High-Throughput NanoBiT-Based Screening for Inhibitors of HIV-1 Vpu and Host BST-2 Protein Interaction.

Boye Li,Xiaoli Wang,Yishu Yang,Tian Chen,Wenmei Zhang,Qin Hu,Xiaoxiao Dong,Xiayan Wang,Boyang Yu,Wenyue Zhao

doi:10.3390/ijms22179308

Boye Li, Xiaoli Wang + Show 8 more

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https://doi.org/10.3390/ijms22179308

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Abstract

Bone marrow stromal cell antigen 2 (BST-2), also known as CD317 or tetherin, has been identified as a host restriction factor that suppresses the release of enveloped viruses from host cells by physically tethering viral particles to the cell surface; however, this host defense can be subverted by multiple viruses. For example, human immunodeficiency virus (HIV)-1 encodes a specific accessory protein, viral protein U (Vpu), to counteract BST-2 by binding to it and directing its lysosomal degradation. Thus, blocking the interaction between Vpu and BST-2 will provide a promising strategy for anti-HIV therapy. Here, we report a NanoLuc Binary Technology (NanoBiT)-based high-throughput screening assay to detect inhibitors that disrupt the Vpu-BST-2 interaction. Out of more than 1000 compounds screened, four inhibitors were identified with strong activity at nontoxic concentrations. In subsequent cell-based BST-2 degradation assays, inhibitor Y-39983 HCl restored the cell-surface and total cellular level of BST-2 in the presence of Vpu. Furthermore, the Vpu-mediated enhancement of pesudotyped viral particle production was inhibited by Y-39983 HCl. Our findings indicate that our newly developed assay can be used for the discovery of potential antiviral molecules with novel mechanisms of action.

Highlights

Acquired immunodeficiency syndrome (AIDS) is a malignant infectious disease caused by human immunodeficiency virus (HIV), which causes severe destruction of immune function and eventually leads to opportunistic infections and tumors
viral protein U (Vpu) and Bone marrow stromal cell antigen 2 (BST-2) proteins were fused to LgBiT and SmBiT, respectively, and co-expressed in HEK293T
When Vpu binds to BST-2, LgBiT and SmBiT subunits are brought together, resulting in the formation of NanoLuc luciferase (NLuc), which can be quantitatively measured and is applicable to high-throughput screening (Figure 1A)

Summary

Introduction

Acquired immunodeficiency syndrome (AIDS) is a malignant infectious disease caused by human immunodeficiency virus (HIV), which causes severe destruction of immune function and eventually leads to opportunistic infections and tumors. Current antiretroviral drugs are often classified into six main classes: chemokine receptor antagonists, fusion inhibitors, nucleosides/non-nucleoside reverse transcriptase inhibitors, protease inhibitors, and integrase inhibitors. The advent and continuous optimization of combination antiretroviral therapy (cART) has significantly reduced the mortality and morbidity due to HIV infection [1,2], many challenges remain to be overcome, including incomplete adherence to ART, adverse effects, and drug resistance [3]. The HIV-1 viral protein U (Vpu) protein is a type I transmembrane protein expressed in the late stage of the viral life cycle [4,5] and a multifunctional accessory protein that facilitates viral egress. Vpu is found to counteract bone marrow stromal cell antigen

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