Protein-protein Interaction Networks Among Structural Proteins Of Bombvx Mori Nucleopolyhedrovirus (Budded Virus) And Characterization Of Interactions Between GP64 And Its Associated Proteins

Baculoviruses are a type of enveloped virus with double-stranded circular DNA containing a genome size of 80-180 kb and specially infect arthropods.One characteristic of baculoviruses that distinguishes them from other viruses is that they have two distinct viral particle morphologies:occlusion-derived virus(ODV)and budded virus(BV).The former are responsible for the oral infection.The occluded viral particles were released from the occlusion body in the alkaline environment of the arthropods intestine and infect the midgut epithelial cells to achieve primary infection;the latter mainly mediate the infection between cells and cells.Both of them contain a large number of structural proteins,of which the structure of BV is simpler than ODV.A large number of structural proteins work together to form complete virus particles,and these interactions also have other functions other than structural supporting.In this study,Bombyx mori nucleopolyhedrovirus(BmNPV)was used as a model to screen the interactions among BV structural proteins using the yeast two-hybrid system and some interactions were validated by co-immunoprecipitation.To further analyze the roles of viral envelope and capsid play in the process of viral infection based on the interactions of viral structural proteins,we constructed a dual-fluorescent virus that simultaneously labeled its envelope and capsid.In addition,the interaction characters of GP64(the most important envelope fusion protein of BV)related interactions were analyzed with GP64 truncated variants.The main conclusions are as follows:1.The networks of protein-protein interactions among BV structural proteins were detection.The yeast two hybrid(Y2H)system was used to evaluate the interactions of 27 viral genes products.Fifty-seven interactions were identified with 51 binary interactions and 6 self-associations.VP39,38 K,and FP were identified to interact with most of the viral proteins based on the related studies up to now.Thus,they may form major structural elements of the viral architecture.In addition,each envelope protein was detected to interact with more than one capsid protein.Five interaction networks were formed based on these assays.These results help us to understand the structural conformation of baculovirus and its infection mechanism.2.A dual fluorescent recombinant virus with simultaneously labeled envelope and capsid was successfully constructed.In order to further explore the interactions between the viral envelope preteins and capsid proteins of BV to reveal the effects of the viral envelope and capsid in the process of viral infection,a dual-fluorescences labeled virus was constructed.In this study,both of vp39 and gp64 genes of BmNPV genome were knocked out by "?-red homologous recombination system".Then,vp39 with three mCherry genes were repaired to the viral genome by the same homologous recombinant system,and gp64 fused with egfp were transported to the downstream of polyhedrin promoter by"Bac-to-bac baculovirus expression system".Our results showed that GP64 fused with EGFP and VP39 fused with 3 mCherrys were successfully integrated into each progeny virion.The C-terminal egfp fused with gp64 had no effect on the viral production,but the N-terminal fused egfp led a significant decrease in viral production.This indicating that the gp64 N-terminal domain plays an important role in its function.This virus can be used for real-time tracking of individual virion.Antibodies of EGFP and mCherry tags can be used for immunofluorescence to amplify viral fluorescence signals when needed.It can also be directly used for protein fluorescence colocalization or subcellular localization.3.The characters of the interaction between the major envelope fusion protein GP64 and the important capsid protein VP39 were clarified.GP64 is the major envelope protein of BV,and VP39 is the major capsid protein.Both of them are the most structural proteins in the virus.Therefore,an in-depth analysis of the interaction between GP64 and VP39 is of great significance for understanding the relationship between the viral envelope and capsid.A series of GP64 truncated viriants were constructed according to the division of the GP64 functional domains.Through the yeast two-hybrid and Co-IP validation,1-78aa of the GP64 protein was identified as a key functional domain for the interaction with VP39.In addition,immunofluorescence was used to observe the sub-localization and co-localization of GP64 full-length and truncated viriants with VP39 in the cells.Full-length GP64 and VP39 were co-localized in cytoplasm and cellular membrane at 24 hp.i.and 48 h p.i.At 72 h p.i.,GP64 forms a ring structure on the nuclear membrane and VP39 gradually entered the nucleus.This suggestted that GP64 may be involved in the translocation of VP39 to the nucleus.At 48 h p.i.,there was a circular co-localization of GP64(1-523 aa)with VP39 on the nuclear membrane,but the co-localization of both GP64 and cytoplasm was transferred from the nuclear membrane to the cell membrane as the N-terminal retention of GP64 decreasing.This showed that the interaction between VP39 and GP64 has an important effect on their localization in the cells.4.the characteristics of interaction between the major envelope fusion protein GP64 and FP,PKIP and 38K were clarified.Whereas VP39,FP,and 38K(all of them are capsid proteins)are the core proteins in viral protein interactions,and PKIP is the structural associated protein that interacts with the most structural proteins.To further explore the interactions between these proteins,the interactions between GP64 and FP,PKIP,and 38K was further analyzed.The truncated viriants of GP64 and FP,PKIP and 38K were analyzed by a direct Y2H assays,respectively.The results showed that the key interaction domains of GP64 and these three proteins were located at its N-terminal 1-78 aa.The fluorescent co-localization assays revealed that GP64 with the N-terminal 1-78 aa can till colocalize with PKIP and FP,which is consistent with the results of Y2H assays.The co-localization of GP64 partial truncated viriants with PKIP at 24 hp.i.occurred at the nuclear membrane,which is similar to the co-localization of GP64 and VP39,suggesting that GP64 may be involved in the transport of PKIP into the nucleus.