Characterization Of32kDa Virus Attachment Protein Of Lymphocystis Disease Virus From Paralichthys Olivaceus

Lymphocystis disease virus (LCDV), the causative agent of the lymphocystisdisease, has a worldwide geographical distribution, infecting more than140species offreshwater and marine fish belonging to42families. Since the lymphocystis diseasefirstly broke out in flounderParalichthys olivaceus in Weihai of Shandong province in1997, the viral disease was rampant in some coastal provinces in China and hasbrought tremendous economic loss to the aquaculture. LCDV is one of large DNAviruses with envelope. Single or multiple virus attachment proteins on the envelopeare employed by enveloped virus to specifically bind to the receptor proteins in thesurface of target cells, mediating the structual change of proteins of both the virus andthe target cell and accelerating the process of membrane fusion, which facilitate viralgenetic materials entering into the target cell. The study on virus attachment proteinswill give insights into molecular mechanisms of the virus-host interactions and viruspathogenesisIn previous research using co-immunoprecipitation, a27.8kDa protein inflounder P. olivaceus gill (FG) cells was identified as the putative receptor specific forLCDV infection, and monoclonal antibodies (MAbs) designated as2G11and3D9against the27.8kDa protein were developed, which were able to efficientlyinhibit LCDV infection to FG cells. Meanwhile, a32kDa protein of LCDVspecifically binding to the27.8kDa receptor protein was found by far-western blottingcoupled with Mabs2G11and3D9, and Mass spectrographic analysis showed that the32kDa protein was encoded by open reading frame (ORF)038in LCDV-C. In thispaper, the biological characterization of32kDa virus protein is analyzed, indicatingthat the protein is virus attachment protein which plays pivotal role in virus infectionupon FG cells. This study lays a solid foundation for the LCDV pathogenesis as wellas effective methods to block virus infection. The details are as follows: The expression plasmids of pET-32a-ORF038was constructed and transformedinto Escherichia coli strain BL-21(DE3), and recombinant protein with molecularweight50kDa was obtained by induction of IPTG and purified by Ni-NTA column.Polyclonal antibodies against the recombinant protein were prepared by immunizationof rabbits. ELISA results showed that polyclonal antibodies could specifically bind tothe recombinant protein; In Western blotting, the antiserum could strongly react withthe recombinant protein as well as a32kDa protein in LCDV, which indicate that theantiserum possessed high specificity.Colloidal gold marked polyclonal antibody against ORF038recombinant proteinwas used as a probe to locate the32kDa protein encoded by ORF038in LCDV. TheImmunoelectron microscopy results showed that gold particles were located at theoutermost surface of LCDV particles, indicating that the32kDa protein was theenvelope protein of LCDV.In an immunofluorescence assay, the recombinant proteintagged with FITC was incubated with FG cells and positive signals were detectedprimarily on the cell membrane, indicating that the recombinant protein could bind tothe membrane protein of FG cells.Blocking ELISA results showed that the blocking rate of MAb against27.8kDareceptor protein was63.2%; In a neutralization assay, pre-incubation of LCDV withthe polyclonal antibodies against the recombinant protein could largely impair thevirulence of LCDV and inhibit LCDV infection to FG cells in vitro, showing adose-dependent blocking effect, which further indicate that the32kDa protein was thevirus attachment protein that was able to bind to27.8kDa receptor protein on thesurface of FG cells and play a pivotal role in invasing target cells.