Screening Of Anti-HIV-1 Inhibitors Targeting Vif-CBF? Interaction

AIDS(Acquired Immunodeficiency Syndrome)is an infectious disease caused by the human immunodeficiency virus(HIV)that specifically infects and kills primary lymphocytes of human body.It has spread rapidly around the world since reported in the 1980s.Individuals infected with HIV are prone to get opportunistic infection and malignant tumor due to the destruction of the immune system.AIDS has a high mortal-ity rate,and cannot be completely cured currently.Highly active antiretroviral therapy(HAART)is the main method used in the clinical treatment of AIDS at present,but its disadvantage is that patients need to take drugs for life and it will produce side effects and drug resistance.Therefore,there is an urgent need to develop new drugs and find new targets.In addition,functional cure for AIDS is also rapidly developing as a novel treatment method.Gene therapy that genetically modify hematopoietic stem cells to continuously express antiviral genes in patients is expected to achieve the effect of one-time treatment for life.In the development of AIDS drugs,the interaction of HIV-1 accessory proteins with host restriction factors has become a new target.Its main advantage is that drugs designed for host restriction factors can avoid escape mutations of the HIV-1 genome and reduce drug resistance.Among the host restriction factors,the APOBEC3 family is a class of cytosine deaminase,of which(APOBEC3G)A3G and(APOBEC3F)A3F have a significant inhibitory effect,which causes lethal mutations by mutating G to A in the HIV-1 genome,and interfere with viral reverse transcription and genome integra-tion.HIV-1 Vif induces the degradation of APOBEC3 family proteins by forming a Vif-CBF?-Elo B-Elo C-Cullin5 E3 ubiquitin ligase complex,of which CBF?is an essential molecular chaperone.CBF?can enhance the production of Vif,stabilize Vif protein and promote the degradation of APOBEC3s.There are already some HIV-1 inhibitors tar-geting Vif-E3 complexes,and most of them target Vif-A3G and Vif-EC interface.The advantage of selecting Vif-CBF?interaction as the target to inhibit HIV-1 is that it can release all APOBEC3 family proteins and the proteins of E3 ubiquitin ligase complex(Elo B,Elo C,Cullin5,etc.)related to normal cell metabolism.Therefore,we designed the screening of drugs that could applied for HAART ther-apy and gene therapy based on the target of Vif-CBF?interaction.The first one is to utilize the structure-based virtual screening method,in which a database of small mo-lecular compounds docking to Vif-CBF?target to screen out small molecular com-pounds that can block their interaction.The antiviral effect and active mechanism of candidate compounds were then validated.The crystal structure of the Vif-CBF?-Cul5-EB-EC complex has been resolved in 2014(PDB ID:4N9F),and there are many reports that the N-terminus of Vif is an important region for interaction with CBF?.It was subsequently found that residue W5 of Vif,CBF?residues I55 and F68 formed a hy-drophobic pocket with three amino acid interactions.Therefore,in the virtual screening process,the interface of residues 5-11 of Vif with CBF?was defined to be docking pocket.The molecular docking software was Discovery Studio 2.5 and the 3D structure data of small molecules were obtained from the Alfa-Aesar database on the ZINC web-site,which could be used directly for molecular docking.After molecular docking ac-cording to our strategy,we selected small molecules with high scores and manually chose seven candidate compounds that form hydrogen bonds with CBF?for subsequent evaluation.Firstly,CV-3 was obtained by verifying the anti-HIV-1 activity,ability to restore A3G protein,and cytotoxicity.CV-3 can specifically inhibit HIV-1 replication in non-permissive lymphocytes(IC₅₀=8.16?M),which proves that its antiviral effect is dependent on APOBEC3.We then demonstrated that in the present of Vif,the addi-tion of CV-3 can protect h A3G,h A3C,and h A3F in 293T cells,promote the packaging of A3G into HIV-1 virus particles,reduce the virus infectivity,and induce G to A mu-tations in the HIV-1 genome.Finally,yeast surface display technology(YSD),fluores-cence resonance energy transfer(FRET),and co-immunoprecipitation(Co-IP)experi-ments showed that CV-3 can block Vif-CBF?interaction.It was further domenstrated by Co-IP experiment that CV-3 weakened the interaction between Cullin5 and Vif by interrupting the binding of Vif-CBF?.In order to explore the mechanism of CV-3,we analyzed the way CV-3 binds to CBF?,and found four CBF?residues that may interact with CV-3.Using FRET assay,we demonstrated that CV-3 blocks the Vif-CBF?inter-action by binding to Q67,I102,and R131 residues of CBF?simultaneously.The above results showed that CV-3 weakened the assembly of E3 complex by blocking the inter-action of Vif-CBF?,thereby inhibiting the degradation of APOBEC3s mediated by Vif and achieving the anti-HIV-1 effect.The second method is to use yeast surface display technology to express Vif mu-tant libraries,and Vif mutants with high affinity of CBF?were sorted by flow cytometry.The mutants should be able to competitively bind CBF?to inhibit the function of wild-type Vif.We initially selected sixteen mutants containing high-frequency mutation sites and mutated the key sites PPLP to AALA so that it could not oligomerize or bind to APOBEC3G.Through functional evaluation,we found that Vif-6M can antagonize the function of wild-type Vif at protein level,restore h A3G,h A3C,h A3F,and AGM A3G,promote the packaging of A3G into HIV-1 particles,reduce the infectivity of HIV-1,and produce G-to-A hypermutation in HIV-1 genome.We then demonstrated that Vif-6M had a high affinity for CBF?through YSD experiments.FRET and Co-IP assay showed that Vif-6M blocked the interaction of Vif and CBF?by competitively binding CBF?and can impacted the assembly of Vif-E3 complexes.Finally,in order to explore the possibility of applying Vif-6M in gene therapy,we transduced it into CEM cells and tested the viral long-term replication ability after infection with HIV-1 NL4-3.It was proved that Vif-6M can effectively inhibit the newly infected HIV-1 replication,and the amounts of produced virus was lower than 50%of EGFP control group at 15 days.Similarly,Vif-6M was transduced into H9 cells that were continuously and stably in-fected with HIV-1 HXB2,and Vif-6M was found to have a good inhibitory effect on virus replication in lymphocytes that already infected with HIV-1.However,the repli-cation of HIV-1 in CEM-SS cells was not affected by the expression of Vif-6M,indi-cating that Vif-6M inhibits the replication of HIV-1 by restoring the function of APO-BEC3.The above results showed that Vif-6M inhibited HIV-1 replication in T lympho-cytes by competitively inhibiting Vif-CBF?interaction,and has a good prospect for application in gene therapy.In summary,although the large interaction interface between Vif and CBF?makes the screening of small molecule inhibitors more difficult,we identified the first small molecule HIV-1 inhibitor,CV-3,targeting Vif-CBF?interaction through virtual screen-ing.It released the host restriction factor APOBEC3 family proteins by antagonizing the function of HIV-1 Vif,thereby inhibiting HIV-1 replication.CV-3 can be used as a lead compound,and can be used as a new drug in HAART treatment after further opti-mization.At the same time,we displayed a series of Vif mutanst library on the surface of yeast cells,and sorted Vif mutants with CBF?-high affinity by flow cytometry.Fian-lly,Vif-6M was screened out after functional verification.Vif-6M was verified to inter-fere with the function of wild-type Vif by competitively binding CBF?,and can also release APOBEC3 family proteins to achieve anti-HIV-1 effect.More importantly,transduction of Vif-6M into human T lymphocytes can effectively inhibit the replication of newly infected and infected HIV-1.The Vif-6M gene can be used in gene therapy as an antiviral protein(AVP)to treat AIDS patients.The effectiveness of the above two antiviral drugs targeting Vif-CBF?proves that the interaction between Vif and CBF?can be used as a novel target for HIV-1,providing a new development direction for the treatment of AIDS.