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
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.
Available from: sciencedirect.com
- "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 . 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  . 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.
Biochemical and Biophysical Research Communications 01/2015; 457(3). DOI:10.1016/j.bbrc.2015.01.001 · 2.30 Impact Factor
Available from: Xiao-Fang Yu
- "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 . 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.
Retrovirology 01/2014; 11(1):4. DOI:10.1186/1742-4690-11-4 · 4.19 Impact Factor
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ABSTRACT: HIV has several accessory proteins (Vif, Vpu, Vpr, Vpx, and Nef) along with structural /enzymatic (Gag, Pol, and Env) and gene-expression regulatory proteins (Tat and Rev) essential for viral replication. The accessory proteins are neither required in some kinds of cells and nor all conserved between HIV-1 and HIV-2. For these reasons, their functional roles and mechanisms had been unclear. However, since a finding of Vif's neutralizing function against host restriction factor APOBEC3G, it has been elucidated that the accessory proteins play critical roles to antagonize host intrinsic antiviral activity. So far, in addition to Vif-APOBEC3, Vpu-BST-2/Tetherin and Vpx-SAMHD1 have been identified as such examples. Here, we summarize the biological functions and features on HIV accessory proteins in terms of antagonizing factors against the host antiviral factors.
Uirusu 01/2013; 63(2):187-98. DOI:10.2222/jsv.63.187
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