Virus-host interactions essential for genome replication and transcription of Lassa virus
Bioinformatics and Theoretical Biology
Structural Biology
Final Report Abstract
Lassa virus (LASV), as well as other members of the family Arenaviridae, represent a high risk for the human health due to the high pathogenicity and the lack of vaccination and treatment possibilities according to the WHO. The viral L protein is the key player in viral transcription and genome replication and thus of central importance for the viral infection cycle. In order to identify cellular interaction partners involved in the functions carried out by the L protein, a combination of Affinity Purification (AP-) Mass Spectrometry (MS) studies with arenaviral L proteins and a genetic knockout screening with LASV was applied and additionally the feasibility of proximity dependent biotinylation studies was examined. The AP-MS studies with purified arenaviral L proteins and recombinant Lassa viruses carrying a StrepII tag in the L protein identified 991 cellular proteins as potential interaction partners and 231 cellular proteins were also identified during viral infection. Using a recombinant LASV producing red fluorescence in in infected cells, 106 cellular genes essential for LASV were identified in a genetic knockout screen, 19 of which were also shown in the AP-MS studies. To prioritize candidates for experimental validation, we developed AlphaPulldown, a software pipeline that streamlines high-throughput structure prediction of protein-protein interactions using AlphaFold. We applied it to all L-host protein pairs identified by AP-MS and, by integrating structural data with AP-MS and genetic screens, shortlisted interactors for validation. We confirmed the proteinprotein interaction between LASV L and the DDX helicases DDX5, DDX17, DDX19 and DDX24, as well as eukaryotic translation initiation factor eIF4G2, part of eIF4F complex, and NCBP1, a subunit of the cellular cap-binding complex CBC. The interaction of LASV L with DDX17 and DDX24 was RNA-independent and the complex could be reconstituted in vitro as detected by mass photometry demonstrating direct protein-protein interaction. Studies in the minireplicon system suggest a rather proviral role for DDX17 and DDX24 and a rather antiviral role for eIF4G2.
Publications
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The Cap-Snatching Mechanism of Bunyaviruses. Trends in Microbiology, 28(4), 293-303.
Olschewski, Silke; Cusack, Stephen & Rosenthal, Maria
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Reduced Nucleoprotein Availability Impairs Negative-Sense RNA Virus Replication and Promotes Host Recognition. Journal of Virology, 95(9).
Nilsson-Payant, Benjamin E.; Blanco-Melo, Daniel; Uhl, Skyler; Escudero-Pérez, Beatriz; Olschewski, Silke; Thibault, Patricia; Panis, Maryline; Rosenthal, Maria; Muñoz-Fontela, César; Lee, Benhur & tenOever, Benjamin R.
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Validation of Inactivation Methods for Arenaviruses. Viruses, 13(6), 968.
Olschewski, Silke; Thielebein, Anke; Hoffmann, Chris; Blake, Olivia; Müller, Jonas; Bockholt, Sabrina; Pallasch, Elisa; Hinzmann, Julia; Wurr, Stephanie; Neddersen, Neele; Rieger, Toni; Günther, Stephan & Oestereich, Lisa
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AlphaPulldown—a python package for protein–protein interaction screens using AlphaFold-Multimer. Bioinformatics, 39(1).
Yu, Dingquan; Chojnowski, Grzegorz; Rosenthal, Maria & Kosinski, Jan
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RNA to Rule Them All: Critical Steps in Lassa Virus Ribonucleoparticle Assembly and Recruitment. Journal of the American Chemical Society, 145(51), 27958-27974.
Sänger, Lennart; Williams, Harry M.; Yu, Dingquan; Vogel, Dominik; Kosinski, Jan; Rosenthal, Maria & Uetrecht, Charlotte
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AlphaPulldown2—a general pipeline for high-throughput structural modeling. Bioinformatics, 41(3).
Molodenskiy, Dmitry; Maurer, Valentin J.; Yu, Dingquan; Chojnowski, Grzegorz; Bienert, Stefan; Tauriello, Gerardo; Gilep, Konstantin; Schwede, Torsten & Kosinski, Jan
