In vitro neutralization of nervous necrosis virus by a nanobody binding to the protrusion domain of capsid protein

A system has been established to produce infectious RNA transcripts for Striped jack nervous necrosis virus (SJNNV), the type species of the betanodaviruses, which infect fish. An enzymological analysis suggested that both RNA1 and RNA2 of SJNNV have a 5′ cap. Both RNAs were largely resistant to 3′ polyadenylation and ligation, suggesting the presence of an interfering 3′ structure, while a small quantity of viral RNAs were polyadenylated in vitro. The complete 5′ and 3′ non-coding sequences of both segments were determined using the rapid amplification of cDNA ends method. Based on the terminal sequences obtained, RT–PCR was carried out and plasmid clones containing full-length cDNA copies of both RNAs, positioned downstream of a T7 promoter, were constructed. These plasmids were cleaved at a unique restriction site just downstream of the 3′ terminus of each SJNNV sequence and were transcribed in vitro into RNA with a cap structure analogue. A mixture of the transcripts was transfected into the fish cell line E-11. Using indirect immunofluorescence staining with anti-SJNNV serum, fluorescence was observed specifically in these transfected cells; this culture supernatant exhibited pathogenicity to striped jack larvae. Northern blot analysis of E-11 cells infected with the recombinant virus or SJNNV showed small RNA (ca. 0·4 kb) that was newly synthesized and corresponded to the 3′-terminal region of RNA1. Finally, the complete nucleotide sequences of these functional cDNAs (RNA1, 3107 nt; RNA2, 1421 nt) were determined. This is the first report of betanodavirus cDNA clones from which infectious genomic RNAs can be transcribed.

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Screening for the Proteins That Can Interact with Grouper Nervous Necrosis Virus Capsid Protein

Viruses ◽

10.3390/v12090985 ◽

2020 ◽

Vol 12 (9) ◽

pp. 985

Author(s):

Po-Yu Huang ◽

Han-Chia Hsiao ◽

Szu-Wen Wang ◽

Shao-Fu Lo ◽

Ming-Wei Lu ◽

...

Keyword(s):

Optic Nerve ◽

Capsid Protein ◽

Protein Interactions ◽

Ribosomal Proteins ◽

Nervous Necrosis Virus ◽

Structural Protein ◽

Necrosis Virus ◽

Ion Regulation ◽

Protein Protein Interaction ◽

Atp Generation

Nervous necrosis virus (NNV) can infect many species of fish and has an 80–100% mortality rate. NNV capsid protein (NNVCP) is the only structural protein of NNV, but there are few studies on the protein–protein interaction between NNVCP and the host cell. To investigate NNV morphogenesis, native NNV capsid protein (NNVCP) was used to screen for protein–protein interactions in this study. The results identified that 49 grouper optic nerve proteins can interact with NNVCP and may function as putative receptor or co-receptor, cytoskeleton, glucose metabolism and ATP generation, immunity, mitochondrial ion regulation, and ribosomal proteins. Creatine kinase B-type (CKB) is one of those 49 optic nerve proteins. CKB, a kind of enzyme of ATP generation, was confirmed to interact with NNVCP by far-Western blot and showed to colocalize with NNVCP in GF-1 cells. Compared to the control, the expression of CKB was significantly induced in the brain and eyes infected with NNV. Moreover, the amount of replication of NNV is relatively high in cells expressing CKB. In addition to providing the database of proteins that can interact with NNVCP for subsequent analysis, the results of this research also verified that CKB plays an important role in the morphogenesis of NNV.

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Capsid protein from red-spotted grouper nervous necrosis virus induces incomplete autophagy by inactivating the HSP90ab1-AKT-MTOR pathway

动物学研究 ◽

10.24272/j.issn.2095-8137.2021.249 ◽

2021 ◽

Vol 43 (0) ◽

pp. 1-13

Author(s):

Wan-Wan Zhang ◽

◽

Peng Jia ◽

Xiao-Bing Lu ◽

...

Keyword(s):

Capsid Protein ◽

Mtor Pathway ◽

Nervous Necrosis Virus ◽

Necrosis Virus

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GHSC70 Is Involved in the Cellular Entry of Nervous Necrosis Virus

Journal of Virology ◽

10.1128/jvi.02523-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 61-70 ◽

Cited By ~ 31

Author(s):

Jui-Shin Chang ◽

Shau-Chi Chi

Keyword(s):

Capsid Protein ◽

Fish Species ◽

Receptor Protein ◽

Economic Losses ◽

Nervous Necrosis Virus ◽

Specific Receptor ◽

Necrosis Virus ◽

Content Type ◽

Voltage Dependent ◽

Selective Channel

ABSTRACTNervous necrosis virus (NNV) is a devastating pathogen of cultured marine fish and has affected more than 40 fish species. NNV belongs to the betanodaviruses ofNodaviridaeand 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 inEscherichia colifor 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.

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