Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host-directed antivirals

Abstract

Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded, RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed a small interfering RNA (siRNA) screen to identify those interactors that have functional roles in authentic LASV infection. As proof of principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small-molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet-to-be-defined host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses.

Combating Lassa Fever in West African Sub-Region: Progress, Challenges, and Future Perspectives

“…For example, GSPT1 cellular protein currently identified might be a potential drug target, as CC-90009, a drug that specifically targets and degrades GSPT1, exhibited antiviral activity against LASV without cytotoxicity. Currently, CC-90009 is in phase 1 clinical development for the treatment of acute myeloid leukemia, raising the possibility of its repurposing as an antiviral against LASV [ 165 ]. Additionally, about 30 vaccines have been reported to be in the pre-clinical stage while 4 of them are currently undergoing clinical trials.…”

Chemical and Biological Strategies for Profiling Protein‐Protein Interactions in Living Cells

“…Thus, TurboID (35 kDa) and miniTurbo (28 kDa) obtained through directed evolution of BioID have been developed to allow efficient proximity labeling within 10‐mins [77] . Recently, TurboID is used to identify high‐confidence interactors of the Lassa virus polymerase, shedding light on the potential druggable target for host‐directed antivirals development [78] . However, possibly due to the higher activity of TurboID than that of miniTurbo, low‐level labeling often occurred even without addition of exogenous biotin into cells, indicating that endogenous biotin can still serve as the substrate for labeling.…”

“… [77] Recently, TurboID is used to identify high‐confidence interactors of the Lassa virus polymerase, shedding light on the potential druggable target for host‐directed antivirals development. [78] However, possibly due to the higher activity of TurboID than that of miniTurbo, low‐level labeling often occurred even without addition of exogenous biotin into cells, indicating that endogenous biotin can still serve as the substrate for labeling. In this sense, TurboID is not a suitable tool for the study that needs to be carried out in a precisely defined labeling time window.…”

Development and therapeutic potential of GSPT1 molecular glue degraders: A medicinal chemistry perspective

“…Fang et al 115 combined proximity proteomics with the use of a noninfectious cell-based LASV minigenome system to reproduce the molecular processes involved in LASV replication. Based on the combined approach, 42 host proteins were identified that play a role in the replication and diffusion of LASV.…”