The Role Of Bombyx Mori Nucleopolyhedrovirus Bm65 Protein In The Repair Of UV-induced DNA Damage

Insect baculoviruses are characterized by host specificity that it does not infect humans and other vertebrates.In recent years,more and more studies have been performed to develop the environmentally friendly biological insecticides.With the pollution of atmospheric environment,ultraviolet radiation is becoming a risk that it can cause covalently bond adjacent bases of DNA to produce cyclobutane pyrimidine dimers(CPD).Baculovirus biopesticides are susceptible to UV,resulting in decreased virulence.As we all know,the baculovirus will lose most of its activity after exposure to sunlight for 24 hours.The endonuclease in the process of nucleotide excision repair can specifically cleave the 5’-end glycosidic bond of the pyrimidine dimer to repair UVdamaged DNA.Bombyx mori nuclear polyhedrosis virus Bm65 protein belonges to the GIY-YIG endonuclease superfamily and is predicted to have a function in the repair of UV-damaged DNA.In this study,Bm65 proteins were expressed and purified.It was confirmed that Bm65 had endonuclease activity in vitro and could specifically cleave UV-incuced DNA.At the same time,the GIY-YIG domain was identified as the key functional sequence of Bm65 protein.The interaction between virus and host in the repair of UV-induced DNA damage was explored by studying the host protein BmRPL13 that interacted with Bm65.The main results in the study are as follows:1.Bm65 protein has endonuclease activity.We expressed and purified Bm65 protein in vitro.Endonuclease activity analysis showed that Bm65 protein could specifically cleave UV-induced DNA.2.GIY-YIG domain of Bm65 protein is a key functional domain.The truncated mutants of Bm65 were expressed and purified.The endonuclease activity of the truncated mutants were further analyzed.It was found that Bm65(1-36AA),the GIYYIG domain,was the key functional domain for endonuclease activity.The Escherichia coli expressing Bm65(1-36AA)has stronger resistance to ultraviolet radiation than the control group.Compared with the full-length Bm65 protein,the enzyme activity of Bm65(1-36AA)decreased.3.The Bm65 protein interacts with the host protein BmRPL13.(1)We expressed and purified BmRPL13 and then verified the direct interaction between Bm65 and BmRPL13 by pull-down.(2)The subcellular localization results found that BmRPL13 and Bm65 were co-localized in the nucleus.(3)Using a local UV irradiation technique and immunofluorescence staining,Bm65 and BmRPL13 were shown to accumulate at the sites of UV-induced DNA damage.(4)BmRPL13 protein may be related to the repair of UV-induced damage.The E.coli expressing BmRPL13 had stronger resistance to UV radiation than the control group.At present,although the discovery of photorepair enzymes were related to the repair of UV-induced DNA damage in a few baculoviruses,it is still unclear what pathways are used in most baculoviruses to repair UV-induced damage.This study proves that Bombyx mori nuclear polyhedrosis virus Bm65 protein is an endonuclease that can specifically cleave UV-damaged DNA.So,it is speculated that a nucleic acid excision repair pathway may be existed in baculovirus,and further studies should be conducted to confirm the hypothesis.