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 negative-sense RNA viral genome in the cytoplasm of infected cells. We hypothesized that interactions with an array of cellular proteins may enable LASV polymerase to execute distinct viral RNA biosynthetic processes. To investigate this hypothesis, we applied proximity proteomics to define the interactome of LASV polymerase in cells, under conditions that recreate viral transcription and replication. We engineered a LASV polymerase-biotin ligase TurboID fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed us to identify 42 high-confidence hits that interact with LASV polymerase. We performed an siRNA screen to evaluate the role of the identified interactors in LASV infection, which uncovered six host factors for which their depletion affected LASV infection. We found that one polymerase interactor, eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), physically and functionally associated with LASV polymerase, exhibiting proviral activity. Accordingly, pharmacological targeting of GSPT1 resulted in strong inhibition of LASV infection. In summary, our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet to be defined, host-pathogen interactomes, which can reveal new biology and uncover novel targets for the development of antivirals against LASV. Significance Statement Lassa virus (LASV), the causative agent of Lassa fever (LF), poses an important public health problem in Western Africa, and current therapeutic interventions are very limited. Due to its limited genome coding capacity, LASV proteins are often multifunctional and orchestrate complex interactions with cellular factors to execute the steps required to complete its viral life cycle. LASV polymerase is essential for the replication and expression of the viral genome, and it is consequently an attractive target for antiviral intervention. Here we present the first host interactome of LASV polymerase, which can guide the identification of novel druggable host cellular targets for the development of cost-effective antiviral therapies against LF.