Targeting Heat Shock Protein 70 as an antiviral strategy against grass carp reovirus infection.

Grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus (GCRV), has been a serious problem in grass carp aquaculture for several decades. Characterization of the primary host factors associated with host-virus interaction is critical for understanding how a virus infects its host cell and these host factors can be antiviral targets. This study aimed to screen host factors that interacted with GCRV in the C. idella kidney (CIK) cells and used them as antiviral targets. Twelve proteins were identified by virus overlay protein binding assay and LC-MS-MS. Among these twelve proteins, Heat Shock Protein 70 (HSP70) was outstanding. Results of flow cytometry and immunofluorescence assay indicated that HSP70 was on the cell membrane. HSP70 was expressed at low levels preceding GCRV infection, but its expression was induced upon GCRV infection. Inhibition of HSP70's function by inhibitors (VER155008 and pifithrin-μ) maintained HSP70 on the cell surface in infected cells, however GCRV quantity was decreased in the CIK cells (compared with the control group, the maximum inhibition rate of the treatment group was close to 85%), suggesting that fully functional HSP70 was required for GCRV infection. Moreover, GCRV showed a dose dependent reduction by inhibiting the entry stage of the viral life cycle following treated with VER155008 and pifithrin-μ. VER + PIF (1:1) were used at 15 μM and the expression of GCRV-VP6 downregulated nearly to 90%, which revealed that HSP70 played an important role in GCRV entering into CIK cells. This work speculated that HSP70 might be a host factor in the process of GCRV infecting CIK cells, therefore, it might be a potential antiviral target for GCRV infection.