GHSC70 Is Involved in the Cellular Entry of Nervous Necrosis Virus

Abstract

Nervous necrosis virus (NNV) is a devastating pathogen of cultured marine fish and has affected more than 40 fish species. NNV belongs to the betanodaviruses of Nodaviridae and is a nonenveloped icosahedral particle with 2 single-stranded positive-sense RNAs. To date, knowledge regarding NNV entry into the host cell remains limited, and no NNV-specific receptor protein has been published. Using grouper fin cell line GF-1 and purified NNV capsid protein in a virus overlay protein binding assay (VOPBA), grouper heat shock cognate protein 70 (GHSC70) and grouper voltage-dependent anion selective channel protein 2 (GVDAC2) were investigated as NNV receptor protein candidates. We cloned and sequenced the genes for GHSC70 and GVDAC2 and expressed them in Escherichia coli for antiserum preparation. Knockdown of the expression of GHSC70 and GVDAC2 genes with specific short interfering RNAs (siRNAs) significantly downregulated viral RNA expression in NNV-infected GF-1 cells. By performing an immunoprecipitation assay, we confirmed that GHSC70 interacted with NNV capsid protein, while VDAC2 did not. Immunofluorescence staining and flow cytometry analysis revealed the presence of the GHSC70 protein on the cell surface. After a blocking assay, we detected the NNV RNA2 levels after 1 h of adsorption to GF-1 cells; the level was significantly lower in the cells pretreated with the GHSC70 antiserum than in nontreated cells. Therefore, we suggest that GHSC70 participates in the NNV entry of GF-1 cells, likely functioning as an NNV receptor or coreceptor protein. IMPORTANCEFish nodavirus has caused mass mortality of more than 40 fish species worldwide and resulted in huge economic losses in the past 20 years. Among the four genotypes of fish nodaviruses, the red-spotted grouper nervous necrosis virus (RGNNV) genotype exhibits the widest host range. In our previous study, we developed monoclonal antibodies with high neutralizing efficiency against grouper NNV in GF-1 cells, indicating that NNV-specific receptor(s) may exist on the GF-1 cell membrane. However, no NNV receptor protein has been published. In this study, we found GHSC70 to be an NNV receptor (or coreceptor) candidate through VOBPA and provided several lines of evidence demonstrating that GHSC70 protein has a role in the NNV entry step of GF-1 cells. To the best of our knowledge, this is the first report identifying grouper HSC70 and its role in NNV entry into GF-1 cells.

Grouper voltage-dependent anion selective channel protein 2 is required for nervous necrosis virus infection

“…GVDAC2 short-interfering RNAs of GVDAC2 (GVDAC2-siRNAs) and negative control nonsilencing siRNA (NS-siRNA) were prepared in our previous study [27]. Table 1 shows the siRNA sequences.…”

“…NNV was proliferated in GF-1 cells with a multiplicity of infection (MOI) of 1, and infected cells were incubated in L-15 medium with 2% FBS at 28 C. After a complete cytopathic effect (CPE) was observed, the culture supernatant was collected through centrifugation at 12,000Â g for 10 min and then titrated in GF-1 cells. The NNV RdRp and GVDAC2-specific polyclonal antibodies were prepared as described in our previous studies [27,31].…”

“…GVDAC1 was reported to induce apoptosis when overexpressed in fathead minnow cells and participates in antibacterial immune response [26]. GVDAC2 was initially identified in our previous study, and downregulation of GVDAC2 reduced NNV RNA replication [27]. However, the underlying mechanism of GVDAC2 siRNA-induced reduction of NNV RNA synthesis remains unclear.…”

Evidence that Hsc70 Is Associated with Cucumber Necrosis Virus Particles and Plays a Role in Particle Disassembly

“…Due to the ability of Hsc70 homologs to disaggregate stable oligomeric complexes (18), viruses have evolved to recruit these cellular disassembling machines to uncoat their capsids for the establishment of infection. Hsc70 homologs have been found to promote the uncoating processes of several animal viruses, such as polyomaviruses, papillomaviruses, reoviruses, adenoviruses, nodaviruses, rotaviruses, and Nervous necrosis virus (19)(20)(21)(22)(23)(24)(25). However, there are no reports regarding the involvement of cellular Hsc70 in the disassembly of plant viruses, although the virally encoded HSP70 homolog of Beet yellows virus has been hypothesized to play a role in disassembly of the helical capsid (26).…”

Genetic Characterization of Viral Nervous Necrosis Infects Tilapia (Oreochromis sp.) in Indonesia

“…A specific host on the most viral infections are usually controlled by the interaction with a viral surface protein receptors on the surface of target cells precisely [31], so long as the tilapia have receptors that can bind to the attachment site (s) virus particles, then certainly virus will entry into host cells (tilapia), because it does not directly host cell have facilitated the virus to enter the cell [32]. The tilapia have such receptors HSC70, as well as groupers [33], which specifically alleged that the virus can perform attachment and involved in an interaction that could cause the virus to enter the body tilapia. Interactions between viruses and cellular receptors is a dynamic process chain that allows the entry of the virus into the cell [34] [35].…”