Interaction of Infectious Spleen and Kidney Necrosis Virus ORF119L with PINCH Leads to Dominant-Negative Inhibition of ILK and Cardiovascular Defects in Zebrafish

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus, Iridoviridae family, causing a severe systemic disease with high mortality to mandarin fish (Siniperca chuatsi) in China and South-East Asia. Up to now, the pathogenesis of ISKNV infection is still not fully understood. Based on a genome-wide bioinformatics analysis of ISKNV-encoded proteins, we found that ISKNV open reading frame 119L (ORF119L) is predicted to encode a three ankyrin-repeats (3ANK) domain-containing protein, which shows high similarity to the dominant-negative form of integrin-linked kinase (ILK), i.e., viral ORF119L lacks the ILK kinase domain. Thus, we speculated that viral ORF119L might affect the host ILK complex. Here, we demonstrated that viral ORF119L directly interacts with particularly interesting Cys-His-rich protein (PINCH) and affects the host ILK-PINCH interaction in vitro in Fathead minnow (FHM) cells. In vivo ORF119L overexpression in zebrafish (Danio rerio) embryos resulted in myocardial dysfunctions with disintegration of sarcomeric Z-disk. Importantly, ORF119L overexpression in zebrafish highly resembles the phenotype of endogenous ILK inhibition, either by over-expressing a dominant-negative form of ILK or by injecting an ILK antisense morpholino. Intriguingly, ISKNV-infected mandarin fish develop disorganized sarcomeric Z-disk in cardiomyocytes. Furthermore, phosphorylation of AKT, a downstream effector of ILK, was remarkably decreased in ORF119L overexpressing zebrafish embryos. As such, we show that ISKNV ORF119L acts as a domain-negative inhibitor of the host ILK, providing a novel mechanism for the megalocytivirus pathogenesis. IMPORTANCE: Our work is the first to show the role of a dominant-negative inhibitor of the host ILK from ISKNV (an Iridovirus). Mechanistically, the viral ORF119L directly binds to the host PINCH, attenuates the host PINCH-ILK interaction, and thus impairs the ILK signalling. Intriguingly, ORF119L-overexpressing zebrafish embryos and ISKNV-infected mandarin fish develop similar disordered sarcomeric Z-disk in cadiomyocytes. These findings provide a novel mechanism for megalocytivirus pathogenesis.