Study On Interaction Of Host Proteins HSC70 And PHB With Glycoprotein Of Spring Viraemia Carp Virus And Their Roles In Virus Infection

Spring viremia of carp(SVC)is an infectious disease caused by the Spring viremia of carp virus(SVCV),which seriously affects common carp and cyprinoid species.SVCV-infected fish can cause serious tissue damage,including internal organ bleeding and peritonitis,and the mortality rate can be as high as 70%.SVCV virions Glycoprotein(G)on the surface of the ascus membrane mediates virus invasion,assembly and release through binding to different host protein.Consequently,the comprehensive examination of the interplay between SVCV G protein and host protein is of immense scientific importance for the thorough examination of G protein’s purpose,the analysis of SVCV replication and pathogenesis,and the development of novel antiviral drugs.Based on previous studies,the host proteins HSC70 and PHB interacting with SVCV G protein were further identified in this study,and the molecular mechanisms and functional effects of their interactions were further analyzed as follows:1.Study on the interaction between SVCV G protein and HSC70In this study,the interaction between HSC70 and SVCV G protein was verified by co-immunoprecipitation and indirect immunofluorescence experiments.It was found that HSC70 overexpression dramatically inhibited SVCV replication,whereas its loss of functions elicited opposing effects on SVCV replication.Mechanistic studies indicate that HSC70 induces lysosomal degradation of ubiquitinated-SVCV G protein.This study further demonstrates that Membrane-associated RING-CH 8(MARCH8),an E3 ubiquitin ligase,is critical for SVCV G protein ubiquitylation and leads to its lysosomal degradation.Furthermore,the MARCH8 mediated ubiquitylation of SVCV G protein required the participation of HSC70 through forming a multicomponent complex.Taken together,these results demonstrate that HSC70 serves as a scaffold for MARCH8 and SVCV G,which leads to the ubiquitylation and degradation of SVCV G protein and thus inhibits viral replication.These findings have established a novel host defense mechanism against SVCV.2.Prohibitin mediates the cellular invasion of Spring viremia of the carp virusThe interaction between prohibitin(PHB)with the SVCV G protein was confirmed by immunoprecipitation and immunofluorescence assays.Treatment with PHB-specific inhibitors or knockdown of the expression of PHB by si RNAs resulted in a marked reduction in binding and entry of SVCV on host cells,while overexpression of PHB increased SVCV attachment and invasion.Furthermore,binding of SVCV to ZF4 and FHM cells was inhibited by pre-incubating the virus with recombinant PHB protein(r PHB)or blocking the cell surface PHB with its polyclonal antibodies.In addition,overexpression of PHB on SVCV-nonpermissive Grouper spleen cells(GSs)conferred susceptibility to SVCV infection.In vivo,treatment of r PHB could significantly inhibit SVCV propagation within zebrafish and benefit the survival rate of SVCV-infected zebrafish.These results demonstrate that PHB plays a crucial role in both the attachment and entry stages of SVCV infection.