Study On The Interactions Between Tetherin /SAMHD1 And Equine Infectious Anemia Virus

Equine infectious anemia virus(EIAV)and Human immunodeficiency virus(HIV-1)belong to the Retroviridae lentiviruses,and it is an important animal model to study pathogenicity and immune mechanism of the lentivirus.Lentivirus-induced disease,a serious threat to animal and human health,which AIDS among people with HIV-1 infection caused by the most serious.Vaccine is an important mean of prevention and control of infectious diseases,but the highly variable features of lentivirus antigens,making vaccine research difficult.Especially for the study of HIV-1 vaccine,even though the world has invested enormous human and material resources,but it has not yet made a key breakthrough.In EIAV vaccine research,in the 1970 s,researchers of Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences,had successfully developed the EIAV attenuated vaccine by inoculate the virus to dissimilar animals and passaged in vivo/in vitro.Laboratory and terrain applied had proved that equine animals had immunized the EIAV attenuated vaccine can induce a strong protective immunity,and has effectively blocded the prevalent of EIAV for its powerful immunoprotective effect in China.To clarify the mechanisms of virulence weakening and immunity protection,will be able to find the key factors of lentivirus-induced immune protection,and to provide a reference for HIV vaccine development.An important scientific question when exploring EIAV vaccine to induce an immune protection mechanism and the weakening of the process,is why via different animals or in vitro cell passage can cause virus attenuated? The scientific community did not give a clear mechanism of the phenomenon described for a long time.Studies have shown that virus invaded the host will induce host cells to produce a strong innate immune response and secondary immune response.Meanwhile,there are many innate immunity restriction factors in the host cells,they can respond quickly when the virus invaded host cells,and effected on virus directly or indirectly to inhibit viral replication and spread of the virus and thus protect the host against viral infection.Currently,more extensive study of the host innate immunity restriction factors have APOBEC3 family,TRIM family,Tetherin / BST2,SAMHD1,MOV10 etc.These factors can inhibite the viral replication process in various stages through a variety of mechanisms.Virulence weakened of EIAV attenuated vaccine,mainly in the in vitro subculture process.Since this procedure is missing a host specific immune response role,we hypothesized that the innate immune response of host innate immune cells in particular,the restriction factors may play an important role in viral virulence attenuated process.To verify the above hypothesis,in this study,Tetherin and SAMHD1 were chosen to evaluatetheir interaction with EIAV and the restriction role in EIAV attenuated process.Firstly,basic biological functions and anti-virus capabilities of equine Tetherin and donkey Tetherin were compared by sequence analysis,protein structure analysis,cellular localization,protein expression analysis,cell transfection,Western Blotting experimental techniques.Experimental results shown that the amino acid sequence,molecular topology,advanced cellular localization and molecular structures of the equine Tetherin and donkey Tetherin were very similar,but there were both individual amino acid mutation sites,which lead to donkey Tetherin stronger than equine Tetherin in the ability for restricting EIAV virus budding.Furthermore,protein-protein interactions and other molecular biology experiments have been developed to evaluate the interaction between donkey Tetherin and EIAV strains for different degrees of evolution.The results shown that there are different capacity for donkey Tetherin to inhibite the budding of EIAV strains for different degrees of evolution,this difference is reflected in that the weaker of virus virulence the stronger of inhibit effect for Tetherin;there are some differences among different Env genes of EIAV strains,they all can antagonise the do-Tetherin,and the weaker of virus virulence the weaker for antagonism.On this basis,we have studied the interaction between SAMHD1 and EIAV.Firstly,the sequences of human SAMHD1 and equine SAMHD1 have been compared,and then we established the U937 cell lines which can stabley expressed the SAMHD1 gene as a model of infection,and then the three-plasmids system packaged fake virus(HIV-1,SIV,EIAV)infected the cell lines to evaluate the antiviral capacity of the equine SAMHD1.Experimental results shown that the equine SAMHD1 has the similar amino acid sequences and structures to human SAMHD1,and has a broad-spectrum anti-retroviral effect,and HD domains exert its antiviral function of the main functional domains.Finally,we have proofed the interaction between equine SAMHD1 and EIAV by protein-protein interactions and other experimental methods,the results shown that EIAV can antagonist with equine SAMHD1 by the Rev protein.There were different abilities for Rev of different strains to antagonistic equine SAMHD1,namely the ability to EIAV-Rev-LN antagonistic equine SAMHD1 stronger than EIAV Rev of other strains,tips EIAV-Rev antagonistic equine SAMHD1 have species differences,but the specific mechanism of EIAV-Rev antagonistic equine SAMHD1 is unclear.In this study,we have firstly studied the mechanism for different/unsusceptible hosts passaged attenuated pathogen infection with Chinese EIAV attenuated system as a study mode.From the perspective of the interaction between host innate immunity factors and pathogen,to explore the reasons for the weakening of pathogen virulence.The results confirmed the scientificity of our hypothesis,it will help to clarify the pathogenic and immune mechanisms of lentivirus,and to promote the related studies of lentivirus immunology and prevention,on the basis of deeply digging the mechanism of innate restriction factors antiretroviral and expand its significance.