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Principal Investigator: Siyuan Ding
Organization: WASHINGTON UNIVERSITY
Fiscal Year: 2022
Award: $393,750
Funding agency: National Institute of Allergy and Infectious Diseases
Project Summary
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><Adsorption><Anti-Viral Response><Antiviral Response><Assay><Binding><Binding Proteins><Bioassay><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><Cessation of life><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><Data><Data Set><Dataset><Death><Diarrhea><Disease><Disorder><E3 Ligase><E3 Ubiquitin Ligase><Early Endosome><Ectopic Expression><Elongation Factor><Emesis><Endocytosis><Enteral><Enteric><Epithelial><Epithelial Cells><Evolution><Foundations><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><Infection><Integration Host Factors><Interferons><Intestinal><Intestines><Intracellular Communication and Signaling><Knock-out><Knockout><Knowledge><LOC51191><Label><Letters><Libraries><Ligand Binding Protein><Ligand Binding Protein Gene><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><Mutation><Neonatal><Non-Polyadenylated RNA><Non-structural Protein><Nonstructural Protein><Organoids><Outcome><Patients><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 Intervention><Transcription><Ubiquitin Protein Ligase><Ubiquitin-Protein Ligase Complexes><Ubiquitin-Protein Ligase E3><Viral><Viral Antigens><Viral Diseases><Viral Pathogenesis><Virus><Virus Diseases><Virus Replication><Vomiting><antagonist><attenuation><base><biological signal transduction><bound protein><bowel><burden of disease><burden of illness><cell type><cross-link><crosslink><disease burden><enteric viral infection><enteric virus infection><fluorophore><gain of function><genome mutation><human pathogen><improved><in vivo><intervention therapy><intestinal epithelium><mRNA><major vault protein><mortality><mutant><novel><overexpress><overexpression><pox virus><prevent><preventing><rational design><response><reverse genetics><screening><siRNA><small bowel><social role><suckling><therapeutic agent development><therapeutic development><ubiquitin-protein ligase><vaccine candidate><viral RNA><viral infection><viral multiplication><viral replication><virus RNA><virus antigen><virus infection><virus multiplication><virus pathogenesis><virus-induced disease><years of life lost to disability><years of life lost to disease><youngster>