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.

Public Health Relevance

): Ebola virus causes high mortality rates with extreme risk of person-to-person transmission. The last outbreak in West Africa resulted in more than 28,000 patients and over 10,000 fatal cases, highlighting that there is still insufficient knowledge to develop effective antiviral therapies. The goals of this proposal are to understand the mechanisms that mediate Ebola virus replication by the host ubiquitin system, which will serve as the basis for the development of novel antiviral approaches.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI134907-01
Application #
9426133
Study Section
Virology - B Study Section (VIRB)
Program Officer
Repik, Patricia M
Project Start
2018-05-16
Project End
2023-04-30
Budget Start
2018-05-16
Budget End
2019-04-30
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Texas Med Br Galveston
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
800771149
City
Galveston
State
TX
Country
United States
Zip Code
77555