Identification Of Receptor And Cellular Entry Pathway Of Grass Carp Reovirus

Production of grass carp(Ctenopharyngodonidellus) constitutes the largest aquaculture industry of freshwater fish in China. However, a hemorrhagic disease frequently reported in grass carp has severely affected the production of aquaculture. Hemorrhagic disease, caused by the Grass Carp Reovirus(GCRV), is one of the major diseases of grass carp in China. GCRV is a dsRNA virus with a double-layered protein capsid. The outer GCRV capsid is composed of 200 trimers of VP5-VP7 heterodimers. Identifying factor(s) involved in GCRV, adhereing to host cell, is crucial for interpreting pathogenesis and disease development of GCRV. For this purpose, we performed series of experiments. 1.Cell surface Laminin receptor(LamR)is an adhesin-recepor of GCRV.We performed yeast two-hybrid screen for potential interaction partner(s) with outer capsid protein VP5 of GCRV, in which the LamR was identified to bind to VP5. We cloned and sequenced the gene encoding grass carp LamR. Viral attachment assay through confocal immunofluorescence microscopy demonstrated the involvement of membrane-associated LamR in GCRV entry. GST-tagged LamR was expressed and purified from E.coli., and solid-phase overlay assays demonstrated that GCRV interacted with GST-tagged LamR in vitro. VP5 and another viral outer capsid protein VP7 were also expressed and purified as GST-tagged recombinant protein respectively; in contrast to VP7, recombinant VP5 was shown to associate with LamR in both pulldown and solid-phase blot overlay assays. With the reduction of LamR expression in host cells achieved by RNAi, remarkably reduced infection efficiency of GCRV was observed. CIK cells pretreated with polyclonal antibody against LamR resulted in dosedependent inhibition of GCRV infection with 10-100 fold reduction of viral yield. These results collectively indicated that in the cellular attachment of GCRV, grass carp LamR was involved by interacting with viral outer capsid protein VP5. 2. Identification of EGCG as a potential agent for blocking infection of GCRV.EGCG((-)-epigallocatechin-3-gallate) has also been shown to target to the cell surface receptor LamR. Surface plasmon resonance experiments demonstrated the binding of EGCG to LamR with a Kd value in the nanamolar range. It’s of our interest to test the potential of EGCG in blocking infection of GCRV, which might function through inhibiting viral attachment. We provided evidence to show that adhesion to CIK cell surface by GCRV particles was inhibited dose-dependently by EGCG, as well as the crude extract of green tea. We also evaluated the safety of EGCG and green tea extract to CIK cells, which supported EGCG as a promising compound that may be developed as a plant-derived small molecular therapeutic agent against grass hemorrhagic disease caused by GCRV infection. 3.GCRV enters CIK cells by clathrin-dependent receptor-mediated endocytosis.In this study, the entry mechanism of GCRV was investigated in detail by applying endocytosis inhibition assays. We used various specific inhibitors to block different entry pathways to investigate the early stages of GCRV infection in CIK cells. Our results indicate that GCRV infection was inhibited by Chlorpromazine but not Filipin III, Nystatin, or LY294002. Moreover, GCRV infection of CIK cells depended on acidification of the endosome. This was indicated by significant inhibition following prophylactic treatment with the lysosomotropic drugs Chloroquine and Ammonium chloride. In addition, the disturbance of dynamin activity also blocked GCRV entry. The majority of GCRV virions internalized into CIK cells were colocalized with endogenous clathrin. Our findings suggest that GCRV might enter CIK cells via clathrin-mediated endocytosis in a pH-dependent manner. Additionally, dynamin is critical for efficient viral entry.