Identification Of Interaction Between Hantaanvirus Gn Cytoplasma Tail And Host ESCRT In Virus Budding

?Background?Hantaanvirus(HTNV)is a Bunyavirales-Hantaviridae-Orthohantavirus virus with negative,segmented RNA strands,whose pathogenic mechanism is still unclear,especially the late stage of virus life cycle,in which the budding stage is least studied.The mature and infective virons assembled at the late stage of virus life cycle need to be released through budding process to infect new hosts.Most of the negative strand RNA viruses rely on their matrix proteins to bud from host cells,but the matrix protein still need the host protein network ESCRT(Endosomal sorting complex required for transport)to support budding and complete fission.The matrix protein possess some specific conservative domains that can bind to ESCRT linker molecules and hijack the complex to facilitate budding,because these conservative domains usually take effect in the late stage of virus life cycle,so they are called late domains(L-domain).For the Bunyavirales,they don' t have matrix proteins,but their glycoprotein can act as matrix protein in their budding process,and more over,the glycoprotein itself can form virus like particle(VLP),whichresembles matrix protein.Then we compared the amino acid sequences among Orthohantaviruses HTNV Gn-CTs and found a conservative sequence Y-R-T-L,which is similar to the known late domain Y-P-x-L.In order to make clear of the role of this conservative sequence in HTNV release,we constructed mutants based on the Y-R-T-L sequence and in the meantime screened out the ESCRT factors participating the budding stage of HTNV,then tried to find the link between HTNV glycoprotein and ESCRT,explaining how the HTNV bud from host cells.?Objective?Our research would study the role of the Y-R-T-L sequence in HTNV budding and tried to clarify whether and how host ESCRT participate in HTNV budding.?Methods and results?1.Studying the influence of HTNV Gn-CT Y-R-T-L on VLP releaseWe found the conservative sequence Y-R-T-L among Hantaviridae using sequence aliment,and based on this finding,we constructed three HTNV Gn-CT truncation covering the Y-T-R-L sequence as well as point mutants of the Y-R-T-L to alanine respectively.The mutated HTNV Gn-CTs were loaded into pCDNA3.1 vector and named Gn?C14,Gn?C27,Gn?C42,?Gn-CT,GnY615 A,GnR616A,GnT617 A and GnL618 A respectively.These truncation and mutants were verified using double enzyme cleavage and sequencing.The HTNV Gn-CT truncation and mutants were transfected to HEK293 T cells and VLP in the cell culture supernatant were collected by ultracentrifuge and amount and of VLP were determined by Western-blotting.We found the Y-R-T-L sequence's importance in VLP release: when the amino acids tyrosine and valine were mutated to alanine,there was a significant drop in supernatant VLP compared with wild type.The morphology and distribution of VLP were determined by transmission electron microscope and immunofluorescene.VLP aggregation was observed by tasnmission electron microscopy and altered Gn distribution was observed by immunofluorescene.2.Clarification of host ESCRT factors participating HTNV buddingAccording to the assembly process and the structure of ESCRT,we designed four setsof siRNA targeting the four key linker molecules of ESCRT: ALIX,TGS101,Nedd4 and Vps4 A.siRNA containing lentivirus targeting the four molecules were constructed and verified by Western-blotting,these lentiviruses were able to knock down ALIX,TGS101,Nedd4 or Vps4 A effectively.Lentiviruses were used to knock down ALIX,TGS101,Nedd4 and Vps4 A before transfection of HTNV M segment or HTNV infection.VLP in the cytoplasma as well as HNTV NP in infected cells were detected by transmission electron microscopy,in-cell Western and immunofluorescene.The results showed that VLP aggregated in cytoplasma,the amount and distribution of HTNV antigen detected by in-cell Western and immunofluorescene showed aggregation in cells.In the mean time,ALIX,TGS101,Nedd4 and Vps4 A were knocked down before or after HTNV infection,then the supernatant of the infected cells was transferred to new uninfected cells,HTNV antigen was detected by ELISA.Analysis of HTNV antigen in second round infected cells showed significant reduction compared with control group.3.Clarification of the interaction between HTNV Gn-CT and host ESCRT in buddingWe used co-immunoprecipitation to detect the interaction between Gn and ESCRT key molecules ALIX,TGS101,Nedd4 and Vps4 A and screened out ALIX,which could interact with Gn directly,in the meantime we verified this interaction by immunofluorescene,which showed co-localization of Gn and ALIX.ALIX were then to determine the site of interaction with Gn by co-immunoprecipitation with HTNV Gn-CT mutants.The site of interaction was further verified by co-immunoprecipitation with HTNV Gn-CT mutants under ALIX overexpression.ALIX showed its ability to bind Gn directly and and the binding site in Gn was the Y-R-T-L sequence,either ALIX was overexpressed or not.The role of the key ESCRT molecule in HTNV budding was further determined ELISA and Western-blot detecting HTNV antigen or VLP release under ALIX overexpression,ALIX overexpression could promote HTNV release and ALIX overexpression could also rescue VLP release to a certain degree when HTNV Gn-CT waslost or Y-R-T-L mutated.?Conclusion?In this research we found the consistence sequence Y-R-T-L of Gn cytoplasma was critical for HTNV VLP's release,and the Y-R-T-L sequence was the binding site of host ESCRT factor ALIX.HTNV budding process relies on host ESCRT and interact specifically with ALIX to hijack ESCRT.Our work shed light to the late stage of HTNV budding process and partially explained how HTNV completes its life cycle.Our work would provide a foothold for further research and shed light on antiviral drug development.