Differential Effects Of Species-specific Vif On Virus Production And Mechanism Of Conserved Interaction Of Lentiviral Vif Molecules With HIV-1 Gag

Open reading frame virion infectivity factor(Vif)is conserved among most lentiviruses.Former studies illuminated that Vif molecules contribute to viral replication in non-permissive cells by inactivating host anti-viral factors,the apolipoprotein B m RNA-editing catalytic polypeptide-like(APBOBC)cytidine deaminases.The host's anti-viral APOBEC proteins can induce lethal mutations in the viruses and be packaged into viral progeny.The HIV-1 Vif counteracts host anti-viral factors APBOBC by recruiting host Cullin 5,Elo B/C(CRL5)E3 ubiquitin ligase to induce APOBEC3 polyubiquitination and proteasome-mediated degradation in virus-producing cells.However,various species of lentiviral Vif proteins have evolved different strategies for overcoming host APOBEC.During lentiviral evolution,the Vif-APOBEC3 interaction has been mainly been species-specific: BIV Vif cannot induce the degradation of human APOBEC3 proteins,and HIV-1 Vif cannot overcome bovine APOBEC3 proteins.Whether different species of lentiviral Vif proteins still preserve certain common features has not been reported.In this study,we used cell biological,molecular biological and virology technologies to discover for the first time that even without anti-viral APOBEC proteins,BIV Vif rather than HIV-1 Vif can suppress the production and infectivity of HIV-1.It was indeed a discovery of significance,as BIV Vif can be seen as an exogenous anti-HIV-1 factor and it has no functional relation with HIV-1 Vif.This can be viewed as a new idea of anti-HIV treatment.On one hand,BIV Vif can act as a brand new anti-viral factor,on the other hand,it brought more question about how BIV Vif counteracted HIV-1? Figuring this out might provide more detail of anti-viral mechanisms.Next,we studied on the underlying mechanisms by analyzing the full profile of secreted viruses.Firstly,BIV Vif can partially suppress the secretion of newly produced viruses.However,this suppress cannot fully explain the suppression of HIV infect ability by BIV Vif.Secondly,which is more important,BIV Vif can directly suppress newly produced HIV-1's infectivity.Thirdly,BIV Vif suppressed the cleavage of the HIV-1 Gag precursor polyprotein(Pr55Gag)in secreted viral particles.This will interrupt one of the most important processes in viral life cycle,the maturation.The core step of viruses' maturation is the proper cleavage of Pr55 Gag.Following strictly controlled order,the Pr55 Gag will be cleavage into matrix,capsid,nucleocapsid,p6,p2 and p1.Not only the successful cleavage of each part is crucial,but also the proper order of cleavage is necessary.BIV Vif suppressed the first step of Pr55 Gag cleavage,the cut between NC/p2.Through this interruption of virus maturation,BIV Vif can remarkably suppress the infectivity of HIV-1.This explains why BIV Vif could counteract HIV-1 infectivity.The following question is that why BIV Vif rather than HIV or SIV Vif can counteract HIV-1? Here,we show for the first time that BIV Vif has an enhanced interaction with Pr55 Gag when compared to HIV-1 Vif.BIV Vif mutations which are defective for the Pr55 Gag interaction lost their abilities in inhibiting HIV-1.This explained that although HIV-1,SIV and BIV Vif all interact with Pr55 Gag,only BIV Vif can remarkably suppress HIV-1.Furthermore,diverse lentiviral Vif molecules maintain the ability to interact with the HIV-1 Pr55 Gag.This conservation of interaction implies that virus might need this interaction to serve certain important function.As the interaction between Vif-Pr55 Gag is the core in the suppression of HIV-1 by BIV Vif.Meanwhile,the conservation of different species of Vif in interacting with Pr55 Gag implied the underlying importance of this interaction to virus survival or replication.Thus we next use molecular cloning and co-immunoprecipitation to identify the crucial regions of Pr55 Gag and Vif for interacting with each other.According to our results,the C-terminal region of CA and the p2 region in Pr55 Gag,which are both important for virus assembly and maturation,were involved in the interaction.Furthermore,both our single amino acid scanning mutagenesis and Vif truncation strategies identified the N-terminal region,especially amino acids Y30 and R/H33,as being important for the Vif-Gag interaction.This identification of the important regions for Vif and Pr55 Gag to interact with each other provides more details for further study on the application of BIV Vif and the function of Vif-Gag interaction.BIV Vif can remarkably suppress HIV-1 in HEK 293 T cells.Whether BIV Vif can also suppress HIV-1 in HIV-1 targeting CD4 positive T cells? According to our results,transduction of CD4 positive T cells with BIV Vif can also blocked HIV-1 replication.To sum up,this study used modern biological technology firstly discovered that BIV Vif can remarkably suppress the production and infectivity of HIV-1.We further explored and discussed the underlying mechanisms: BIV Vif can efficiently interact with HIV-1 Pr55 Gag and interrupt the viruses' maturation after virus budding.Meanwhile,we found that HIV-1,SIV and BIV Vif all can interact with Pr55Gag;this is the first report of this interaction.We further identified the crucial regions for Vif and Pr55 Gag to interact with each other.This study provides important details for the development of anti-viral strategy and provide new potential target of counteracting HIV-1.