The C-terminal domain of the MERS coronavirus M protein contains a trans -Golgi network localization signal

International audienceCoronavirus M proteins represent the major protein component of the viral envelope. They play an essential role during viral assembly by interacting with all the other structural proteins. Coronaviruses bud into the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), but the mechanisms by which M proteins are transported from their site of synthesis, the ER, to the budding site remain poorly understood. Here, we investigated the intracellular trafficking of the Middle East respiratory syndrome coronavirus (MERS-CoV) M protein. Subcellular localization analyses revealed that the MERS-CoV M protein is retained intracellularly in the trans-Golgi network (TGN), and we identified two motifs in the distal part of the C-terminal domain as being important for this specific localization. We identified the first motif as a functional diacidic DxE ER export signal, since substituting Asp-211 and Glu-213 with alanine induced retention of the MERS-CoV M in the ER. The second motif, 199 KxGxYR 204 , was responsible for retaining the M protein in the TGN. Substitution of this motif resulted in MERS-CoV M leakage toward the plasma membrane. We further confirmed the role of 199 KxGxYR 204 as a TGN retention signal by using chimeras between MERS-CoV M and the M protein of infectious bronchitis virus (IBV). Our results indicated that the C-terminal domains of both proteins determine their specific localization, namely, TGN and ERGIC/cis-Golgi for MERS-M and IBV-M, respectively. Our findings indicate that MERS-CoV M protein localizes to the TGN because of the combined presence of an ER export signal and a TGN retention motif

A graph-based approach identifies dynamic H-bond communication networks in spike protein S of SARS-CoV-2

We apply graph-based approaches to identify H-bond clusters in protein complexes. Three conformations of spike protein S have distinct H-bond clusters at key sites. Hydrogen-bond clusters could govern structural plasticity of spike protein S. Protein S binds to ACE2 receptor via H-bond clusters extending deep across interface.Corona virus spike protein S is a large homo-trimeric protein anchored in the membrane of the virion particle. Protein S binds to angiotensin-converting-enzyme 2, ACE2, of the host cell, followed by proteolysis of the spike protein, drastic protein conformational change with exposure of the fusion peptide of the virus, and entry of the virion into the host cell. The structural elements that govern conformational plasticity of the spike protein are largely unknown. Here, we present a methodology that relies upon graph and centrality analyses, augmented by bioinformatics, to identify and characterize large H-bond clusters in protein structures. We apply this methodology to protein S ectodomain and find that, in the closed conformation, the three protomers of protein S bring the same contribution to an extensive central network of H-bonds, and contribute symmetrically to a relatively large H-bond cluster at the receptor binding domain, and to a cluster near a protease cleavage site. Markedly different H-bonding at these three clusters in open and pre-fusion conformations suggest dynamic H-bond clusters could facilitate structural plasticity and selection of a protein S protomer for binding to the host receptor, and proteolytic cleavage. From analyses of spike protein sequences we identify patches of histidine and carboxylate groups that could be involved in transient proton binding.PSI COVID19 Emergency Science FundSpanish Ministry of Science, Innovation and Universities RTI2018-098983-B-I00Excellence Initiative of the German Federal and State Governments via the Freie Universitat BerlinGerman Research Foundation (DFG) SFB 107

Identification of a Severe Acute Respiratory Syndrome Coronavirus-Like Virus in a Leaf-Nosed Bat in Nigeria

Bats are reservoirs for emerging zoonotic viruses that can have a profound impact on human and animal health, including lyssaviruses, filoviruses, paramyxoviruses, and severe acute respiratory syndrome coronaviruses (SARS-CoVs). In the course of a project focused on pathogen discovery in contexts where human-bat contact might facilitate more efficient interspecies transmission of viruses, we surveyed gastrointestinal tissue obtained from bats collected in caves in Nigeria that are frequented by humans. Coronavirus consensus PCR and unbiased high-throughput pyrosequencing revealed the presence of coronavirus sequences related to those of SARS-CoV in a Commerson’s leaf-nosed bat (Hipposideros commersoni). Additional genomic sequencing indicated that this virus, unlike subgroup 2b CoVs, which includes SARS-CoV, is unique, comprising three overlapping open reading frames between the M and N genes and two conserved stem-loop II motifs. Phylogenetic analyses in conjunction with these features suggest that this virus represents a new subgroup within group 2 CoVs

Requirement of Bardet-Biedl syndrome proteins for leptin receptor signaling

Obesity is a major public health problem in most developed countries and a major risk factor for diabetes and cardiovascular disease. Emerging evidence indicates that ciliary dysfunction can contribute to human obesity but the underlying molecular and cellular mechanisms are unknown. Bardet-Biedl syndrome (BBS) is a genetically heterogeneous human obesity syndrome associated with ciliary dysfunction. BBS proteins are thought to play a role in cilia function and intracellular protein/vesicle trafficking. Here, we show that BBS proteins are required for leptin receptor (LepR) signaling in the hypothalamus. We found that Bbs2−/−, Bbs4−/− and Bbs6−/− mice are resistant to the action of leptin to reduce body weight and food intake regardless of serum leptin levels and obesity. In addition, activation of hypothalamic STAT3 by leptin is significantly decreased in Bbs2−/−, Bbs4−/− and Bbs6−/− mice. In contrast, downstream melanocortin receptor signaling is unaffected, indicating that LepR signaling is specifically impaired in Bbs2−/−, Bbs4−/− and Bbs6−/− mice. Impaired LepR signaling in BBS mice was associated with decreased Pomc gene expression. Furthermore, we found that BBS1 protein physically interacts with the LepR and that loss of BBS proteins perturbs LepR trafficking. Our data indicate that BBS proteins mediate LepR trafficking and that impaired LepR signaling underlies energy imbalance in BBS. These findings represent a novel mechanism for leptin resistance and obesity

Molecular mechanism of inhibiting the SARS-CoV-2 cell entry facilitator TMPRSS2 with camostat and nafamostat

The entry of the coronavirus SARS-CoV-2 into human lung cells can be inhibited by the approved drugs camostat and nafamostat. Here we elucidate the molecular mechanism of these drugs by combining experiments and simulations. In vitro assays confirm that both drugs inhibit the human protein TMPRSS2, a SARS-Cov-2 spike protein activator. As no experimental structure is available, we provide a model of the TMPRSS2 equilibrium structure and its fluctuations by relaxing an initial homology structure with extensive 330 microseconds of all-atom molecular dynamics (MD) and Markov modeling. Through Markov modeling, we describe the binding process of both drugs and a metabolic product of camostat (GBPA) to TMPRSS2, reaching a Michaelis complex (MC) state, which precedes the formation of a long-lived covalent inhibitory state. We find that nafamostat has a higher MC population than camostat and GBPA, suggesting that nafamostat is more readily available to form the stable covalent enzyme-substrate intermediate, effectively explaining its high potency. This model is backed by our in vitro experiments and consistent with previous virus cell entry assays. Our TMPRSS2-drug structures are made public to guide the design of more potent and specific inhibitors

Drug Weaponry to Fight Against SARS-CoV-2

The current outbreak of SARS-CoV-2 virus has caused a large increase in mortality and morbidity associated with respiratory diseases. Huge efforts are currently ongoing to develop a vaccine against this virus. However, alternative approaches could be considered in the fight against this disease. Among other strategies, structural-based drug design could be an effective approach to generate specific molecules against SARS-CoV-2, thus reducing viral burden in infected patients. Here, in addition to this structural approach, we also revise several therapeutic strategies to fight against this viral threat. Furthermore, we report ACE-2 genetic polymorphic variants affecting residues involved in close contacts with SARS-CoV-2 that might be associated to different infection risks. These analyses could provide valuable information to predict the course of the disease

Ready, Set, Fuse! The Coronavirus Spike Protein and Acquisition of Fusion Competence

Coronavirus-cell entry programs involve virus-cell membrane fusions mediated by viral spike (S) proteins. Coronavirus S proteins acquire membrane fusion competence by receptor interactions, proteolysis, and acidification in endosomes. This review describes our current understanding of the S proteins, their interactions with and their responses to these entry triggers. We focus on receptors and proteases in prompting entry and highlight the type II transmembrane serine proteases (TTSPs) known to activate several virus fusion proteins. These and other proteases are essential cofactors permitting coronavirus infection, conceivably being in proximity to cell-surface receptors and thus poised to split entering spike proteins into the fragments that refold to mediate membrane fusion. The review concludes by noting how understanding of coronavirus entry informs antiviral therapies

Cleavage of the SARS Coronavirus Spike Glycoprotein by Airway Proteases Enhances Virus Entry into Human Bronchial Epithelial Cells In Vitro

Background: Entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike) of the severe acute respiratory syndrome coronavirus (SARS-CoV) to cleavage by various airway proteases. Methodology/Principal Findings: Purified triSpike proteins were readily cleaved in vitro by three different airway proteases: trypsin, plasmin and TMPRSS11a. High Performance Liquid Chromatography (HPLC) and amino acid sequencing analyses identified two arginine residues (R667 and R797) as potential protease cleavage site(s). The effect of protease-dependent enhancement of SARS-CoV infection was demonstrated with ACE2 expressing human bronchial epithelial cells 16HBE. Airway proteases regulate the infectivity of SARS-CoV in a fashion dependent on previous receptor binding. The role of arginine residues was further shown with mutant constructs (R667A, R797A or R797AR667A). Mutation of R667 or R797 did not affect the expression of S-protein but resulted in a differential efficacy of pseudotyping into SARS-CoVpp. The R667A SARS-CoVpp mutant exhibited a lack of virus entry enhancement following protease treatment. Conclusions/Significance: These results suggest that SARS S-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of SARS S-protein upon ACE2 binding. These data have direct implications for the cell entry mechanism of SARS-CoV along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against SARS-CoV. © 2009 Kam et al.published_or_final_versio

The neurology of COVID-19 revisited: A proposal from the environmental neurology specialty group of the world federation of neurology to implement international neurological registries

A comprehensive review of the neurological disorders reported during the current COVID-19 pandemic demonstrates that infection with SARS-CoV-2 affects the central nervous system (CNS), the peripheral nervous system (PNS) and the muscle. CNS manifestations include: headache and decreased responsiveness considered initial indicators of potential neurological involvement; anosmia, hyposmia, hypogeusia, and dysgeusia are frequent early symptoms of coronavirus infection. Respiratory failure, the lethal manifestation of COVID-19, responsible for 264,679 deaths worldwide, is probably neurogenic in origin and may result from the viral invasion of cranial nerve I, progressing into rhinencephalon and brainstem respiratory centers. Cerebrovascular disease, in particular large-vessel ischemic strokes, and less frequently cerebral venous thrombosis, intracerebral hemorrhage and subarachnoid hemorrhage, usually occur as part of a thrombotic state induced by viral attachment to ACE2 receptors in endothelium causing widespread endotheliitis, coagulopathy, arterial and venous thromboses. Acute hemorrhagic necrotizing encephalopathy is associated to the cytokine storm. A frontal hypoperfusion syndrome has been identified. There are isolated reports of seizures, encephalopathy, meningitis, encephalitis, and myelitis. The neurological diseases affecting the PNS and muscle in COVID-19 are less frequent and include Guillain-Barré syndrome; Miller Fisher syndrome; polyneuritis cranialis; and rare instances of viral myopathy with rhabdomyolysis. The main conclusion of this review is the pressing need to define the neurology of COVID-19, its frequency, manifestations, neuropathology and pathogenesis. On behalf of the World Federation of Neurology we invite national and regional neurological associations to create local databases to report cases with neurological manifestations observed during the on-going pandemic. International neuroepidemiological collaboration may help define the natural history of this worldwide problem

Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner

Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion