Abstract
Clathrins, composed of clathrin heavy chains (CHCs) and clathrin light chains (CLCs), are usually hijacked by viruses for infection. However, the role of CLCs, especially in regulating fish virus infection, remains poorly understood. Here, two isoforms of CLCs were cloned from the red-spotted grouper (Epinephelus akaara) (EaCLCa and EaCLCb). Both EaCLC transcripts were expressed in all examined tissues, and the expression of EaCLCa was much higher than that of EaCLCb. Over-expressing EaCLCa-W119R mutant significantly reduced Singapore grouper iridovirus (SGIV) infectivity. However, no effect of EaCLCb-W122R on SGIV infection was observed. The detailed steps were further studied, mainly including virus attachment, entry and the following transport to early endosomes. EaCLCa-W119R mutant notably inhibited internalization of SGIV particles with no effect on SGIV attachment. Furthermore, EaCLCa-W119R mutant obviously impaired the delivery of SGIV to early endosomes after virus internalization. In addition, the EaCLCa-W119R mutant markedly reduced the colocalization of SGIV and actin. However, EaCLCb is not required for such events during SGIV infection. Taken together, these results demonstrate for the first time that EaCLCa and EaCLCb exerted different impacts on iridovirus infection, providing a better understanding of the mechanisms of SGIV infection and opportunities for the design of new antiviral strategies.
Highlights
Clathrin-coated vesicles (CCVs) are major carriers for cargo trafficking at the plasma membrane, the trans-Golgi network (TGN), and the endosomal system and often carry proteins, lipids and even pathogens[1]
From the N terminus to the C terminus, other features shared by the two clathrin light chains (CLCs) include an EF-hand that is responsible for binding to calcium (Ca2+), the heavy-chain-binding region (HC), neuronally expressed inserts (N), and a calmodulin-binding domain (CBD)
Phylogenetic analysis indicated that EaCLCa and EaCLCb are both sorted into the Osteichthyes branch, which is separate from amphibian and mammals (Supplementary Fig. S1)
Summary
Clathrin-coated vesicles (CCVs) are major carriers for cargo trafficking at the plasma membrane, the trans-Golgi network (TGN), and the endosomal system and often carry proteins, lipids and even pathogens[1]. The uptake of G-protein-coupled receptors does depend on CLCs7 Another important role of CLCs is to regulate clathrin-mediated trafficking between the TGN and the endosomal system by acting as recruitment proteins for huntingtin-interacting protein 1-related (HIP1R), enabling HIP1R to regulate the interactions of clathrin-coated structures with the actin cytoskeleton[6]. Clathrin is the factor most commonly used for pathogen internalization into the host cell Numerous viruses, such as influenza virus, African swine fever virus and bovine ephemeral fever virus, hijack clathrin-mediated endocytosis as the primary means of entry[8,9,10]. Most studies focus on the interactions of clathrin with viruses, and the role of CLCs in virus infection remain largely unknown. There are two isoforms of CLCs in all metazoans, CLCa and CLCb, encoded by different genes They share approximately 60% protein sequence identity and are expressed at characteristically different levels in all tissues. EaCLCb showed no measurable effect on SGIV infection
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