Core-Binding Factor β Increases the Affinity between Human Cullin 5 and HIV-1 Vif within an E3 Ligase Complex

Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry , 601 Elmwood Avenue, Box 712, Rochester, New York 14642, United States.
Biochemistry (Impact Factor: 3.02). 10/2012; 51(44). DOI: 10.1021/bi301244z
Source: PubMed


HIV-1 Vif masquerades as a receptor for a cellular E3 ligase harboring Elongin B, Elongin C, and Cullin 5 (EloB/C/Cul5) proteins that facilitate degradation of the antiretroviral factor APOBEC3G (A3G). This Vif-mediated activity requires human core-binding factor β (CBFβ) in contrast to cellular substrate receptors. We observed calorimetrically that Cul5 binds tighter to full-length Vif((1-192))/EloB/C/CBFβ (K(d) = 5 ± 2 nM) than to Vif((95-192))/EloB/C (K(d) = 327 ± 40 nM), which cannot bind CBFβ. A comparison of heat capacity changes supports a model in which CBFβ prestabilizes Vif((1-192)) relative to Vif((95-192)), consistent with a stronger interaction of Cul5 with Vif's C-terminal Zn(2+)-binding motif. An additional interface between Cul5 and an N-terminal region of Vif appears to be plausible, which has therapeutic design implications.

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    • "Increasing evidence has indicated that knocking down endogenous CBF-b decreases the stability of HIV-1 Vif and that CBF-b overexpression enhances the steady-state level of Vif, thus promoting its function [31]. Some researchers have hypothesized that CBF-b might interact with HIV-1 Vif to decrease its proteasome-dependent degradation, thereby increasing its steady-state level [15] [25]. To evaluate this hypothesis, we transfected HIV-1 Vif expression vectors in the presence or absence of CBF-b overexpression or the proteasome inhibitor MG132 in 293T cells. "
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    ABSTRACT: The Vif (viral infectivity factor) protein of human immunodeficiency virus type-1 (HIV-1) is critical for HIV-1 infectivity. CBF-β is required for HIV-1 Vif function, as it increases the steady-state level of the HIV-1 Vif protein to promote host restriction factor APOBEC3 degradation. However, the precise mechanism by which CBF-β promotes HIV-1 Vif levels remains unclear. In the present study, we provided evidences that CBF-β promoted steady-state levels of HIV-1 Vif by inhibiting the degradation of HIV-1 Vif through the proteasome pathway. Our results reveal a new mechanism by which a cellular protein supports viral infectivity by inhibiting viral protein degradation. Copyright © 2015. Published by Elsevier Inc.
    Preview · Article · Jan 2015 · Biochemical and Biophysical Research Communications
    • "The c value is the product of the equilibrium association constant, K A , the receptor concentration in the cell at the start of the experiment, [M], and the stoichiometry of ligand binding to the receptor, N: c ¼ K A Á M ½ ŠÁN Large c values approaching 500 will result in a very steep inflection point, which precludes a proper fit of K A . Here, values for N and ΔH can still be measured reliably, which is an important consideration if one is assessing heat capacity (ΔC p ) changes (Salter et al., 2012; Sokoloski, Dombrowski, & Bevilacqua, 2012). By contrast, c values less than 10 are associated with flat, featureless curves that do not possess the information necessary to extract K A , N, or ΔH accurately. "
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    ABSTRACT: Riboswitches regulate genes by binding to small-molecule effectors. Isothermal titration calorimetry (ITC) provides a label-free method to quantify the equilibrium association constant, KA, of a riboswitch interaction with its cognate ligand. In addition to probing affinity and specific chemical contributions that contribute to binding, ITC can be used to measure the thermodynamic parameters of an interaction (ΔG, ΔH, and ΔS), in addition to the binding stoichiometry (N). Here, we describe methods developed to measure the binding affinity of various preQ1 riboswitch classes for the pyrrolopyrimidine effector, preQ1. Example isotherms are provided along with a review of various preQ1-II (class 2) riboswitch mutants that were interrogated by ITC to quantify the energetic contributions of specific interactions visualized in the crystal structure. Protocols for ITC are provided in sufficient detail that the reader can reproduce experiments independently, or develop derivative methods suitable for analyzing novel riboswitch-ligand binding interactions.
    No preview · Article · Nov 2014 · Methods in enzymology
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    • "We also showed that a N-terminal truncated (amino acids 99–192) Vif couldn’t precipitate endogenous Cul5 in 293 T cells, although others have demonstrated that a N-terminal truncated Vif has a high binding affinity for Cul5, in vitro[11,33]. However, a group recently reported Cul5 binds to full length Vif with greater affinity when compared to a C-terminal half fragment containing both the HCCH domain and cullin box [12]. Taken together, these reports suggest that Cul5 may interact with or require additional Vif residues in the N-terminus. "
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    ABSTRACT: HIV-1 Vif promotes the degradation of host anti-retroviral factor family, APOBEC3 proteins via the recruitment of a multi-subunit E3 ubiquitin ligase complex. The complex is composed of a scaffold protein, Cullin 5 (Cul5), RING-box protein (Rbx), a SOCS box binding protein complex, Elongins B/C (Elo B/C), as well as newly identified host co-factor, core binding factor beta (CBF-beta). Cul5 has previously been shown to bind amino acids within an HCCH domain as well as a PPLP motif at the C-terminus of Vif; however, it is unclear whether Cul5 binding requires additional regions of the Vif polypeptide. Here, we provide evidence that an amino terminal region of full length Vif is necessary for the Vif-Cul5 interaction. Single alanine replacement of select amino acids spanning residues 25-30 (25VXHXMY30) reduced the ability for Vif to bind Cul5, but not CBF-beta or Elo B/C in pull-down experiments. In addition, recombinant Vif mutants had a reduced binding affinity for Cul5 compared to wild-type as measured by isothermal titration calorimetry. N-terminal mutants that demonstrated reduced Cul5 binding were also unable to degrade APOBEC3G as well as APOBEC3F and were unable to restore HIV infectivity, in the presence of APOBEC3G. Although the Vif N-terminal amino acids were necessary for Cul5 interaction, the mutation of each residue to alanine induced a change in the secondary structure of the Vif-CBF-beta-Elo B/C complex as suggested by results from circular dichroism spectroscopy and size-exclusion chromatography experiments. Surprisingly, the replacement of His108 to alanine also contributed to the Vif structure. Thus, it is unclear whether the amino acids contribute to a direct interaction with Cul5 or whether the amino acids are responsible for the structural organization of the Vif protein that promotes Cul5 binding. Taken together, we propose a novel Vif N-terminal motif that is responsible for Vif recruitment of Cul5. Motifs in Vif that are absent from cellular proteins represent attractive targets for future HIV pharmaceutical design.
    Full-text · Article · Jan 2014 · Retrovirology
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