Functional Study Of Heat Shock Cognate Protein 70-4 And Its Acetylation In Bombyx Mori Baculovirus Infection

Sericulture is a traditional characteristic industry with regional advantage in Zhejiang province as well as one of top 10 leading industries in Zhejiang agriculture,and plays essential roles in increasing agricultural benefit and farmers’ income in major producing area.However,Bombyx mori nucleopolyhedrovirus(BmNPV)infection is one of main reasons causing huge economic damage for sericulture.Because the interaction mechanism between virus and host remains unknown until now,thus,this is one of the research hot topics in virology field all the time.In our previous baculovirus-infected BmN cell acetylome,we found that HSC70-4 had 5 lysines(K77,K100,K246,K524,K557)with acetylated dynamics.Previous study indicated that HSC70-4 would accumulate in nucleus and assemble into ODV and BV structure.Therefore,studying HSC70-4 special lysine posttranslational modification influence for baculovirus would contribute to deeply elucidate interacting molecular mechanism between host cell and BmNPV as well as providing new ideas for breeding antiviral silkworm.In this study,we initially cloned partial gene encoding silkworm HSC70-4(300-600aa.)protein and constructed prokaryotic expression vector for inducing and purifying target protein followed by immunizing New Zealand rabbit for acquiring specific polyclonal antibody with high potency.Then,BmNPV genome replication and progeny BVs production were reduced by inhibiting HSP/HSC70 activity.On the contrary,overexpressing HSC70-4 promoted BmNPV genome copies and BVs release In order to further explore different lysine acetylation of HSC70-4 to affect BmNPV duplication,we applied overlapping PCR to site-specifically mutate certain lysine by acetylation-mimic K/Q and deacetylation-mimic K/R.Next,the results analyzed by qPCR had shown that K77 deacetylation could significantly elevate viral DNA copies while acetylation decreasing that.By means of lase confocal microscopy,K77 acetylation disturbed HSC70-4 nuclear import during BmNPV infection.Meanwhile,yeast two hybrid and cellular colocalization assay exhibited that K77 acetylation blocked the interaction between HSC70-4 and CHIP,inferring that this residue posttranslational modification may influence HSC70-4 executing protein degradation function and lead to the HSC70-4 nuclear import failure,ultimately declining BmNPV genome replication.Finally,we applied proteasome inhibitor MG132 to treat BmN cell,and found that intact proteasome is essential for BmN VP proliferation as well as hindering HSC70-4 nuclear import and posttranslational modification-mediated viral genome copies.Conclusion of this study:HSC70-4 K77 posttranslational modification change is able to significantly regulate BmNPV genome replication and assembly pathway.This study will lay the foundation for deeply understanding interaction mechanism between silkworm host and BmNPV,and provide new insights for developing robust antiviral silkworm breeding.