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Principal Investigator: Ricardo Rajsbaum
Organization: UNIVERSITY OF TEXAS MED BR GALVESTON
Fiscal Year: 2020
Award: $395,000
Funding agency: National Institute of Allergy and Infectious Diseases
The most recent outbreak of Ebola virus (EBOV) in West Africa reached alarming numbers of infections and
deaths, prompting extensive efforts to develop antiviral strategies; however, none have thus far been approved
for use in humans. The development of antiviral drugs is mostly based on designing compounds that target
viral proteins, but this approach may select for viral escape mutants, and reversion to virulence can occur. In
contrast, the identification of host cellular factors required for virus replication may lead to novel therapeutic
strategies with a minimal inherent risk of escape mutants. The host ubiquitin system is a conserved cellular
pathway important in many functions, including innate immune signaling. Viruses are known to manipulate the
ubiquitin system for their own advantage; therefore, the goal of this proposal is to define the molecular
mechanisms of EBOV replication involving the ubiquitin system with the long-term goal of applying this
knowledge in the development of effective antiviral approaches.
EBOV VP35 is a critical viral protein contributing to pathogenesis because it serves as an essential cofactor
of the viral polymerase, thereby promoting efficient replication. Further, VP35 potently blocks innate immunity
by inhibiting the host antiviral type-I interferon (IFN-I) system. However, regulation of VP35 function by cellular
post-translational modifications is not understood. Our preliminary data suggest that EBOV replication is
regulated by a novel mechanism involving VP35 ubiquitination by TRIM6, a host E3-ubiquitin ligase with known
antiviral functions. Based on our data, we hypothesize that VP35 hijacks TRIM6 to promote EBOV replication
through ubiquitination, while also blocking TRIM6 innate immune signaling activity. By using in vitro and in vivo
approaches, including our newly generated TRIM6-knockout mice, we will test how TRIM6 contributes to
EBOV replication and pathogenesis. We propose the following specific aims: 1) Determine the mechanistic role
of VP35 ubiquitination in EBOV replication. We will use already available EBOV VP35 viral mutants that lack
ubiquitination sites and test their function in relation to innate immune antagonism or viral polymerase function.
2) Determine the molecular mechanism by which TRIM6 regulates EBOV replication. We will examine how
TRIM6 regulates EBOV replication by using TRIM6 knockout cell lines and in vitro ubiquitination and binding
assays. We will also demonstrate that poly-Ub chains synthesized by TRIM6 in vivo promote EBOV replication,
using our TRIM6-/- mice and mouse-adapted WT EBOV and the VP35 mutants that lack ubiquitination.
The outcome of these studies is significant because it will provide fundamental knowledge about the
mechanism of EBOV replication, which may apply to other viruses. Thus, a common approach for the
development of broad-spectrum antivirals may be revealed. Importantly, this work will inform whether a host
cellular factor, TRIM6, may be used as a target for the development of novel therapeutic strategy against one
of the most lethal viruses of mankind.
Terms: <APF-1><ATP-Dependent Proteolysis Factor 1><Africa><Anti-Viral Response><Antiviral Agents><Antiviral Drugs><Antiviral Response><Antiviral Therapy><Antivirals><Area><Assay><BSL-4 facility><BSL4 facility><Binding><Bioassay><Biochemical><Biologic Assays><Biological Assay><Biology><Case Fatality Rates><Category A pathogen><Category A priority pathogen><Cell Body><Cell Function><Cell Line><Cell Process><Cell physiology><CellLine><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cessation of life><Containment><Data><Death><Development><Disadvantaged><Disease Outbreaks><Double-Stranded RNA><E3 Ligase><E3 Ubiquitin Ligase><EBOV><Ebola><Ebola virus><Epidemic><Escape Mutant><FLJ11330><Failure><Family><Filoviridae><Filovirus><Goals><HMG-20><High Mobility Protein 20><Host Factor><Host Factor Protein><Human><IFN><Immune><Immune signaling><Immunes><In Vitro><Infection><Innate Immunity><Integration Host Factors><Interferon Type I><Interferons><KO mice><Kinases><Knock-out><Knock-out Mice><Knockout><Knockout Mice><Knowledge><L-Lysine><Laboratories><Lead><Lysine><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mediating><Mice><Mice Mammals><Modern Man><Molecular><Molecular Interaction><Murine><Mus><NF-Kb-Activating Kinase Gene><NIAID><National Institute of Allergy and Infectious Disease><Native Immunity><Natural Immunity><Nipah Virus><Non-Specific Immunity><Nonspecific Immunity><Null Mouse><Outbreaks><Outcome Study><Pathogenesis><Pathogenicity><Pathway interactions><Patients><Pb element><Persons><Phosphotransferase Gene><Phosphotransferases><Polymerase><Polyubiquitin><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Protein Modification><Proteins><RBCC/TRIM Motif><Recombinants><Regulation><Reporter><Reporting><Risk><Role><Severities><Site><Strains Cell Lines><Subcellular Process><System><T2K><TBK1><TBK1 gene><TRIM Motif><Testing><Therapeutic Intervention><Transmission><Transphosphorylases><Tripartite Motif><Ubiquitilation><Ubiquitin><Ubiquitin Protein Ligase><Ubiquitin-Protein Ligase Complexes><Ubiquitin-Protein Ligase E3><Ubiquitination><Ubiquitinoylation><Vaccines><Viral><Viral Burden><Viral Diseases><Viral Gene Products><Viral Gene Proteins><Viral Hemorrhagic Fevers><Viral Load><Viral Load result><Viral Pathogenesis><Viral Proteins><Virulence><Virus><Virus Diseases><Virus Replication><Work><anti-viral agents><anti-viral drugs><anti-viral therapy><anti-virals><base><biosafety level 4 facility><cofactor><cultured cell line><design><designing><developmental><drug development><dsRNA><ebolavirus><heavy metal Pb><heavy metal lead><hemorrhagic fever><in vivo><intervention therapy><member><mortality><mutant><new drug treatments><new drugs><new therapeutics><new therapy><next generation therapeutics><non-human primate><nonhuman primate><novel><novel drug treatments><novel drugs><novel therapeutics><novel therapy><pathogen><pathogenic virus><pathway><phase 3 trial><phase III trial><poly-ubiquitin><public health relevance><response><social role><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><transmission process><ubiquination><ubiquitin conjugation><ubiquitin-protein ligase><viral infection><viral infectious disease treatment><viral multiplication><viral pathogen><viral replication><virus infection><virus multiplication><virus pathogenesis><virus protein><virus-induced disease>