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Principal Investigator: Christopher F Basler
Organization: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
Fiscal Year: 2022
Award: $217,459
Funding agency: National Institute of Allergy and Infectious Diseases
The Middle East respiratory syndrome coronavirus (MERS-CoV), is a highly pathogenic, zoonotic, non-segmented, positive-sense RNA virus related to the severe acute respiratory syndrome coronavirus (SARS CoV) and SARS-CoV-2 that can spread from person to person. The potential threat posed by zoonotic coronaviruses is demonstrated by the emergence of SARS-CoV-2 and the subsequent COVID-19 pandemic. Because MERS CoV remains a threat, an understanding of mechanisms of MERS CoV replication, particularly as these related to pathogenesis and therapeutic development, remains critical. ORF4b (4b) is notable among the MERS CoV accessory proteins because it strongly localizes to the nucleus, despite virus replication occurring in the cytoplasm, and it exerts innate immune evasion functions. These include inhibition of interferon beta (IFNβ) and IFNλ production and inhibition of NF-κB-dependent cytokine production. The 4b protein also inhibits the 2’, 5’ oligoadenylate synthetase (OAS)-RNase L pathway, an activity attributed to its C-terminal phosphodiesterase domain. ORF4b is a nuclear protein and both transfection- and infection-based assays indicate that mutation of the apparent ORF4b nuclear localization signal (NLS) impairs affects inhibition of innate immune evasion functions. One notable study found that 4b blocks NF-ĸB-dependent responses and this correlated with the capacity of 4b to outcompete the p65 subunit of NF-ĸB for IMPA3 binding. We have undertaken X ray crystallography studies of the 4b-IMPA interaction. Our Preliminary Data demonstrate that 4b has uniquely bypassed canonical rules of NLS recognition and does not contain a Lys residue at a binding site formerly thought to be critical for NLS function. Further, the proposed specificity of 4b for IMPA3 is not fully supported by our data. We found that the NLS region of 4b binds IMPA2 and with an interface that is more extensive than IMPA3. Thus, the specificity that has been proposed is unlikely to be mediated by this simple interaction interface. Furthermore, if 4b is able to bind a greater range of IMPA isoforms than had previously been proposed, this MERS-CoV protein is likely to be able to competitively inhibit the nuclear import of other innate immune transcription factors such as IRF3 and STAT1. Consistent with such a model, Preliminary Data of crystal structures of the p50 NF-κB NLS bound to IMPA2 and IMPA3 demonstrate that these regions overlap with MERS ORF4b. Based on these observations, we propose to solve structures of full-length and NLS peptides of MERS- CoV and bat Merbecovirus ORF4b proteins in complex with nuclear receptor IMPA isoforms and define interaction interfaces. .This will provide a structural basis for the specificity of ORF4b binding and nuclear import. To compare the structural data obtained for ORF4b and IMPAs, we will determine the structures of IMPA isoforms in complex with NF-κB to establish a structural basis for the immune evasion. Finally. we will test the hypothesis that ORF4b inhibits NF-κB signaling and IFN production by competing for p50-IMPA interaction in transfection-based and virus infection studies.
Terms: <2',5'-Oligo(A) Polymerase><2',5'-Oligo(A) Synthetase><2',5'-Oligoadenylate Polymerase><2',5'-Oligoadenylate Synthetase><2-5A Synthetase><2019 novel corona virus><2019 novel coronavirus><2019-nCoV><ATP-(2'-5')oligo(A)adenylyltransferase><Affect><Affinity><Assay><Basal Transcription Factor><Basal transcription factor genes><Bats><Binding><Binding Sites><Bioassay><Biologic Assays><Biological Assay><Bp50><Bypass><C-terminal><CD40><CDW40><COVID crisis><COVID epidemic><COVID pandemic><COVID-19 crisis><COVID-19 epidemic><COVID-19 global health crisis><COVID-19 global pandemic><COVID-19 health crisis><COVID-19 pandemic><COVID-19 public health crisis><COVID-19 virus><COVID19 crisis><COVID19 epidemic><COVID19 global health crisis><COVID19 global pandemic><COVID19 health crisis><COVID19 pandemic><COVID19 public health crisis><COVID19 virus><Cell Communication and Signaling><Cell Nucleus><Cell Signaling><Chiroptera><CoV emergence><CoV-2><CoV2><Combining Site><Complex><Coronaviridae Infections><Coronavirus Infections><Crystallization><Cytoplasm><Dangerousness><Data><Endogenous Interferon Beta><Enzyme Gene><Enzymes><Exhibits><Fibroblast Interferon><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Genetic Alteration><Genetic Change><Genetic Polymorphism><Genetic Transcription><Genetic defect><Genetics-Mutagenesis><Human><IFN><IFN-Beta><IFN-regulatory factor 3><IFN-β><IFNb><IRF-3 protein><IRF3><IRF3 gene><Immune><Immune Evasion><Immune Precipitation><Immune response><Immune signaling><Immunes><Immunological response><Immunoprecipitation><Impairment><Importin-alpha><Importin-α><Infection><Innate Immunity><Interferon Regulatory Factor 3><Interferon-beta><Interferon-β><Interferons><Intracellular Communication and Signaling><Investigation><Isoforms><Length><Ligase><Ligase Gene><MERS><MERS corona virus><MERS coronavirus><MERS coronavirus disease><MERS virus><MERS-CoV><MERS-CoV disease><MGC9013><Measures><Mediating><Merbecovirus><Mice><Mice Mammals><Middle East Respiratory Syndrome><Middle East Respiratory Syndrome CoV disease><Middle East Respiratory Syndrome Corona Virus><Middle East Respiratory Syndrome Coronavirus><Middle East Respiratory Syndrome Virus><Middle East Respiratory Syndrome coronavirus disease><Middle East Respiratory Syndrome-CoV><Middle Eastern Respiratory Syndrome><Middle Eastern Respiratory Syndrome CoV disease><Middle Eastern Respiratory Syndrome Corona virus><Middle Eastern Respiratory Syndrome Coronavirus><Middle Eastern Respiratory Syndrome Virus><Middle Eastern Respiratory Syndrome coronavirus disease><Middle Eastern Respiratory Syndrome-CoV><Modeling><Modern Man><Molecular Interaction><Murine><Mus><Mutagenesis><Mutagenesis Molecular Biology><Mutation><N-terminal><NH2-terminal><NLS Peptide><NLS Receptor><NLS-Binding Protein><NLSBP><Native Immunity><Natural Immunity><Natural Interferon Beta><Natural human interferon beta><Non-Specific Immunity><Nonspecific Immunity><Nuclear><Nuclear Import><Nuclear Localization Sequence Receptor><Nuclear Localization Signal><Nuclear Localization Signal Peptide><Nuclear Localization Signal-Binding Protein><Nuclear Protein><Nuclear Receptors><Nuclear Translocation><Nucleus><Pathogenesis><Pathogenicity><Pathway interactions><Peptide Signal Sequences><Persons><Phosphodiesterases><Play><Position><Positioning Attribute><Production><Protein Import><Protein Isoforms><Proteins><RNA Expression><RNA Nucleases><RNA Viruses><RNase><Reactive Site><Reporting><Ribonuclease Family Protein><Ribonucleases><Role><SARS Virus><SARS corona virus><SARS corona virus 2><SARS coronavirus><SARS-Associated Coronavirus><SARS-CO-V2><SARS-COVID-2><SARS-CoV><SARS-CoV-1><SARS-CoV-2><SARS-CoV-2 epidemic><SARS-CoV-2 global health crisis><SARS-CoV-2 global pandemic><SARS-CoV-2 pandemic><SARS-CoV2><SARS-CoV2 epidemic><SARS-CoV2 pandemic><SARS-Related Coronavirus><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-coronavirus-2 epidemic><SARS-coronavirus-2 pandemic><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><STAT1><STAT1 gene><STAT91><Severe Acute Respiratory Coronavirus><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV 2 epidemic><Severe Acute Respiratory Syndrome CoV 2 pandemic><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 epidemic><Severe acute respiratory syndrome coronavirus 2 pandemic><Severe acute respiratory syndrome related corona virus 2><Signal Peptide><Signal Sequences><Signal Transduction><Signal Transduction Systems><Signaling><Single Crystal Diffraction><Site><Specificity><Structure><Synthetases><TNFRSF5><TNFRSF5 gene><Testing><Therapeutic Intervention><Transcription><Transcription Factor Proto-Oncogene><Transcription factor genes><Transfection><Tumor Necrosis Factor Receptor Superfamily Member 5 Gene><Variant><Variation><Viral><Viral Diseases><Viral Gene Products><Viral Gene Proteins><Viral Proteins><Virulence><Virus><Virus Diseases><Virus Replication><Wuhan coronavirus><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><Zoonoses><Zoonotic><Zoonotic Infection><alpha Karyopherins><base><beta CoV><beta coronavirus><betaCoV><betacoronavirus><biological signal transduction><corona virus disease 2019 epidemic><corona virus disease 2019 pandemic><corona virus emergence><coronavirus disease 2019 crisis><coronavirus disease 2019 epidemic><coronavirus disease 2019 global health crisis><coronavirus disease 2019 global pandemic><coronavirus disease 2019 health crisis><coronavirus disease 2019 pandemic><coronavirus disease 2019 public health crisis><coronavirus disease 2019 virus><coronavirus disease crisis><coronavirus disease epidemic><coronavirus disease pandemic><coronavirus disease-19 global pandemic><coronavirus disease-19 pandemic><coronavirus disease-19 virus><coronavirus emergence><cytokine><emergent CoV><emergent corona virus><emergent coronavirus><emerging CoV><emerging corona virus><emerging coronavirus><genome mutation><hCoV19><host response><immune system response><immunoresponse><improved><inhibitor><innate immune pathways><intervention therapy><mutant><nCoV><nCoV2><new CoV><new corona virus><new coronavirus><novel CoV><novel corona virus><novel coronavirus><oligoadenylate><p50><p65><pathogen><pathway><phosphoric diester hydrolase><polymorphism><preference><prevent><preventing><protein function><protein signal sequence><response><severe acute respiratory syndrome coronavirus 2 global health crisis><severe acute respiratory syndrome coronavirus 2 global pandemic><severe acute respiratory syndrome-CoV><social role><structural biology><therapeutic agent development><therapeutic development><transcription factor><viral infection><viral multiplication><viral replication><virus infection><virus multiplication><virus protein><virus-induced disease><zoonotic CoV><zoonotic coronavirus><β CoV><β coronavirus><βCoV>