Mandarin Fish Caveolin 1 Interaction with Major Capsid Protein of Infectious Spleen and Kidney Necrosis Virus and Its Role in Early Stages of Infection

Abstract

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus from the family Iridoviridae.ISKNV is one of the major agents that cause mortality and economic losses to the freshwater fish culture industry in Asian countries, particularly for mandarin fish (Siniperca chuatsi). In the present study, we report that the interaction of mandarin fish caveolin 1 (mCav-1) with the ISKNV major capsid protein (MCP) was detected by using a virus overlay assay and confirmed by pulldown assay and coimmunoprecipitation. This interaction was independent of the classic caveolin 1 scaffolding domain (CSD), which is responsible for interacting with several signaling proteins and receptors. Confocal immunofluorescence microscopy showed that ISKNV MCP colocalized with mCav-1 in the perinuclear region of virus-infected mandarin fish fry (MFF-1) cells, which appeared as soon as 4 h postinfection. Subcellular fractionation analysis showed that ISKNV MCP was associated with caveolae in the early stages of viral infection. RNA interference silencing of mCav-1 did not change virus-cell binding but efficiently inhibited the entry of virions into the cell. Taken together, these results suggested that mCav-1 plays an important role in the early stages of ISKNV infection. I ridoviruses, with large, icosahedral, and double-stranded DNA (dsDNA), can infect both invertebrates (particularly insects), and poikilothermic vertebrates (fish, amphibians, and reptiles), leading to systematic diseases (1). To date, more than 100 iridovirus strains have been isolated, and the entire genomes of 20 strains have been completely sequenced. The Iridoviridae family is composed of five genera, namely, Iridovirus, Chloriridovirus, Ranavirus, Lymphocystivirus, and Megalocytivirus, according to viral particle size, host range, DNA cross-hybridization, the presence of a methyltransferase, and the major capsid protein (MCP) sequence (1, 2). Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus (2, 3). The ISKNV genome is 111,362 bp in length, consisting of 124 potential open reading frames (ORFs) with encoding capacities ranging from 40 to 1,208 amino acids (aa) (3). Proteomic analysis of ISKNV shows that 38 viral structural proteins are present in ISKNV virions (4). Fifteen highly abundant ISKNV structural proteins were identified. ISKNV MCP is the main structural component of the virus particle, comprising 40% to 45% of the total particle polypeptide (4, 5). ISKNV is one of the fatally infectious agents rendering tremendous economic losses in Chinese mandarin fish cultures (3). Control of ISKNV infection is desperately needed. A better understanding of the ISKNV infection process is the first step toward viral control and prevention.An increasing number of studies have shown that lipid rafts, liquid-ordered, plasma membrane microdomains, also known as detergent-resistant membranes (DRMs) or caveolae, are involved in virus internalization (6), in addition to signal transduction (...

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