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Principal Investigator: Don Brad Gammon
Organization: UT SOUTHWESTERN MEDICAL CENTER
Fiscal Year: 2024
Award: $205,000
Funding agency: National Institute of Allergy and Infectious Diseases
Project Summary
Bats are important reservoirs for diverse viral pathogens affecting humans. However, we have a poor
understanding of the key bat innate immunity factors that restrict virus replication. Functional assays that can
identify bat factors that are truly relevant to combating viruses are needed to understand the innate immune
mechanisms that ultimately define bat susceptibility to viral infection. While historically such functional screens
have relied on genome-wide genomic editing (e.g. CRISPR-Cas9)- or RNA interference (RNAi)-based
techniques, such platforms are unavailable for most bat species. Thus, new methods for uncovering functionally-
relevant components of the bat immune response to virus infection are needed. To address this need, we have
developed an innovative arbovirus "rescue" assay wherein immune evasion proteins (IEPs) encoded by
mammalian pathogens can be expressed in bat cells and one can assay for changes in bat cell susceptibility to
arbovirus infection. Enhancement of arbovirus replication after expression of a candidate IEP indicates that the
IEP likely inhibits bat immunity mechanisms that normally restrict arbovirus replication. Using these IEPs as
"tools", one can then identify the bat immunity factors these IEPs target. Thus, this screening methodology
provides a mechanism to both identify novel IEPs and functionally-relevant components of the bat immune
response. To discover IEPs that promote arbovirus replication in bat cells, we will screen an expression library
encoding ~200 bacterial effector proteins. Bacterial effectors are proteins secreted by pathogenic bacteria into
eukaryotic hosts cells that modulate or inhibit various eukaryotic cellular processes to promote bacterial
replication. Many bacterial pathogens that replicate in the cytoplasm of eukaryotic host cells encode effectors
that function as IEPs. Thus, we hypothesize that some effectors may suppress immune responses that restrict
both bacteria and cytoplasmic viruses such as arboviruses. Indeed, our initial screens have identified four
effectors that promote the replication of four different arboviruses when expressed in bat cells. We have
characterized one of these effector screen "hits" as a novel ubiquitin ligase that targets an uncharacterized Ring
Finger (RNF) Domain-containing protein for degradation in eukaryotic cells. Importantly, RNAi depletion of this
RNF factor in human and bat cells promotes arbovirus replication, suggesting that it may be a novel component
of human and bat immune responses. These results suggest that we can use bacterial effectors as tools to both
inhibit, and identify, functionally-relevant immunity factors in bats. Our study has the following specific aims: 1)
Identify bacterial effector proteins that promote arbovirus replication in bat cells;; 2) Identify bat proteins
interacting with effector “hits” from our arbovirus rescue assays;; and 3) Determine which bat host factors
interacting with effector protein hits affect viral replication. Our long-term goal is to use this model system to
define the key bat innate immune mechanisms that restrict arbovirus replication.
Terms: <Address><Affect><Arboviral><Arboviral infections><Arbovirus Infections><Arboviruses><Arthropod-Born Viral Infection><Arthropod-Borne Viruses><Assay><Attention><Bacteria><Bacterial Infections><Bacteriophages><Bats><Bioassay><Biologic Models><Biological Assay><Biological Models><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cell Function><Cell Physiology><Cell Process><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cellular biology><Chiroptera><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Cytoplasm><Data><Eukaryotic Cell><Expression Library><Family><Filoviridae><Filovirus><Genes><Glean><Goals><Grant><Host Factor><Host Factor Protein><Human><Immune><Immune Evasion><Immune response><Immune system><Immunes><Immunity><Immunochemical Immunologic><Immunologic><Immunologic Factors><Immunological><Immunological Factors><Immunological response><Immunologically><Immunologics><Immunology><Immunosuppression><Immunosuppression Effect><Immunosuppressive Effect><Infection><Innate Immune Response><Innate Immunity><Insecta><Insects><Insects Invertebrates><Integration Host Factors><Libraries><Metabolic Protein Degradation><Methodology><Methods><Model System><Modern Man><Native Immunity><Natural Immunity><Non-Specific Immunity><Nonspecific Immunity><Pathogenesis><Pathogenicity Factors><Pathway interactions><Phages><Population><Post-Transcriptional Gene Silencing><Posttranscriptional Gene Silencing><Predisposition><Protein Secretion><Protein Turnover><Proteins><Proteomics><Public Health><RNA Interference><RNA Silencing><RNA interference screen><RNAi><RNAi screen><RNAi-based screen><Regulatory Protein Degradation><Research><Rhabdoviridae><Rhabdoviruses><Ring Finger><Ring Finger Domain><Ring Finger Motif><Ring-Type Zinc Finger Domain><S flexneri><S. flexneri><Sequence-Specific Posttranscriptional Gene Silencing><Shigella flexneri><Subcellular Process><Susceptibility><System><Techniques><Testing><Therapeutic><Togaviridae><Togaviruses><Transmission><Two Hybrid><Ubiquitin Ligase Component Gene><Ubiquitin Ligase Gene><Vaccines><Viral Diseases><Viral reservoir><Virulence Factors><Virus><Virus Diseases><Virus Replication><Virus reservoir><Work><Yeast One Hybrid System><Yeast One/Two-Hybrid System><Yeasts><arthropod-borne infection><arthropodborne infection><bacteria infection><bacteria pathogen><bacterial disease><bacterial pathogen><bacterial virus><bullet shaped virus group><cell biology><cell type><comparative genomics><genome editing><genome profiling><genome scale><genome-wide><genomewide><genomic editing><genomic profiling><host response><immune evasive><immune suppression><immune suppressive activity><immune suppressive function><immune system response><immunologic substance><immunological substance><immunoresponse><immunosuppressive activity><immunosuppressive function><immunosuppressive response><innate immune mechanisms><innovate><innovation><innovative><insight><novel><pathogen><pathogenic bacteria><pathogenic virus><pathway><protein degradation><protein function><respiratory virus><screening><screenings><tool><transcriptome profiling><transcriptomic profiling><transmission process><ubiquitin ligase><viral infection><viral multiplication><viral pathogen><viral replication><virus infection><virus multiplication><virus pathogen><virus-induced disease><yeast two hybrid system>