Structure Study Of Phlebovirus Envelope Glycoproteins Gn And Gc

Bunyaviruses are a kind of negative sense single strand viruses with segmented genome,which can infect animals,plants and human beings,and cause great harm to animal husbandry,agriculture and human health.Phlebovirus is one of the most important genera of Bunyavirale,which includes several important viruses that can cause human diseases,like severe fever with thrombocytopenia syndrome virus(SFTSV),Heartland virus(HRTV)and Rift Valley fever virus(RVFV).SFTSV was first isolated in 2011 in China.This virus has spread in several provinces in our country.It can infect human beings and cause fatal disease,and the average fatality rate is about 12%.Gene sequence analysis shows that this is a new bunyavirus,belonging to Phlebovirus genus.HRTV was first isolated in America.This virus has spread in America and infected human beings.Gene sequence analysis shows that it also belongs to Phlebovirus and is closely related to SFTSV.RVFV is also an important virus in Phlebovirus genus,which can infect human beings and livestocks,and cause tremendous harmfulness.In 2016,the first imported case of Rift Valley fever was reported in China.Phlebovirus genomes contain three gene segments,large(L),medium(M)and small(S)according to the length.The L and S segments encode polymerase and nucleoprotein of the virus,and the M segment encodes a polypeptide which is cleaved into two glycoproteins Gn and Gc by the enzyme of the host cells.Phleboviruses are enveloped viruses,and the glycoproteins on the envelope have important roles in the process of virus to infect host cells.Therefore,my research has used two phleboviruses SFTSV and HRTV as research objects to study the struct-ures of glycoproteins of phlebovirus utilizing structural biology method.Firstly,we want to explore the structure of HRTV Gc protein.We expressed Gc protein ectodomain in vitro by insect cell expression system and obtained the crystal structure of Gc protein.The structure showed that consistent with previous research results,Gc protein was a type ? fusion protein and had typical type ? fusion protein structure.Furthermore,this structure is obtained in low pH condition,and the structure showed post-fusion conformation,suggesting that this protein has the same acidic induced conformation change fusion mechanism as other type ? fusion protein.By comparing this structure to other Gc protein structures in Bunyavirale,we showed that HRTV Gc structure was almost identical to RVFV and SFTSV Gc structures which also belong to the Phlebovirus,but different from hantaan virus(HTNV)and Puumala virus(PUUV)Gc structures which belong to another genus Orthohantavirus in Bunyavirale.These results indicate that Gc structure is conserved within Phlebovirus and Orthohantavirus.Additionally,we explored Gn protein structure of SFTSV.We also expressed the ectodomain of Gn protein in vitro by insect cell expression system,and found that Gn protein had monomer and dimer forms.The dimer was linked by disulfide bonds.C terminal of the protein could be removed after treating with trypsin.N terminal of the protein could not form dimer.Then,we obtained crystal structure of N terminal head domain of Gn,and Gn head domain folded into triangular shape architecture which consists of three domains.A Dali search within the Protein Data Bank(PDB)failed to identify any existing structures to the Gn head domain of SFTSV,suggesting that it is a novel fold.In addition,we used point mutation and biochemical experiment methods and found that in the Gn C terminal domain,430,447 and 435,438 cysteines formed two disulfide bonds,which have an important role in the dimerization of Gn protein.in summary,our study solves the crystal structures of glycoproteins of phlebovirus,and provide structure basis to study the infection mechanism of these viruses.Also,the structures of the glycoproteins provides the basis for the antivirus strategy.