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1.
Proc Natl Acad Sci U S A ; 119(30): e2201208119, 2022 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-35858434

RESUMEN

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.


Asunto(s)
Acetamidas , Antivirales , Isoindoles , Virus Lassa , Factores de Terminación de Péptidos , Piperidonas , ARN Polimerasa Dependiente del ARN , Proteínas Virales , Acetamidas/farmacología , Acetamidas/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , Línea Celular Tumoral , Humanos , Isoindoles/farmacología , Isoindoles/uso terapéutico , Fiebre de Lassa/tratamiento farmacológico , Virus Lassa/efectos de los fármacos , Factores de Terminación de Péptidos/metabolismo , Piperidonas/metabolismo , Piperidonas/farmacología , Piperidonas/uso terapéutico , Mapas de Interacción de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteoma , Proteómica , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo
2.
Cell Rep ; 38(12): 110544, 2022 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-35320713

RESUMEN

Ebola virus (EBOV) critically depends on the viral polymerase to replicate and transcribe the viral RNA genome in the cytoplasm of host cells, where cellular factors can antagonize or facilitate the virus life cycle. Here we leverage proximity proteomics and conduct a small interfering RNA (siRNA) screen to define the functional interactome of EBOV polymerase. As a proof of principle, we validate two cellular mRNA decay factors from 35 identified host factors: eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1) and up-frameshift protein 1 (UPF1). Our data suggest that EBOV can subvert restrictions of cellular mRNA decay and repurpose GSPT1 and UPF1 to promote viral replication. Treating EBOV-infected human hepatocytes with a drug candidate that targets GSPT1 for degradation significantly reduces viral RNA load and particle production. Our work demonstrates the utility of proximity proteomics to capture the functional host interactome of the EBOV polymerase and to illuminate host-dependent regulation of viral RNA synthesis.


Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Ebolavirus/genética , Interacciones Huésped-Patógeno , Humanos , Proteómica , ARN Helicasas/genética , ARN Mensajero/metabolismo , ARN Viral/genética , Transactivadores , Replicación Viral
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