Abstract
White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, including crayfish. However, the molecular mechanism underlying its cellular entry remains elusive due to the lack of shrimp cell lines for viral propagation. Crayfish hematopoietic tissue (Hpt) cell culture was recently established as a good model for WSSV infection study. Here, we showed that multiple endocytic routes, including clathrin-mediated endocytosis (CME), macropinocytosis and caveolae-mediated endocytosis, were indispensably employed for the viral entry into Hpt cell of the crayfish Cherax quadricarinatus. Intriguingly, cellular autophagic activity was positively correlated with efficient viral entry, in which a key autophagy-related protein, γ-aminobutyric acid receptor-associated protein (Cq-GABARAP), that not only localized but also co-localized with WSSV on the Hpt cell membrane, strongly facilitated WSSV entry by binding to the viral envelope VP28 in a CME-dependent manner that was negatively regulated by Cq-Rac1. Furthermore, cytoskeletal components, including Cq-β-tubulin and Cq-β-actin, bound to both recombinant rCq-GABARAP and WSSV envelope proteins, which likely led to viral entry promotion via cooperation with rCq-GABARAP. Even under conditions that promoted viral entry, rCq-GABARAP significantly reduced viral replication at an early stage of infection, which was probably caused by the formation of WSSV aggregates in the cytoplasm.
Highlights
White spot syndrome virus (WSSV), the only member of the genus Whispovirus in the novel Nimaviridae family, is a large enveloped virus (~70–167 nm × 210–380 nm) with double-stranded DNA (~300 kb)
Because WSSV entry was promoted by pre-incubation of the hematopoietic tissue (Hpt) cell with rCq-GABARAP (Fig. 6D), we investigated the interaction of rCq-GABARAP with the Hpt cell by performing a protein pull-down assay to isolate the putative proteins that might bind to rCq-GABARAP with the aim of obtaining molecular insights into how rCq-GABARAP affected Hpt cell to promote the viral entry, for example whether rCq-GABARAP could interact with cytoskeleton in consideration to that the transport of GABAA receptor and κopioid receptor mediated by rCq-GABARAP is achieved by its interaction with cytoskeleton[35,36]
We focused on the early viral entry events during WSSV infection in crayfish Hpt cell and found that multiple endocytic pathways, including clathrin-mediated endocytosis (CME), macropinocytosis and caveolae-mediated endocytosis, were all utilized for viral entry
Summary
White spot syndrome virus (WSSV), the only member of the genus Whispovirus in the novel Nimaviridae family, is a large enveloped virus (~70–167 nm × 210–380 nm) with double-stranded DNA (~300 kb). The suppression of WSSV entry via gene knockdown of Cq-dynamin was determined from the relative quantification of the viral envelope protein VP28 at 1 hpi by immunoblotting (lower panel). We focused on investigating the mechanism through which WSSV entered Hpt cell based on TEM combined with different pharmacological inhibitor assays for blocking different endocytic pathways, including CME, macropinocytosis and caveolae-mediated endocytosis, and autophagy. The role of CME and autophagy in WSSV entry was further validated by gene silencing of the key components of CME, such as Cq-CLC, Cq-AP50 (medium subunit of clathrin-associated adaptor protein complexes) The impact of Cq-GABARAP on the regulation of WSSV entry into Hpt cell was investigated with regard to its possible roles in autophagy, regulation of CME activity, interactions with both virion and Hpt cell, and subsequent effects on viral replication of the internalized viruses
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