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Principal Investigator: Siyuan Ding
Organization: WASHINGTON UNIVERSITY
Fiscal Year: 2023
Award: $77,750
Funding agency: National Institute of Allergy and Infectious Diseases
Project Summary
From the parent grant: Rotaviruses (RVs) are a medically important human pathogen and the
predominant cause of severe gastroenteritis, vomiting, and diarrhea in infants and young children worldwide.
RVs are also a great model to interrogate the antiviral responses at the host mucosal surfaces. Our overall
objectives are to better understand RV-host interactions and to use that information to develop improved RV
vaccines and therapeutic interventions, thereby preventing and treating enteric virus infections. The host
interferon (IFN) signaling underlies the basis of RV host range restriction and suppresses the replication of
RVs not native to that species in vivo. However, the specific IFN-mediated antiviral effectors are not known and
the associated molecular mechanisms remain unclear. To bridge this gap in knowledge, we sought to define
the most highly induced IFN-stimulated genes (ISGs) in primary human intestinal epithelial cells (IECs). Using
an IEC-specific ISG gain-of-function screening approach, we identified several novel host factors that restrict
RV replication, including sterile alpha motif domain-containing 9 (encoded by SAMD9). Intracellular viral RNA
levels and virus progeny production were significantly enhanced in SAMD9 CRISPR knockout cells. In parallel,
we also made the exciting discovery that RV encodes non-structural protein 1 (NSP1) to target SAMD9 for
proteasomal degradation. In this R01 application, using a set of novel, powerful, and tractable model systems,
we will test the hypotheses that SAMD9 confines early RV replication in an epithelial cell-specific manner and
that RV NSP1 functions to overcome SAMD9 restriction to promote viral replication and pathogenesis in vivo.
In Aim 1, we will examine the mechanistic basis underlying SAMD9 inhibition of RV replication in vitro using
several newly available fluorophore-labeled RVs and a recently developed RV reverse genetics system. We
will test these findings in a physiologically relevant human small intestinal organoid culture derived from
healthy individuals and SAMD9-mutation patients. In Aim 2, we will examine how RV NSP1 binds to SAMD9
via a novel recognition motif and induces its degradation. We will determine whether NSP1 degrades SAMD9
in IECs in vivo and if this process contributes to successful RV intestinal replication using a novel neonatal rat
model. Collectively, we expect these studies on SAMD9-RV interactions to have a substantial impact on
elucidating the basic biology of ISG mode of action, identifying new viral innate immune evasion mechanisms,
and laying the scientific foundation for the rational design of new RV vaccine candidates based on targeted
NSP1 attenuation.
Terms: <0-11 years old><Accountability><Adsorption><Amino Acids><Anti-Viral Response><Antiviral Response><Assay><Binding><Binding Proteins><Bioassay><Biochemical><Biochemistry><Biologic Assays><Biologic Models><Biological Assay><Biological Chemistry><Biological Models><Biology><CEB1 Gene><CRISPR><CRISPR/Cas system><CURL><Cell Body><Cell Communication and Signaling><Cell Signaling><Cells><Cellular biology><Cessation of life><Chemical Fractionation><Child><Child Youth><Children (0-21)><Chronic diarrhea><Clinical><Clustered Regularly Interspaced Short Palindromic Repeats><Common Rat Strains><Compartment of the Uncoupling Receptors and Ligands><Complex><Cryo-electron Microscopy><Cryoelectron Microscopy><Data><Data Set><Death><Development><Diarrhea><Disease><Disorder><E3 Ligase><E3 Ubiquitin Ligase><Early Endosome><Ectopic Expression><Electron Cryomicroscopy><Elongation Factor><Emesis><Endocytosis><Enteral><Enteric><Epithelial Cells><Epithelium><Expression Library><FRACN><Foundations><Fractionation><Fractionation Radiotherapy><Gastroenteritis><Gene Transcription><Generations><Genes><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic defect><Genetics-Mutagenesis><Goals><HERC5><HERC5 Gene><Host Factor><Host Factor Protein><House mice><Human><IFN><Immune Evasion><Immune Precipitation><Immunoprecipitation><In Vitro><Individual><Infant><Integration Host Factors><Interferons><Intestinal><Intestines><Intracellular Communication and Signaling><Knock-out><Knockout><Knowledge><LOC51191><Label><Letters><Ligand Binding Protein><Ligand Binding Protein Gene><Maps><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mediating><Medical><Messenger RNA><Model System><Modeling><Modern Man><Modification><Molecular><Molecular Interaction><Morbidity><Morbidity - disease rate><Mucosa><Mucosal Tissue><Mucous Membrane><Mus musculus><Mutagenesis><Mutagenesis Molecular Biology><Mutate><Mutation><NTPase><Neonatal><Non-Polyadenylated RNA><Non-structural Protein><Nonstructural Protein><Nucleic Acids><Nucleoside Triphosphate Phosphohydrolase><Nucleosidetriphosphatase><Organoids><Patients><Penetration><Persons><Physiologic><Physiological><Poxviridae><Poxviruses><Predisposition><Process><Production><Protein Binding><Proteins><Proteomics><Protocol><Protocols documentation><RNA><RNA Expression><RNA Gene Products><Rat><Rats Mammals><Rattus><Ribonucleic Acid><Role><Rotavirus><Rotavirus Infections><Rotavirus Vaccines><SAM Domain><Severities><Short interfering RNA><Side><Signal Transduction><Signal Transduction Systems><Signaling><Site-Directed Mutagenesis><Site-Specific Mutagenesis><Small Interfering RNA><Small Intestines><Sterile Alpha Motif><Sterile Alpha Motif Domain><Structure><Surface><Susceptibility><Symptoms><Syndrome><System><Targeted DNA Modification><Targeted Modification><Testing><Therapeutic><Therapeutic Intervention><Transcription><Ubiquitin Protein Ligase><Ubiquitin-Protein Ligase Complexes><Ubiquitin-Protein Ligase E3><Viral><Viral Antigens><Viral Pathogenesis><Virus><Virus Replication><Vomiting><aminoacid><antagonism><antagonist><attenuation><biological signal transduction><bound protein><bowel><burden of disease><burden of illness><cell biology><cell type><cofactor><crosslink><cryo-EM><cryoEM><cryogenic electron microscopy><developmental><disease burden><enteric viral infection><enteric virus infection><fluorophore><gain of function><genome mutation><human pathogen><immune evasive><improved><in vivo><intervention therapy><intestinal epithelium><kids><mRNA><major vault protein><mortality><mutant><novel><nucleoside triphosphatase><overexpress><overexpression><parent grant><pox virus><prevent><preventing><rational design><response><reverse genetics><screening><screenings><siRNA><small bowel><social role><suckling><superresolution microscopy><ubiquitin-protein ligase><vaccine candidate><viral RNA><viral multiplication><viral replication><virus RNA><virus antigen><virus multiplication><virus pathogenesis><years of life lost to disability><years of life lost to disease><youngster>