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

Abstract

African swine fever virus (ASFV) I73R encodes a protein consisting only of a Zalpha (Zα) domain. Here, we studied its biochemical properties. Complementary approaches demonstrated that I73R is a multifunctional Zα domain that combines Z-binding, B-to-Z conversion, and liquid–liquid phase separation (LLPS) induction activities. I73R proved to be a better converter than human ADAR1 Zα, and it retained conversion activity with Y48I mutation of its Z-binding site. Next, we investigated I73R biological functions, leading to the following findings: (i) Ectopic expression of I73R or mutated I73R with deficient Z-binding both inhibited arsenite-induced stress granules to a similar extent, and none of these proteins localized within these organelles. (ii) I73R formed cytosolic condensates with dsRNA, Z-DNA, and Z-RNA in ASFV-infected cells. (iii) Rescuing of ASFV I73R deletion by sequences encoding natural, mutated, and chimeric forms of Zα domains demonstrated that I73R mediated both Z-binding-dependent and -independent biological functions through induction of LLPS. This conclusion was further supported by the effect of the LLPS inhibitor 1,6-hexanediol on the replication of ASFV I73R recombinant strains. This study further demonstrates the functional diversity that exists among Zα domains. It unravels new outlooks targeting Zα domain-mediated LLPS for the development of pharmaceutical molecules able to regulate viral and cellular Zα domain-containing proteins.