The Study On The Interaction Between BST-2and Vpu By BRET Assay And The Screening Of Small Molecular Compound Inhibitors

Host restriction factors, produced by host cells, which inhibit viral replication or dissemination, perform as a barrier to the host defense against virus invasion. The viruses also develop some corresponding strategies in the evolutionary process to overcome these barriers. So the interaction between host and virus has been a research hotspot in the virology field in recent years. BST-2(bone marrow stromal cell antigen2, also referred to as Tetherin, CD317or HM1.24) is a host restriction factor for inhibiting the release of enveloped viruses, and this antiviral effect can be antagonized by HIV-1accessory protein Vpu, via the interaction of the two protein transmembrane domains. However, the molecular mechanism of interaction is still not very clear. Therefore, we utilized the bioluminescence resonance energy transfer (BRET) method to define the interaction of BST-2and Vpu in living cells, and identified the key residues for their interaction through BRET signal changes by joint or single point mutations. In addition, cell line stably co-expressed BST-2and Vpu was established based on BRET method, which is used as a high-throughput screening assay. Based on the high-throughput screening assay, a small molecular compound PR was screened out aiming at the transmembrane domain interaction as a potential drug target, and was carried on the preliminary functional verification. The results are as follows:1. Identification of key residues of transmembrane domain for BST-2and Vpu interactionWe firstly fused BRET donor protein Rluc or acceptor protein EYFP to N or C terminus of BST-2or Vm (Vpu S52,56A), constructing eight fused proteins. The two proteins above (including donor protein Rluc and receptor protein EYFP) were expressed in293T cells, and the best combination (fused Rluc at N terminal of BST-2, Rluc-BST2, and fused EYFP at C terminal of Vm, Vm-EYFP) applied for BRET were determined after BRET signal detection. The saturation and competition assays were carried out to ensure the specificity of BRET method in the study of BST-2and Vpu interaction. Then transmembrane domains of these two proteins were mutated, and transfected into293T cells, then the changed BRET signal was detected. We found that the mutations of I34A, L37A, P40A and L41A in BST-2, and L11A, A18V and W22A in Vpu, decreased BRET ratio significantly, indicating that these sites as the key amino acids of BST-2and Vpu interaction. Among them, P40in BST-2and L11in Vpu were identified by us for the first time. In addition, we also found that some triple amino acids substitution,14-16(All to VAA) and26-28(IIE to AAA) in Vpu TM, not a single mutation of the three, produced meaningful impact on the interaction. Obvious local conformation changes were found by molecular dynamics (MD) simulation for the mutants we identified for the first time, and such conformational changes would possibly dismiss the interaction between BST-2and Vpu. Functional experiments confirmed the mutations of these key amino acids of the two proteins lost the susceptibility of each other, namely affected the ability of Vpu to antagonize the BST-2antiviral effect.2. The screening of small molecular compound inhibitors and preliminary functional verificationThe dual-expression plasmid expressing Rluc-BST2and Vpu-EYFP was constructed, and then the293dual-expression stable cell line was established and utilized as a screening assay for interaction inhibitors. The decreased BRET ratio was deemed to be the screening positive index. After screening12000small molecular compounds,27positive compounds were selected. Most compounds were excluded through the later functional verification, and eventually a compound PR was chosen as the final results of the screening. The recovery of intracellular BST-2expression level and virus release inhibition was found after the compound PR added. That is to say the compound PR could interfere the interaction between BST-2and Vpu, and then affect Vpu antagonist to BST-2antiviral function, thus recovery BST-2inherent restriction factor activity.In summary, our study applied BRET method on the interaction between BST-2and Vpu in living cells for the first time. Key amino acids in BST-2and Vpu transmembrane domains were identified for their interactions, and P40in BST-2, L11in Vpu were reported firstly. A high-throughput screening assay based on BRET was first established aiming at the target of the two proteins interaction. Preliminary functional verification was executed for the small molecular compounds screened positive. The results of our study provide the reliable data to further elucidate the mechanism of interaction between BST-2and Vpu, and provide a new way for screening drugs against HIV-1, as well as a new candidate to the future development of drugs against HIV-1.