Abstract :
[en] The immune system relies on numerous sensors of nucleic acids (NAs). The latter are
detected based on their subcellular localization, sequences, and even their conformation.
Zalpha (Zα) domains predominantly bind to left-handed Z-DNA and Z-RNA. The description of Zα domains in proteins of the immune system, as well as in viral proteins involved in immune evasion mechanisms, demonstrates that the conformation of NA is also analyzed by the immune system as a sign of abnormality. The first cellular protein reported to express a Zα domain was the Adenosine deaminase RNA-specific binding protein 1 (ADAR1). Only its interferon-inducible isoform expresses a Zα domain at its N-terminal end. ADAR1 is an important protein of innate immunity. Its main role is to regulate double-stranded RNA (dsRNA) within cells. Its action is mainly mediated through its deaminase domain leading to sequence modification of RNA interacting with ADAR1. The second cellular protein containing Zα domains is the Z-DNA binding protein 1 (ZBP1). ZBP1 has also been extensively studied in the context of innate immunity, particularly during the induction of cell death in response to detection of NA expressing Z-conformation. Finally, the protein kinase Z (PKZ) is the latest cellular protein reported to contain Zα domains. This protein is a paralog of the Protein kinase RNA-activated (PKR). PKZ has only be reported in certain orders of teleost fish. PKZ is thought to play a similar role to PKR in translation shutdown, but its activation relies on the specific detection of Z-NA rather than dsRNA. Zα domains have also been reported in few viral proteins. The E3 protein of the vaccinia virus (Vv) consists of a Zα domain and a double-stranded RNA binding domain (dsRBD). This protein has been demonstrated to antagonize ZBP1 and PKR, playing an important role in immune evasion. Zα domains have also been identified in the ORF112 of cyprinid herpesvirus 3 (CyHV-3) and, very recently, in the I73R protein of African swine fever virus (ASFV).
The general objective of this thesis was to study the biochemical properties of Zα domains using the CyHV-3 ORF112 as a model, and to contribute to unravel the role of this protein in the biological cycle of CyHV-3. First, genome editing of CyHV-3 demonstrated that ORF112 is essential for viral replication in cell culture. Next, the same approach led to the observation that the expression of only the Zα domain of ORF112 was sufficient for normal
viral replication in cell culture and virulence in carp. These observations revealed the potential of the CyHV-3 model as a unique platform to compare the exchangeability of Zα domains expressed alone in living cells. Attempts to rescue the ORF112 deletion with a broad spectrum of cellular, viral, and artificial Zα domains demonstrated that only those exhibiting Z-binding activity, the ability to induce liquid-liquid phase separation (LLPS), and A-to-Z conversion, could rescue viral replication. For the first time, our results report the ability of some Zα domains to induce LLPS and demonstrate the biological relevance of A-to-Z conversion by Zα domains. This first study expands the functional diversity of Zα domains and stimulates new hypotheses concerning the mechanisms of action of Zα domain-containing proteins (ZBPs).
In a second experimental chapter, we took advantage of the recent discovery of a new
Zα domain encoded by ASFV to test the ability of the CyHV-3 model to identify triple-functional Zα domains. This second study also aimed to investigate the relative importance of these properties during ASFV infection in cell culture. We demonstrated the ability of I73R Zα domain to rescue the deletion of CyHV-3 ORF112. However, we observed a significant reduction of CyHV-3 replication in cell culture and absence of virulence in vivo for the recombinant strain. Based on these observations, we performed complementary experimental approaches to confirm that the I73R expressed the three properties described above for the Zα domain of ORF112. Finally, we showed that among a panel of Zα domains representative of the function diversity of Zα domains, only the Zα domain of ORF112 could compensate for the deletion of I73R. This observation, coupled with the presence of dsRNA within infected cells highlighted the presence of ribonucleoprotein (RNP) complexes during ASFV infection and underscored the importance of LLPS in this context.
In conclusion, this project contributed to the characterization of the biochemical and functional properties of Zα domains. Furthermore, this work highlighted a previously underestimated functional diversity among Zα domains and unravel their importance for the functionality of ZBPs during viral infections. These new insights enhance our understanding of ZBPs and allow us to develop new hypotheses related to the roles of ORF112 and I73R as virulence factors during the biological cycle of CyHV-3 and ASFV.
