Eine neue Rolle von HSP70 Proteinen in der antiviralen Immunantwort

• Antragsteller: Professor Dr. Karl-Klaus Conzelmann (†)
• Fachliche Zuordnung: Virologie ; Immunologie ; Zellbiologie
• Förderung: Förderung von 2017 bis 2022
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 346764907

• Erstellungsjahr: 2022

Zusammenfassung der Projektergebnisse

The rabies virus phosphoprotein P is a potent and multifunctional interferon antagonist. Studies on the mechanisms of P in preventing IRF3 activation and type I IFN transcription suggested the involvement of an unexpected player, the cellular heat shock protein HSP70. Members of the large HSP70 family are chaperones binding their standard clients via a substrate binding domain (SBD) and exert their functions via an ATPase in association with co-chaperones and nucleotide exchange factors (NEFs). By engineering P proteins, we identified the linear motif (180)GPPALEW(186) as sufficient for binding the HSP70 SBD. A P single amino acid exchange mutant W186A was deficient in HSP70 binding and IFN inhibition. Co-IP experiments involving HSP variants and subunits verified P binding to the SBD, while direct association with the ATPase domain was not observed. As overexpression of mutated HSP70 revealed a key role of the ATPase function in suppressing IFN transcription, our hypothesis is that P binds to the standard SBD of HSP70 but blocks its ATPase function directly or by preventing association with NEFs or co-chaperones. Most surprisingly, IFN suppression by P did not only require HSP70 binding, but also nuclear shuttling of P. This was revealed by engineering nuclear localization signals (NLS) in the C-terminal domain (CTD) of the P protein. P proteins unable to shuttle but still competent in HSP binding could not suppress IFN induction. Shuttling and IFN suppression, however, was achived by a mutant lacking the N-terminal domain and replacement of the entire CTD with a short heterologous NLS from SV40 virus. This minimal inhibition competent construct could be used further to identify the complex interplay between RABV P nuclear trafficking, binding of HSP70 and collection of other factors to determine the mechanism of IFN inhibition. Apart from viral IFN antagonists like RABV P it appears worthwhile to further investigate the general IFN suppressive function of heat shock conditions and HSP70 members.

Projektbezogene Publikationen (Auswahl)

• Cryo EM structure of the rabies virus ribonucleoprotein complex. Sci Rep. 2019 Jul 3;9(1):9639
  Riedel, Christiane; Vasishtan, Daven; Pražák, Vojtěch; Ghanem, Alexander; Conzelmann, Karl-Klaus & Rümenapf, Till
  
• Components and Architecture of the Rhabdovirus Ribonucleoprotein Complex. Viruses. 2020 Aug 29;12(9):959
  Riedel, Christiane; Hennrich, Alexandru A. & Conzelmann, Karl-Klaus
  
• Optimization of whole-brain rabies virus tracing technology for small cell populations. Sci Rep. 2021 May 17;11(1):10400
  Roelofs, Theresia J. M.; Menting-Henry, Shanice; Gol, Lieke M.; Speel, Annelijn M.; Wielenga, Vera H.; Garner, Keith M.; Luijendijk, Mieneke C. M.; Hennrich, Alexandru A.; Conzelmann, Karl-Klaus & Adan, Roger A. H.
  
• Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses. Nat Methods. 2022 Jan;19(1):81-89
  Albanese, Manuel; Ruhle, Adrian; Mittermaier, Jennifer; Mejías-Pérez, Ernesto; Gapp, Madeleine; Linder, Andreas; Schmacke, Niklas A.; Hofmann, Katharina; Hennrich, Alexandru A.; Levy, David N.; Humpe, Andreas; Conzelmann, Karl-Klaus; Hornung, Veit; Fackler, Oliver T. & Keppler, Oliver T.
  
• Safe and effective two-in-one replicon-and-VLP minispike vaccine for COVID-19:protection of mice after a single immunization. PLoS Pathogens, 2021 Apr 21;17(4):e1009064
  Hennrich, Alexandru A.; Sawatsky, Bevan; Santos-Mandujano, Rosalía; Banda, Dominic H.; Oberhuber, Martina; Schopf, Anika; Pfaffinger, Verena; Wittwer, Kevin; Riedel, Christiane; Pfaller, Christian K. & Conzelmann, Karl-Klaus