Rift Valley fever virus NSs protein interacts with LC3 family members to inhibit antiviral autophagy

PROJECT SUMMARY/ABSTRACT Rift Valley fever virus (RVFV) is a viral zoonosis that causes severe disease in ruminants and humans [1]. Concerns are heightened regarding the introduction of RVFV to the U.S. as competent mosquito vectors have been identified and increased global commerce makes it probable that RVFV will spread to new areas [12]. Despite its pathogenic potential, there are no FDA-approved therapeutics or vaccines to challenge the global spread of this infectious organism. The nonstructural small (NSs) protein is the main virulence factor of RVFV, making it an attractive antiviral target [15]. This proposal aims to close the gap in knowledge regarding the role that NSs plays in the host autophagy pathway, by identifying the impact of the NSs-LC3 interaction on autophagy, viral replication, and pathogenesis. Bioinformatic analysis identified four putative LC3-interacting region (LIR) motifs in the RVFV NSs protein (LIR1-4), suggesting that NSs interacts with LC3 family members, the host key autophagy proteins. Autophagy is a homeostatic process in which cellular material is degraded and recycled and can be exploited by viruses to facilitate replication or can be antiviral. [31]. Preliminary studies confirmed that NSs interacts with all six LC3-family members in vitro and in RVFV-infected cells. Isothermal titration calorimetry (ITC) demonstrated favorable binding between LIR4 and all six-human LC3 family members, however little to no binding was observed between NSs LIR1-3 and LC3, indicating that LIR4 is mediating interaction between NSs and LC3. Additionally, ITC data and co-immunoprecipitation data in RVFV infected cells demonstrated the importance of F261 within LIR4 for the NSs-LC3 interaction. Crystal structures showed that the predominant NSs-LC3 interactions at the binding interface involve the side chain of LIR4 F261, furthering the importance of LIR4 F261 for NSs-LC3 interaction. Aim 1 will define the autophagy molecular mechanism of the NSs-LC3 interaction and the effect of loss of NSs-LC3 interaction through F261S substitution on autophagy. Aim 2 will determine the impact of NSs-LC3 interaction and regulation of autophagy on viral pathogenesis and survival through mouse studies using RVFV ZH548 wild type vs. F261S viruses. This fellowship will prepare the trainee for a career as a Professional Investigator in Academia through an intensive individualized training program facilitated by my strong sponsor Dr. Kehn-Hall, collaborators, and opportunities provided by Virginia Tech. Taken together, this fellowship will define the mechanism by which NSs and LC3 interact to modulate autophagy and the impact of the NSs-LC3 interaction on RVFV pathogenesis in a mouse model, providing a potential future therapeutic target.