The African swine fever virus I73R protein is a multifunctional Zalpha domain that mediates both Z binding dependent and independent biological functions through induction of phase separation

African swine fever virus (ASFV) I73R encodes a protein consisting only of a Zalpha domain. Deletion of ASFV I73R reduces viral replication in cell culture and virulence in vivo. Here, we studied the biochemical properties of ASFV I73R protein. We demonstrated that in addition to its Z-binding activity it induces both BtoZ conversion and liquidliquid phase separation (LLPS). This conclusion relied on (i) the ability of I73R to rescue the deletion of Cyvirus cyprinidallo 3 ORF112 encoding an essential and triplefunctional Zalpha domain (Zbinding, BtoZ conversion and LLPS induction properties) and (ii) characterization of BtoZ conversion and LLPS induction properties of purified I73R. Of note, I73R proved to be a better converter than human ADAR1 Zalpha, and it retained conversion activity with Y48I mutation of its Z-binding site. Next, we investigated the biological functions of I73R. Ectopic expression of I73R and mutated I73R with deficient Z-binding inhibited comparably arseniteinduced stress granules but none of the proteins co-localized with these membraneless organelles. However, the role of I73R as a driver of LLPS was supported by the observation that it forms cytosolic condensates with dsRNA in ASFV-infected. Rescuing of ASFV I73R deletion by sequences encoding natural, mutated and chimeric forms of Zalpha domains demonstrated that I73R mediated both Zbinding-dependent and -independent biological functions through induction of LLPS. This conclusion was further supported by the effect of the LLPS inhibitor 1,6Hexanediol on the replication of ASFV I73R recombinant strains. This study further demonstrates the functional diversity that exists among Zalpha domains. It unravels new outlooks targeting Zalpha domainmediated LLPS for the development of pharmaceutical molecules able to regulate viral and cellular Zalpha domain containing proteins.