CqFlightless-I promotes the depolymerization of actin cytoskeleton to reduce white spot syndrome virus infection in Cherax quadricarinatus

Abstract

As a member of the actin-associated protein gelsolin family, Flightless-I (FliI) is involved in promoting the depolymerization and rearrangement of the actin cytoskeleton, thus regulating a variety of biological processes, including cell proliferation, differentiation, apoptosis, and immune response. However, the molecular regulation of FliI is poorly understood in its regulation on viral infection in crustaceans. Previously, we found that the transcript of CqFliI was up-regulated in a differentially expressed transcriptome library of the hematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus at 1 h post white spot syndrome virus (WSSV) infection. To reveal the role of CqFliI in WSSV infection in crustacean, the open reading frame of CqFliI gene was cloned with 3870 bp, which encoded 1289 amino acids with eleven leucine-rich repeat (LRR) domains and six gelsolin-like (GEL) domains. Tissue distribution analysis showed that CqFliI was widely expressed in all the tested tissues with the highest expression in muscle, followed by a high expression in Hpt. In addition, the mRNA expression of CqFliI was significantly up-regulated in Hpt cells at 1, 3, 6, 12 and 24 h post WSSV infection. Importantly, the internalization of WSSV was clearly increased at an early infection stage of 1 hpi in Hpt cells by CqFliI gene silencing; meanwhile, the expression of viral immediate early gene IE1 and late viral envelope protein gene VP28 were both significantly increased after gene silencing of CqFliI in Hpt cells post WSSV infection, indicating a key role of CqFliI in reducing WSSV infection. Furthermore, protein pull-down assay revealed that CqFliI could bind with cytoskeletal β-actin through its GEL domains, rather than LRR domains; and the cytoskeleton of HeLa cells was obviously depolymerized after overexpression of GEL domain of CqFliI, implying that CqFliI might reduce the internalization of WSSV probably also the intracellular transport mediated by cytoskeleton via inhibiting actin polymerization. These findings may contribute to further understanding of WSSV infection mechanism which may provide more antiviral targeting design against WSSV disease.