A novel role of HSP70 proteins in antiviral immune response

• Applicant: Professor Dr. Karl-Klaus Conzelmann
• Subject Area: Virology ; Immunology ; Cell Biology
• Term: from 2017 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 346764907

• Final Report Year: 2022

Final Report Abstract

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.

Publications

• Cryo EM structure of the rabies virus ribonucleoprotein complex. Sci Rep. 2019 Jul 3;9(1):9639
  Riedel C, Vasishtan D, Pražák V, Ghanem A, Conzelmann KK, Rümenapf T
  
• Components and Architecture of the Rhabdovirus Ribonucleoprotein Complex. Viruses. 2020 Aug 29;12(9):959
  Riedel C, Hennrich AA, Conzelmann KK
  
• Optimization of whole-brain rabies virus tracing technology for small cell populations. Sci Rep. 2021 May 17;11(1):10400
  Roelofs TJM, Menting-Henry S, Gol LM, Speel AM, Wielenga VH, Garner KM, Luijendijk MCM, Hennrich AA, Conzelmann KK, Adan RAH
  
• 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 AA, Sawatsky B, Santos-Mandujano R, Banda DH, Oberhuber M, Schopf A, Pfaffinger V, Wittwer K, Riedel C, Pfaller CK, Conzelmann K
  
• 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 M, Ruhle A, Mittermaier J, Mejías-Pérez E, Gapp M, Linder A, Schmacke NA, Hofmann K, Hennrich AA, Levy DN, Humpe A, Conzelmann KK, Hornung V, Fackler OT, Keppler OT