Discovering LC3-interacting region (LIR) motifs in hemorrhagic fever viruses: Implications for host autophagy and viral replication.

Abstract

Hemorrhagic fever viruses (HFVs) pose significant global health and economic burdens, yet limited FDA-approved therapeutics exist to combat their spread. Determining alterations in host processes during infection can enhance our understanding of viral replication and lead to potential host-based therapeutic targets. Rift Valley fever virus (RVFV) is a HFV that causes significant disease in humans and livestock and was an initial model HFV for the present study. The RVFV nonstructural small (NSs) protein is the main virulence factor of RVFV and we discovered four novel LC3-interacting region (LIR) motifs within NSs (NSs1-4), indicating that NSs interacts with LC3, the host key autophagy protein. Autophagy is a cellular process that can act anti-virally by enhancing immune responses and promoting viral degradation, or in some cases, be pro-viral, facilitating viral replication. LC3 proteins are critical modulators of autophagy and autophagosome maturation. We hypothesize that HFV proteins interact with LC3-family members via LIR motifs to modulate the host autophagy pathway which represents a target for therapeutic development. The NSs protein of RVFV was selected for analysis due to it being a virulence factor and having an intrinsically disordered region, which are hot spots for LIR motifs. Isothermal titration calorimetry, X-ray crystallography, co-immunoprecipitation, and co-localization experiments confirmed that the C-terminal LIR motif (NSs4) interacts with all six human LC3 proteins. We identified phenylalanine 261 (F261) in NSs4 as essential for the LC3 interaction, nuclear retention, and autophagy inhibition in RVFV-infected cells, highlighting how RVFV inhibits autophagy via the NSs4 LIR motif. Mechanistically, LC3 is located in the nucleus at cellular homeostasis, Sirt1 deacetylates LC3, and DOR then interacts with LC3 to transport it to the cytosol where it interacts with various autophagy proteins to form autophagosomes. We discovered that RVFV NSs interacts with Sirt1, competes with DOR for binding to LC3, and retains unacetylated LC3 in the nucleus therefore inhibiting autophagy and enhancing viral pathogenesis in a mouse model. Building on our success with studying an LIR in RVFV NSs, an AI/ML-driven LIR discovery pipeline was developed to identify and analyze LIR motifs in all HFV proteins. The LIR discovery pipeline identified 42 putative LIR motifs in 166 proteins from 22 HFVs using the iLIR and ELM databases. This list was further narrowed down to 17 HFV proteins that contained a LIR motif in a predicted unstructured region and showed a favorable interaction with LC3 via AlphaFold3 and FoldX analysis. Our LIR discovery pipeline identified a highly favorable interaction between LC3 and the Marburg virus nucleoprotein (MARV NP). Utilizing isothermal titration calorimetry and co-immunoprecipitation we have confirmed the interaction of MARV NP and LCA via a LIR motif, and determined that the interaction modulates autophagy in cellulo, demonstrating the ability of our results to be translated across HFV families. Given the threat of emerging and reemerging HFVs, this research is crucial for public health, exploring LIR motifs as therapeutic targets to disrupt viral replication and mitigate future outbreaks.