Restriktion von RNA-Viren durch ZAP, TRIM25 und ihre Kofaktoren

• Antragstellerin: Dr. Dorota Kmiec
• Fachliche Zuordnung: Virologie
• Förderung: Förderung von 2019 bis 2022
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 428874181

• Erstellungsjahr: 2022

Zusammenfassung der Projektergebnisse

Zinc finger antiviral protein (ZAP) is an important player in innate immunity. Using an unique antiviral defense mechanism, ZAP binds CpG-dinucleotides in viral RNA, targeting them for degradation. While ZAP can recognize foreign transcripts, it relies on multiple cellular cofactors to destroy them. Recent evidence highlights a functional relationship between ZAP, ubiquitin ligase TRIM25 and endonuclease KHNYN, which is important for antiviral immunity against Human Immunodeficiency Virus type 1 (HIV- 1). However, much less is known about the underlying mechanism of their activity and involvement in innate immune responses against other viral pathogens. Through comprehensible structure-function analysis of ZAP’s domains and motifs, we confirmed the importance of RNA-binding domain and CpG-interacting residues in ZAP’s antiviral function. In addition, we found that determinants located in the C-terminal PARP domain are crucial for ZAP’s CpG-specific antiviral activity against HIV-1. These elements were also required for optimal antiviral activity against murine leukamia virus (MLV) and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ZAP’s PARP domain is catalytically inactive, due to a lack of a H-Y-E alternate triad motif required for NAD+ cofactor binding. Surprisingly, restoration of H-Y-E motif in ZAP lead to a partial loss of antiviral activity, suggesting that evolutionary loss of ADP-ribosylation by ZAP was associated with a gain of CpG-specific antiviral function. CaaX box also significantly contributed to ZAP’s antiviral function. This motif is localized at the C-terminus of the PARP domain and mediates S-farnesylation of ZAP-L, which in turn relocalizes from the cytoplasm to intracellular membranes. CaaX motif and PARP domain were also required for optimal interaction with ZAP’s cofactors, TRIM25 and KHNYN, providing a novel role of ZAP’s C-terminus in cofactor recruitment. Furthermore, anchoring of ZAP-L on intracellular membranes might provide a scaffold for antiviral complex assembly, providing an essential effector function in ZAP-L- mediated antiviral activity. Through a screening approach, we have identified 7 new modulators of ZAP’s antiviral activity. These exciting findings are currently being followed up upon and will shed new light on the mechanism of action of the ZAP antiviral complex. Together, my results highlight the relevant role of ZAP in innate immune responses against highly relevant human pathogens such as HIV-1 and SARS-CoV-2. My bioinformatic analyses also suggest that ZAP might be actively contributing to the evolution of these pathogens, driving a decrease in their CpG content to evade ZAP recognition. The main goals of the original project were achieved as proposed, as well as the work schedule was completed as outlined. During my stay at King’s College London, I gained valuable experience in state-of-the-art laboratory techniques such as live-cell imaging and CRISPR/Cas9 gene editing, project management and scientific writing. I also attended helpful courses for young scientists focusing on leadership, statistical methods, grant writing, publishing and public speaking. Furthermore, my first-author project spawned exciting ideas for future research, including the investigation of endonucleases involved in antiviral responses and the role of ZAP in the evolution of zoonotic viruses such as SARS-CoV-2, monkeypox and HIV-2. In summary, this project was highly successful in terms of reaching its proposed goals, identifying novel candidate cofactors of ZAP and unravelling the molecular mechanism of its antiviral activity. The results of the project will provide the basis for compelling future studies on innate immunity and cancer.

Projektbezogene Publikationen (Auswahl)

• SARS-CoV-2 Is Restricted by Zinc Finger Antiviral Protein despite Preadaptation to the Low-CpG Environment in Humans. mBio, 2020; 11(5), e01930-20
  Nchioua R, Kmiec D, Müller JA, Conzelmann C, Groß R, Swanson CM, Neil SJM, Stenger S, Sauter D, Münch J, Sparrer K, Kirchhoff F
  
• Minimal impact of ZAP on lentiviral vector production and transduction efficiency. Mol. Ther. Methods Clin. Dev. 2021;8;28;23:147-157
  Sertkaya H, Hidalgo L, Ficarelli M, Kmiec D, Signell A, Ali S, Parker H, Wilson H, Neil S, Malim M, Vink C, Swanson C
  
• Regulation of KHNYN antiviral activity by the extended di-KH domain and nucleo-cytoplasmic trafficking
  Lista-Brotos MJ, Galão RP, Ficarelli M, Kmiec D, Wilson H, Winstone H, Morris ER, Mischo HE, Wanford J, Youle R, Odendall C, Taylor IA, Neil SJD, Swanson CM
  
• S-farnesylation is essential for antiviral activity of the long ZAP isoform against RNA viruses with diverse replication strategies. PLoS Pathog. 2021;17(10):e1009726
  Kmiec D, Lista MJ, Ficarelli M, Swanson CM, Neil SJD
  
• Omicron: What makes the latest SARS-CoV-2 variant of Concern so concerning? J Virol. 2022 Mar 23;96(6):e0207721
  Jung C, Kmiec D, Koepke L, Zech F, Timo J, Sparrer KMJ, Kirchhoff F