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.
"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. "
"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. "
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Proteins of the ankyrin repeat and SOCS-box (ASB) family act as substrate recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyse the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. ASB multimeric complexes are therefore involved in numerous cell processes and pathways, however, their interactions, assembly and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC) and Cullin 5. Here we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization and ion mobility-mass spectrometry (IM-MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9-EloBC-Cul5NTD quaternary complex is revealed by molecular modelling and is consistent with IM-MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to other CRL systems, and provide a platform for further molecular investigation of this important protein family. Data reported here will also be of use in the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.
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