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Interactions Between HIV-1 Vif and Human Elonginb-Elonginc Are Important for CBF-Β Binding To

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Interactions Between HIV-1 Vif and Human Elonginb-Elonginc Are Important for CBF-Β Binding To Wang et al. Retrovirology 2013, 10:94 http://www.retrovirology.com/content/10/1/94 RESEARCH Open Access Interactions between HIV-1 Vif and human ElonginB-ElonginC are important for CBF-β binding to Vif Xiaodan Wang1, Xiaoying Wang1, Haihong Zhang1, Mingyu Lv1, Tao Zuo1, Hui Wu1, Jiawen Wang1, Donglai Liu1, Chu Wang1, Jingyao Zhang1,XuLi1, Jiaxin Wu1, Bin Yu1, Wei Kong1,2* and Xianghui Yu1,2* Abstract Background: The HIV-1 accessory factor Vif is necessary for efficient viral infection in non-permissive cells. Vif antagonizes the antiviral activity of human cytidine deaminase APOBEC3 proteins that confer the non-permissive phenotype by tethering them (APOBEC3DE/3F/3G) to the Vif-CBF-β-ElonginB-ElonginC-Cullin5-Rbx (Vif-CBF-β-EloB- EloC-Cul5-Rbx) E3 complex to induce their proteasomal degradation. EloB and EloC were initially reported as positive regulatory subunits of the Elongin (SIII) complex. Thereafter, EloB and EloC were found to be components of Cul-E3 complexes, contributing to proteasomal degradation of specific substrates. CBF-β is a newly identified key regulator of Vif function, and more information is needed to further clarify its regulatory mechanism. Here, we comprehensively investigated the functions of EloB (together with EloC) in the Vif-CBF-β-Cul5 E3 ligase complex. Results: The results revealed that: (1) EloB (and EloC) positively affected the recruitment of CBF-β to Vif. Both knockdown of endogenous EloB and over-expression of its mutant with a 34-residue deletion in the COOH-terminal tail (EloBΔC34/EBΔC34) impaired the Vif-CBF-β interaction. (2) Introduction of both the Vif SLQ → AAA mutant (VifΔSLQ, which dramatically impairs Vif-EloB-EloC binding) and the Vif PPL → AAA mutant (VifΔPPL, which is thought to reduce Vif-EloB binding) could reduce CBF-β binding. (3) EloB-EloC but not CBF-β could greatly enhance the folding of full-length Vif in Escherichia coli. (4) The over-expression of EloB or the N-terminal ubiquitin-like (UbL) domain of EloB could significantly improve the stability of Vif/VifΔSLQ/VifΔPPL through the region between residues 9 and 14. Conclusion: Our results indicate that the Vif interaction with EloB-EloC may contribute to recruitment of CBF-β to Vif, demonstrating that the EloB C-teminus may play a role in improving Vif function and that the over-expression of EloB results in Vif stabilization. Keywords: CBF-β, Elongin BC complex, HIV-1, Protein binding, Ubiquitin-protein ligases, Vif Background host proteins (APOBEC3B, APOBEC3DE, APOBEC3G The human immunodeficiency virus type 1/acquired im- and APOBEC3F) [5] are sufficient to confer the non- munodeficiency syndrome (HIV‑1/AIDS) epidemic is a permissive phenotype [6-11]. They possess strong antiviral global health concern. Vif, as an accessory gene of HIV-1, activities based on deamination of retroviral complemen- plays pivotal roles in both the early and late steps of the tary DNA [12]. Vif counteracts the anti-HIV activity mainly HIV-1 life cycle [1-3]. Vif is necessary for efficient viral rep- by recruiting the APOBEC3 proteins (APOBEC3DE, lication in non-permissive cell lines [4], and APOBEC3 APOBEC3G and APOBEC3F) [5], the transcription cofac- tor CBF-β and EloB-EloC to the Cul5-Rbx complex, * Correspondence: [email protected]; [email protected] thereby forming an E3 ubiquitin ligase and ultimately indu- 1National Engineering Laboratory for AIDS Vaccine, College of Life Science, cing the proteasomal degradation of these antiviral host ’ Jilin University, Changchun, Jilin Province, People s Republic of China proteins [10,13-17]. 2Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, College of Life Science, Jilin University, Changchun, Jilin Province, People’s Republic of China © 2013 Wang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Wang et al. Retrovirology 2013, 10:94 Page 2 of 16 http://www.retrovirology.com/content/10/1/94 As the formation of the E3 ubiquitin ligase complex is a protein [49,50]. In the Vif-CBF-β-Cul5 E3 complex, the prerequisite for Vif to neutralize APOBEC3 proteins, the C-terminus of EloB is known to bind to the PPLP motif of functional domains that Vif utilizes to recruit the E3 com- Vif, but without an ascribed function as yet [26]. plex have been well studied [18,19]. The N-terminal re- In this study, we demonstrated that the interactions gion of Vif contains the main sites involved in binding between Vif and EloB-EloC are important for CBF-β re- of CBF-β and APOBEC3 proteins [17,20-25], while its cruitment to Vif. Both knockdown of endogenous EloB (to- C-terminal domain contains a so-called SOCS-box motif, gether with EloC) and over-expression of EloBΔC34, an which is responsible for binding to the EloB-EloC complex EloB mutant with a 34-residue deletion in the COOH- [26-28], and a conserved HCCH zinc coordination site terminal tail, impaired the Vif-CBF-β interaction and stabi- that mediates selective binding to Cul5 [29-32]. Every lized the A3G level. The interaction with EloB-EloC, but component of the Vif-Cul5 E3 ligase is indispensable for not CBF-β, was shown to greatly increase the solubility of degradation of APOBEC3 proteins. Vif acts as a substrate full-length Vif in Escherichia coli, and in cells, both the adaptor molecule to bridge APOBEC3G (A3G) with the introduction of the VifΔSLQ mutant, which dramatically Cul5 E3 ligase [13], and the newly found factor CBF-β is impairs Vif-EloB-EloC binding, and that of the VifΔPPL reported to be a unique regulator of Vif-Cul5 E3 ligase by mutant, which is considered to reduce Vif-EloB binding, promoting folding of Vif [25,33-36]. EloB and EloC are were found to impair Vif-CBF-β binding [33,36]. Moreover, also known as regulatory subunits, whereas Cul5 functions we showed that over-expression of EloB or EloBΔC34 as a scaffold protein [13]. could significantly prevent degradation of Vif/VifΔSLQ/ EloB and EloC are two ubiquitous proteins that form a VifΔPPL, indicating that the EloB N-terminal UbL domain heterodimer complex. They were originally found in the may enhance the stability of Vif in an unknown manner. Elongin (SIII) complex, which was proposed to contain We also found that EloB stabilized Vif mainly through the three subunits: EloA, EloB and EloC [37,38]. EloA is same region (between residues 9 and 14) by which it known as a positive transcriptional elongation factor of interacted with EloC. Overall, our study is helpful in fur- RNA polymerase II, whereas EloB and EloC act as positive ther understanding the interactions among components of regulators of EloA [37,38]. Soon afterward, EloB and EloC the Vif-CBF-β-Cul5 E3 complex, demonstrates that the were identified in many cullin-RING ubiquitin ligase com- C-terminus of EloB, which is not required for EloC binding plexes [13,39-42], forming the cullin E3 core together with and degradation of P53, plays a role in improving Vif cullin family proteins Cul2/Cul5 and a RING finger pro- function and expands the diversity of functions of the UbL tein Rbx [43]. In these E3 ligase complexes, EloB-EloC protein EloB. binds to substrate-targeting subunits via a conserved motif called the BC-box [42-44] in order to mediate poly- Results ubiquitination and ultimately proteasomal degradation Knockdown of endogenous EloB expression impairs of the targeted substrates. These substrate-binding pro- Vif-induced A3G degradation and decreases HIV-1 virion teins, which are characterized by the SOCS-box motif infectivity containing the N-terminal BC-box sequence [42-44], typ- CBF-β and EloB-EloC are reported to regulate Vif function ically include cellular proteins such as von Hippel-Lindau by promoting folding of different domains of Vif (VHL) tumor suppressor [40,45] and the suppressor [25,28,34,51,52], both of which affect Cul5 binding to Vif of cytokine signaling (SOCS) [41,42,46], with correspond- [13,36], while knockdown of CBF-β does not impair EloB- ing substrates HIF-1α [40,45] and Janus kinases (JAKs) EloC binding to Vif [36]. In order to further study the [41,42,46]. The substrate-binding proteins may also be events in the assembly of the Vif-CBF-β-Cul5 complex, we viral proteins, such as adenovirus E4orf6 [39] and HIV-1 attempted, but failed, to obtain a complete knockdown of Vif [13], which target the substrates p53 [39] and the well- EloC (data not shown), similar to the results obtained known APOBEC3 proteins [13], respectively. by Hwang et al. [53]. Therefore, we designed three EloB is a highly conserved cellular factor in humans, EloB-specific small interfering RNAs (siRNAs), designated rats, Drosophila melanogaster and Caenorhabditis elegans siEB1, 2 and 3, to knock down endogenous EloB expres- [47]. As a member of the third class of ubiquitin family sion. Down-regulation of EloB expression in 293 T cells by proteins, it is a 118-amino acid protein composed of an the three siRNAs was confirmed by Western blot analysis 84-amino acid N-terminal (ubiquitin-like) UbL domain 72 h after transfection (Figure 1A). The EloB protein band and a 34-amino acid COOH-terminal tail, both of which intensities in the immunoblot were quantified using are highly conserved [47,48]. The EloB UbL domain is ne- Bandscan software (Glyko, Novato, CA) and normalized cessary and sufficient for binding to EloC [47,48], whereas to tubulin. SiEB1 caused an approximately 60% decrease, its C-terminal tail has been reported to interact with whereas siEB2 caused a modest decrease and siEB3 SOCS2 and SOCS4 proteins, significantly stabilizing the showed nearly no effect, in EloB expression compared SOCS2 E3 complex and assisting folding of the SOCS4 with cells transfected with the negative control siRNA Wang et al.
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