Role and modulation of Zinc-finger antiviral protein and antiviral Regnase-1-like endonucleases

Project Information

EXPL ANTIVIR ENDONUC

Grant agreement ID: 101062524

DOI

10.3030/101062524

Project closed

EC signature date 13 September 2022

Start date 1 November 2022

End date 31 October 2024

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 173 847,36

Coordinated by

UNIVERSITAET ULM
Germany

Periodic Reporting for period 1 - EXPL ANTIVIR ENDONUC (Role and modulation of Zinc-finger antiviral protein and antiviral Regnase-1-like endonucleases)

Reporting period: 2022-11-01 to 2024-10-31

Summary of the context and overall objectives of the project

This project aimed to uncover how components of human innate immunity, Zinc-finger antiviral protein (ZAP) and Regnase-1-like endonucleases protect us from viral infections. Viral infections, such as those caused by Human Immunodeficient Virus, Influenza and SARS-CoV-2, cause significant global health and economic challenges. Current antiviral therapeutic options are limited and often have a selective activity. This highlights the need for innovative approaches that act against a broad range of viruses by harnessing the natural antiviral responses of the human body. The objectives of this project were to characterize the antiviral potential of Regnase-1-like endonucleases and ZAP and identify their targets during virus infection. Furthermore, we investigated the therapeutic potential of pharmacological MALT1 inhibitors to enhance the natural ZAP and endonuclease activity. The pathway to impact involved a four-part work plan: ranking endonuclease antiviral activities, assessing their efficacy against different human viruses, mapping their RNA targets, and evaluating the effect of MALT1 inhibitors on viral infection. Through advanced techniques such as next-generation sequencing, CRISPR-Cas9 genome editing, and primary cell based infection models, the project aimed to uncover immune mechanisms that could inform novel antiviral treatment strategies. The expected impacts include providing a framework for developing broadly acting antiviral therapies and contributing to our understanding of immune regulation, with potential applications in managing autoimmune diseases and cancer. These outcomes address pressing global health challenges and aim to mitigate the substantial burden of respiratory viral diseases.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We confirmed that the human ZAP acts as broadly-active antiviral protein with a potential to target multiple human viruses. Furthermore, we found that it significantly contributes to the restriction of HIV-1 in CD4+ T cells by type I interferon. In addition, we found that all Regnase-1-like endonucleases except for NYNRIN can act antivirally, and this property is generally dependent on their catalytic PIN domain which mediates RNA degradation.
We found that Regnase-1-like endonucleases are upregulated by interferons in human macrophages, but downregulated at both transcript and protein level during T cell activation. This suggests that HIV-1 might at least partially evade the immune factors by preferentially replicating in activated T cells. In addition, we found that primary strains of HIV-1 differ in their sensitivity to Regnase-1-like endonucleases, suggesting potential additional mechanisms of evasion. We identified multiple Regnase-1-like endonucleases that are cleaved and potentially inactivated by MALT-1. Furthermore, pharmacological MALT1 inhibitors inhibited HIV-1 replication in primary CD4+ T cells in vitro.
These findings highlight the relevant role of Regnase-1-like endonucleases in innate immune antiviral response and their suitability as potential targets of new, broadly-active antiviral therapies.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

It remains to be established if this effect is related to enhanced antiviral activity of Regnase-1-like endonucleases, and what roles these proteins have on the cellular transcriptome.
The determinants of Regnase-1-like endonuclease sensitivity are currently investigated.
The project identified ZAP and Regnase-1-like endonucleases as a broadly-active antiviral proteins playing relevant role in response to infection. Furthermore, we characterized the complex dynamics of Regnase-1-like activity through functional association with ZAP, transcriptional modulation during T cell activation and MALT-1 mediated cleavage. Importantly, pharmacological MALT1 inhibitors were found to suppress HIV-1 replication in primary CD4+ T cells in vitro, however the underlying mechanism remains to be established. These findings underscore the pivotal role of Regnase-1-like endonucleases in the innate immune response and highlight their potential as targets for broad-spectrum antiviral therapies. To ensure further uptake and success of this study, future research should investigate the molecular mechanisms governing the activity of Regnase-1-like endonucleases and MALT1 in different viral target cells, characterize the specificity of MALT1 inhibitors and their impact on the cellular state. This approach, aiming to evaluate Regnase-1-like endonucleases as targets of new antiviral therapies, could lead to future pre-clinical and clinical studies and ultimately, aid the development of broadly active antiviral therapies capable of tackling both current and emerging viral threats.