoid diverticulum associated with feline infectious peritonitis.

scholarly journals Amino Acids 1055 to 1192 in the S2 Region of Severe Acute Respiratory Syndrome Coronavirus S Protein Induce Neutralizing Antibodies: Implications for the Development of Vaccines and Antiviral Agents

2005 ◽  
Vol 79 (6) ◽  
pp. 3289-3296 ◽  
Author(s):  
Choong-Tat Keng ◽  
Aihua Zhang ◽  
Shuo Shen ◽  
Kuo-Ming Lip ◽  
Burtram C. Fielding ◽  
...  
Keyword(s):  
Amino Acids ◽  
Severe Acute Respiratory Syndrome ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Antiviral Agents ◽  
Host Cells ◽  
Heptad Repeat ◽  
Antigenic Determinants ◽  
Amino Acid Residues ◽  
S Protein

ABSTRACT The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SΔ10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.

scholarly journals Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells

2018 ◽  
Vol 92 (19) ◽  
Author(s):  
Shutoku Matsuyama ◽  
Kazuya Shirato ◽  
Miyuki Kawase ◽  
Yutaka Terada ◽  
Kengo Kawachi ◽  
...  
Keyword(s):  
Middle East ◽  
Viral Entry ◽  
Cathepsin L ◽  
Inhibitory Effect ◽  
Middle East Respiratory Syndrome ◽  
Cell Entry ◽  
Target Cells ◽  
Furin Cleavage ◽  
S Protein ◽  
Entry Assay

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. In this study, we reexamined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK inhibited not only the enzymatic activity of furin but also those of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin. IMPORTANCE Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be reexamined carefully. Here we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.

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