Design And Evaluation Of HIV-1 Peptide Inhibitors Targeting Vif-CBF-β Binding Site Based On Vif Mutant Library

Human immunodeficiency virus（HIV）can infect the human immune system and cause AIDS.After HIV-1 infects host cells,its accessory protein Vif（viral infectivity factor）will recruit the intracellular ubiquitin-related scaffold protein Cullin5（CRL5）,the linker proteins ElonginB（EloB）and ElonginC（EloC）,and the T-cell chemokine CBF-βto form the E3 ubiquitin ligase complex for ubiquitination and subsequent proteasomal degradation of the intracellular host restriction factor APOBEC3G protein（A3G）,thereby antagonizing the anti-HIV-1 function of A3G.If the binding between Vif and other proteins in the complex is blocked,the formation of the E3 complex will be inhibited,and then the A3G protein will not be degraded and it could be packaged more into the newborn HIV-1 virions to exert its cytosine deaminase activity,causing hypermutations in the HIV-1 genome and inhibiting the replication of the HIV-1.In previous studies,researchers have discovered a variety of anti-HIV-1 drugs by using different methods such as computer-aided drug design,high-throughput screening based on fluorescence resonance energy transfer,and phage surface display technology.Regarding the inhibition of the formation of E3 ubiquitin ligase complex as a target,many studies have been reported on inhibitors blocking Vif oligomerization,blocking Vif binding to A3G,and blocking Vif binding to EloC.Various results show that blocking protein-protein interactions in the E3 complex will inhibit Vif-mediated degradation of A3G and have a certain inhibitory effect on many subtypes of HIV-1virus.Among the multiple protein-protein binding interfaces of the E3 complex,due to the relatively large interface between Vif and CBF-β,few inhibitors blocking this target has been found.Therefore,we used the yeast surface display technology and the Vif protein mutant display library that has been established in previous research to screen Vif mutants with stronger affinity to CBF-βprotein by flow cytometry.Based on the obtained mutation sites,peptide inhibitors were designed to block Vif-mediated A3G degradation and restore its ability to inhibit HIV-1.First,We incubated a previously constructed Vif mutant-displaying library with CBF-βprotein,and then obtained the mutants with stronger affinity to CBF-βcompared with the wild-type Vif protein by three rounds of sorting enrichment.After amplifying the mutant genes and performing sequence alignment,30 mutant sequences were obtained,in which the rate of coincident amino acid sties with reported sites in the Vif protein associated with CBF-βreaches 32.14%,which proves the validity of our screening system.Next,based on the obtained mutation sites,we designed nine small peptides（9-18mer）derived from the Vif mutants.After molecular docking using computer software,six peptides were selected for synthesis,and their abilities to restrict HIV-1 infection and replication were tested.The results showed that peptides 63M and 108M could restrict HIV-1 infection,with IC₅₀ values of 32.7μM and 74.0μM,respectively,and had inhibitive effect on viral replication at a certain concentration.Then,we investigated the cytotoxicity,cell-uptaking capacity,and antiviral mechanisms of these two peptides.The results show that at concentrations below 100μM,63M and 108M have no toxic effect on a variety of cells,and HEK293T cells can effectively take up small peptides.In the process of exploring the anti-HIV-1 mechanisms of 63M and108M,we found that both of them could inhibit the interaction between Vif and CBF-βin vitro and in HEK293T cells,subsequently protecting A3G from degradation in the cells and increasing the amount of A3G packaged in the virions.In summary,we obtained two small peptides with antiviral activities by targeting the Vif-CBF-βinterface.These results might help to broaden the potential targets for development of anti-HIV-1 peptide inhibitors.In subsequent studies,we will further modify and optimize these peptides for the purpose of improving their stability and reducing IC₅₀,hoping to find new peptide inhibitors with stronger inhibitory ability.