Two CqCofilins inhibit WSSV infection by binding with Cqβ-actin cytoskeleton in Cherax quadricarinatus

Cofilin, a key regulator of the cytoskeleton, orchestrates multiple critical cellular processes, including endocytosis, cell migration, and adhesion, by facilitating the depolymerization of actin filaments. Based on our prior transcriptomic library of differentially expressed genes from the hematopoietic tissue (Hpt) cells of Cherax quadricarinatus, the expression profiles of Cofilin1 and Cofilin2 were downregulated following white spot syndrome virus (WSSV) infection. Nonetheless, the role of CqCofilins in the context of WSSV infection remains poorly understood and warrants further investigation. In this study, the full-length cDNA sequences of the CqCofilin1 and CqCofilin2 genes were cloned and characterized. Both genes exhibit an open reading frame (ORF) of 444 bp, which encode a polypeptide of 147 amino acids, and contain an actin-depolymerizing factor (ADF) homology domain. Localization studies revealed that CqCofilin1 and CqCofilin2 were localized in both the nucleus and cytoplasm and were ubiquitously expressed across all examined crayfish tissues, with notably higher expression levels observed in nerve tissue, hemocytes, and Hpt. Gene silencing of either CqCofilin1 or CqCofilin2 led to a notable increase in WSSV internalization and a significant upregulation of viral genes like IE1 and VP28 in Hpt cells. The introduction of recombinant CqCofilin1 or CqCofilin2 proteins resulted in a significant reduction in WSSV replication. Moreover, Alphafold 3 analysis prediction and protein pull-down assay elucidated the interaction between CqCofilin1, CqCofilin2, and Cqβ-actin. These findings suggest that CqCofilins, as members of the actin depolymerization factor family, function conservatively to bind Cqβ-actin and promote cytoskeletal disassembly, a process that might hinder WSSV internalization, and which offers novel insights into host resistance mechanisms against WSSV infection.