Study On The Activity Of CARC Site In The Membrane Fusion Protein GP64 Extracellular Of Bombyx Mori Nucleopolyhedrovirus

Bombyx mori nucleopolyhedrovirus（BmNPV）is an enveloped DNA virus that can produce virions of two phenotypes which are occlusion-derived virions（ODV）and budded virions（BV）in the life cycle.ODV is responsible for the primary infection of the host insect while BV is responsible for the secondary infection.BmNPV is a serious viral pathogen in the silkworm industry.Currently,the mechanism of BV entering host cells is unclear,and there are no effective drugs or specific preventive measures.GP64 is a key membrane fusion protein that mediates BV infection,and it is also an essential protein in the process of virus invasion and budding.Our research team has previously demonstrated that BmNPV infection in host cells is completely dependent on cholesterol,and the cholesterol recognition amino acid consensus motif（CRAC）1 and CRAC2 in GP64,which can bind to cholesterol,play a crucial role in BmNPV infection.CARC which is the mirror structure of CRAC can also interact with cholesterol,but the activity of the CARC site in GP64 in viral infection is unknown.In order to further explore the invasion mechanism of BmNPV,this thesis carried out the following research on the BmNPV GP64 CARC active site.First of all,the biological activity of the predicted CARC motifs in GP64 was analyzed to determine whether it is a necessary active site for BmNPV infection.In order to further explore the cholesterol binding sites in virus GP64,bioinformatics analysis predicted the presence of six CARC motifs in the GP64 extracellular domain.Then the key aromatic amino acids in CARC were mutated at site,and the Bac-to-Bac baculovirus expression system was used to construct the recombinant virus.According to the viral infectivity and protein expression,four CARC motifs（CARC1,CARC2,CARC3,CARC4）were identified as the necessary active motifs for viral infection.Next,multiple transient expression vectors were constructed in the study to explore the effects of CARC mutations on GP64 expression and fusion.The results showed that CARC mutation would not affect the formation of GP64trimer and its location on the cell membrane surface,but would affect the efficiency of GP64mediated membrane fusion.The mutations at CARC1,CARC2,and CARC3 sites directly led to GP64’s loss of membrane fusion activity.Compared with the wild type GP64 membrane fusion activity,mutations at other sites led to a significant reduction in the protein membrane fusion activity.Finally,the CARC polypeptide sequence was designed to more accurately verify that the key CARC region of BmNPV GP64 can bind to Bm N cells,and its binding ability is affected by the content of cholesterol on the cell surface.Secondly,the signal peptide（SP）of BmNPV GP64 did not undergo cleavage in the post translational modification pathway,which is mainly caused by the n-region of the SP.This results in GP64 viruses carrying cell membrane localization（v GP64）compared to GP64viruses carrying cytoplasmic localization(v SP^ΔnGP64)has many features.In this thesis,the CARC activity in SP^ΔnGP64 was analyzed.It was found that after the signal peptide was cut,only CARC2 and CARC3 were the key motifs necessary for virus invasion.This indicates that CARC1 and CARC4 are special CARC sites when signal peptide is not cut,and these two sites have other functions besides binding to cholesterol.Finally,the study analyzed the previously discovered CRAC1 and CRAC2 double mutated viruses(v GP64^Y269&327A).The study compared the activity of CARC motifs in wild-type viruses（v GP64）and mutated viruses(v GP64^Y269&327A),identified two CARC sites necessary for v GP64^Y269&327A invasion,and determined that mutations at non critical sites cannot prevent the recombinant virus from evading cholesterol invasion into cells.This suggests that some CARC motifs have other functions besides binding to cholesterol.This thesis provides a detailed exploration of the biological activity of the CARC motif in BmNPV GP64 during virus infection.By comparing the similarities and differences of three viruses,we can further understand the role of cell surface cholesterol in virus invasion,laying the foundation for further exploration of the invasion mechanism of BmNPV.