A novel HIV-1 inhibitor that blocks viral replication and rescues APOBEC3s by interrupting vif/CBFβ interaction
2020; Elsevier BV; Volume: 295; Issue: 43 Linguagem: Inglês
10.1074/jbc.ra120.013404
ISSN1083-351X
AutoresSizhu Duan, Shiqi Wang, Yanan Song, Nan Gao, Lina Meng, Yanxin Gai, Ying Zhang, Song Wang, Chu Wang, Bin Yu, Jiaxin Wu, Xianghui Yu,
Tópico(s)Hepatitis C virus research
ResumoHIV remains a health challenge worldwide, partly because of the continued development of resistance to drugs. Therefore, it is urgent to find new HIV inhibitors and targets. Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3) are important host restriction factors that inhibit HIV-1 replication by their cytidine deaminase activity. HIV-1 viral infectivity factor (Vif) promotes proteasomal degradation of APOBEC3 proteins by recruiting the E3 ubiquitin ligase complex, in which core-binding factor β (CBFβ) is a necessary molecular chaperone. Interrupting the interaction between Vif and CBFβ can release APOBEC3 proteins to inhibit HIV-1 replication and may be useful for developing new drug targets for HIV-1. In this study, we identified a potent small molecule inhibitor CBFβ/Vif-3 (CV-3) of HIV-1 replication by employing structure-based virtual screening using the crystal structure of Vif and CBFβ (PDB: 4N9F) and validated CV-3's antiviral activity. We found that CV-3 specifically inhibited HIV-1 replication (IC50 = 8.16 µm; 50% cytotoxic concentration >100 µm) in nonpermissive lymphocytes. Furthermore, CV-3 treatment rescued APOBEC3 family members (human APOBEC3G (hA3G), hA3C, and hA3F) in the presence of Vif and enabled hA3G packaging into HIV-1 virions, which resulted in Gly-to-Ala hypermutations in viral genomes. Finally, we used FRET to demonstrate that CV-3 inhibited the interaction between Vif and CBFβ by simultaneously forming hydrogen bonds with residues Gln-67, Ile-102, and Arg-131 of CBFβ. These findings demonstrate that CV-3 can effectively inhibit HIV-1 by blocking the interaction between Vif and CBFβ and that this interaction can serve as a new target for developing HIV-1 inhibitors. HIV remains a health challenge worldwide, partly because of the continued development of resistance to drugs. Therefore, it is urgent to find new HIV inhibitors and targets. Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3) are important host restriction factors that inhibit HIV-1 replication by their cytidine deaminase activity. HIV-1 viral infectivity factor (Vif) promotes proteasomal degradation of APOBEC3 proteins by recruiting the E3 ubiquitin ligase complex, in which core-binding factor β (CBFβ) is a necessary molecular chaperone. Interrupting the interaction between Vif and CBFβ can release APOBEC3 proteins to inhibit HIV-1 replication and may be useful for developing new drug targets for HIV-1. In this study, we identified a potent small molecule inhibitor CBFβ/Vif-3 (CV-3) of HIV-1 replication by employing structure-based virtual screening using the crystal structure of Vif and CBFβ (PDB: 4N9F) and validated CV-3's antiviral activity. We found that CV-3 specifically inhibited HIV-1 replication (IC50 = 8.16 µm; 50% cytotoxic concentration >100 µm) in nonpermissive lymphocytes. 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Harris R.S. Takaori-Kondo A. Small molecules that inhibit Vif-induced degradation of APOBEC3G.Virol. J. 2014; 11 (24986077): 12210.1186/1743-422X-11-122Crossref PubMed Scopus (31) Google Scholar), and Zif-15 (42Pu C. Luo R.H. Zhang M. Hou X. Yan G. Luo J. Zheng Y.T. Li R. Design, synthesis and biological evaluation of indole derivatives as Vif inhibitors.Bioorg. Med. Chem. Lett. 2017; 27 (28754362): 4150-415510.1016/j.bmcl.2017.07.026Crossref PubMed Scopus (15) Google Scholar). Here, we aimed to identify small molecule inhibitors that interrupt the Vif-CBFβ interaction, which can release not only APOBEC3s but also Vif-hijacking proteins in the E3-ubiquitin ligase complex, such as Cul5, EloB, and EloC. By employing virtual screening based on the crystal structure of the Vif-CBFβ complex (PDB: 4N9F), we identified a small molecule compound called CV-3 that inhibited the interaction between Vif and CBFβ. CV-3 was found to inhibit HIV-1 replication (IC50 = 8.16 µm) and have a low cytotoxicity (50% cytotoxic concentration (CC50) > 100 µm). CV-3 can rescue A3G, promote A3G packaging into virions, and induce Gly-to-Ala hypermutation of the viral genome. FRET, yeast surface display, and co-immunoprecipitation (Co-IP) experiments confirmed that CV-3 specifically blocked the binding of Vif-CBFβ. The results indicate that small molecule compound CV-3 can release APOBEC3 proteins by blocking the Vif-CBFβ interaction to achieve an antiviral effect; therefore, it could be used as a potential novel anti-HIV-1 drug candidate. Because the N terminus of Vif is a key region for the interaction between CBFβ and Vif (30Guo Y. Dong L. Qiu X. Wang Y. Zhang B. Liu H. Yu Y. Zang Y. Yang M. Huang Z. Structural basis for hijacking CBF-β and CUL5 E3 ligase complex by HIV-1 Vif.Nature. 2014; 505 (24402281): 229-23310.1038/nature12884Crossref PubMed Scopus (162) Google Scholar) and the tripartite interaction of Ile-55 and Phe-68 of CBFβ and Trp-5 of Vif is critical for Vif-CBFβ binding (33Desimmie B.A. Smith J.L. Matsuo H. Hu W.S. Pathak V.K. Identification of a tripartite interaction between the N-terminus of HIV-1 Vif and CBFβ that is critical for Vif function.Retrovirology. 2017; 14 (28302150): 1910.1186/s12977-017-0346-5Crossref PubMed Scopus (8) Google Scholar), it is desirable to screen for small molecules capable of blocking this region through structure-based virtual screening. The N-terminal of Vif is a linear structure that binds to a shallow pocket on the N-terminal of CBFβ. Based on this structure feature, CBFβ (full sequence) was defined as the receptor. The virtual screening process is described in detail under "Experimental procedures". In brief, the binding of Vif to CBFβ (PDB: 4N9F) was the target of our study. The shallow surface pocket on CBFβ coincides with the position of Vif residues 5–11 in the complex. Thus, the "binding site" was defined as the pocket on CBFβ by the "find sites as volume of selected ligand" tool with residues 5–11 of Vif as the selected ligand. The compound database used for screening, the Alfa Aesar entry in the ZINC database, contains structural information of 34,687 unique chemicals. The LigandFit protocol was run, and molecules that can be docked to CBFβ were then evaluated by consensus score. Then the duplicate structures of the molecules that already obtained a consensus score > 6 were removed and the selected molecules continued to go through the CDOCKER protocol (Fig. 1A). Small molecules with −CDOCKER energy > 40 were selected for visual inspection. Seven compounds that could form hydrogen (H) bonds with Ile-55, Phe-68, or nearby residues of CBFβ were selected for further biological evaluation. The structure, H bonds with CBFβ, and predicted binding energy of the candidate compounds are shown in Fig. 1B. All of the candidates have very low predicted biotoxicity (LD50 > 3.6 g/kg), and most of them form H bonds with residues Gln-67, Phe-69, Arg-83, and Arg-131 of CBFβ. To determine the candidate compounds' ability to block Vif-CBFβ, Vif was displayed on the surface of the yeast cell strain EBY100. The cells were incubated with CBFβ protein for 4 h with a 100 µm concentration of candidate compounds or DMSO. After binding to the CBFβ antibody, the cells were incubated with FITC-IgG antibody. The binding of Vif-CBFβ was analyzed by measuring FITC intensity via flow cytometry (Fig. 2A). The results showed that all of the candidates can inhibit Vif-CBFβ interaction significantly. Next, the protective effect of compounds toward A3G in the cytoplasm was determined. 293T cells were transfected with A3G-YFP and Vif-HA. After 4 h of transfection, 100 µm small molecule compounds or DMSO were added. The expression of A3G-YFP was observed under a fluorescence microscope (Fig. 2B), and protein levels in cell lysates were analyzed by Western blotting (Fig. 2C) 48 h later. For both of these methods, the expression levels of A3G with CV-5 or CV-7 were comparable with the DMSO negative control (Fig. S1). The antiviral activities of the candidates were then examined. After infecting CEM cells with the HIV-1 NL4-3, the cells were treated with 100 µm of candidate compounds or DMSO for 6 days. The viral infectivity was then tested using TZM-bl cells. During 6 days of culture incubation, no additional drugs were added to the culture medium so the inhibition ability detected would be weaker. Only CV-1 and CV-3 significantly decreased virus infectivity (Fig. 2D). To explore the cytotoxicity of the compounds, cell viability was monitored by cell counting after incubating HIV-1-infected CEM cells at different concentrations of CV-1 and CV-3 for 6 days, and the infectivity of the produced virus was determined (Fig. 2E). Treatment with CV-3 did not affect cell viability, and the infectivity was dose-dependently decreased. Virus infectivity only declined at 100 µm CV-1, which suggested that CV-1 reduced the infectivity of HIV-1 due to its cytotoxicity. In addition, the CC50 of CV-1 (87.5 µm) was much lower than that of CV-3 (264.9 µm) in CEM cells (Fig. 2F). Therefore, CV-3 was selected for subsequent investigations. Vif expression is essential for HIV-1 derived from nonpermissive cells, including cell lines H9 and CEM, whereas the virus produced from permissive cells, such as CEM-SS, Jurkat, and SupT1 cells, is fully infectious regardless of the presence of Vif (43Malim M.H. APOBEC proteins and intrinsic resistance to HIV-1 infection.Philos Trans. R Soc. Lond. B Biol. Sci. 2009; 364 (19038776): 675-68710.1098/rstb.2008.0185Crossref PubMed Scopus (210) Google Scholar). To further examine whether the antiviral activity of CV-3 is APOBEC3-dependent, H9 and Jurkat cells stably infected with HIV-1 HXB2 were incubated with 50 µm CV-3 or DMSO for 48 h. The infectivity was assayed using TZM-bl cells. Notably, only the infectivity of virus produced from nonpermissive cell line H9/HXB2 was inhibited by CV-3 (Fig. 3A). This indicated that the antiviral function of CV-3 was related to the expression of APOBEC3s. Moreover, as the concentration of CV-3 became higher in the H9/HXB2 culture system, the viral infectivity gradually weakened, which showed that the antiviral function of CV-3 was dose dependent (Fig. 3B). We next evaluated the effect of CV-3 on viral replication in different human T lymphocyte lines. Equal amounts of NL4-3 or NL4-3ΔVif were utilized to infect permissive cell lines (CEM-SS and SupT1) and nonpermissive cell lines (CEM and SupT1-A3G). After infection, the cells were cultured in the medium with different concentrations of CV-3. In the following 12 days, p24 antigen in the supernatants was quantified to construct virus replication curves (Fig. 3C). Both HIV-1 and HIV-1ΔVif were able to replicate at the same level in permissive cell lines (CEM-SS and SupT1) with or without CV-3. By contrast, in the nonpermissive cell lines CEM and SupT1-A3G, HIV-1ΔVif was suppressed to baseline levels due to the expression of APOBEC3 proteins, which is consistent with previous findings (44Schwedler U. Song J. Aiken C. Trono D. Vif is crucial for human immunodeficiency virus type 1 proviral DNA synthesis in infected cells.J. Virol. 1993; 67 (8331734): 4945-495510.1128/JVI.67.8.4945-4955.1993Crossref PubMed Google Scholar, 45Gabuzda D.H. Lawrence K. Langhoff E. Terwilliger E. Dorfman T. Haseltine W. Sodroski J. Role of vif in replication of human immunodeficiency virus type 1 in CD4+ T lymphocytes.J. Virol. 1992; 66 (1357189): 6489-649510.1128/JVI.66.11.6489-6495.1992Crossref PubMed Google Scholar, 46Kao S. Akari H. Khan M.A. Dettenhofer M. Yu X.-F. Strebel K. Human immunodeficiency virus type 1 Vif is efficiently packaged into virions during productive but not chronic infection.J. Virol. 2003; 77 (12502829): 1131-114010.1128/jvi.77.2.1131-1140.2003Crossref PubMed Scopus (56) Google Scholar). Moreover, HIV-1 showed a high level of replication in nonpermissive cell lines. After the addition of CV-3, HIV-1 replication levels were inhibited in a dose-dependent manner, suggesting that the antiviral activity of CV-3 depends on APOBEC3s. The inhibitory effect of CV-3 on HIV-1 replication was already apparent at a concentration of 5 µm, and HIV-1 replication could still be suppressed at a concentration of 50 µm CV-3 until the 12th day after infection. The results from cell-counting experiments showed that the addition of different concentrations of CV-3 had no effect on the growth of different cell lines, indicating that CV-3 is specific for virus inhibition and does not affect cell growth (Fig. S2). The IC50 of CV-3 detected in CEM cells was ∼8.16 µm (Fig. 3D). In addition, the CC50 detected by the MTT method was greater than 100 µm in all utilized lymphocyte lines (Fig. S3). Blocking the interaction of Vif-CBFβ results in the inability to form the E3 ubiquitin ligase complex; therefore, CV-3 should be able to protect not only A3G but also other proteins of the APOBEC3 family (26Wenyan Z. Juan D. Evans S.L. Yunkai Y. Xiao-Fang Y. T-cell differentiation factor CBF-β regulates HIV-1 Vif-mediated evasion of host restriction.Nature. 2011; 481 (22190036): 376-37910.1038/nature10718PubMed Google Scholar, 28Kim D.Y. Kwon E. Hartley P.D. Crosby D.C. Mann S. Krogan N.J. Gross J.D. CBFβ stabilizes HIV Vif to counteract APOBEC3 at the expense of RUNX1 target gene expression.Mol. Cell. 2013; 49 (23333304): 632-64410.1016/j.molcel.2012.12.012Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 29Fribourgh J.L. Nguyen H.C. Wolfe L.S. Dewitt D.C. Wenyan Z. Xiao-Fang Y. Elizabeth R. Yong X. Core binding factor β plays a critical role by facilitating the assembly of the Vif-cullin 5 E3 ubiquitin ligase.J. Virol. 2014; 88 (24390320): 3309-331910.1128/JVI.03824-13Crossref PubMed Scopus (32) Google Scholar). To verify this, the protective effect of CV-3 on hA3C, hA3F, and hA3G was examined. hA3C-FLAG, hA3F-V5, or hA3G-cmyc was cotransfected with Vif-HA into 293T cells. After incubation with 10 or 50 µm CV-3 for 48 h, the expression of hA3C, hA3F, and hA3G in cells treated with CV-3 was rescued to comparable levels as the DMSO control group in the presence of Vif (Fig. 4, A–C). The feline immunodeficiency virus (FIV) Vif can degrade feline APOBEC3s without CBFβ as a molecular chaperone (47Yoshikawa R. Takeuchi J.S. Yamada E. Nakano Y. Ren F. Tanaka H. Münk C. Harris R.S. Miyazawa T. Koyanagi Y. Sato K. Vif determin
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