Studies On The Structure And The Interaction Mechanism Of Vif-CBF-β-ElonginB-ElonginC-Cullin5E3Complex

HIV-1is the causative agent of the acquired immunodeficiency syndrome (AIDS).As one of the six HIV-1accessory gene, Vif encodes a highly basic23kDaphosphoprotein that functions in both early and late steps of the retroviral life cycle. Vif(virion infectivity factor) is necessary for the production of infectious virus and efficientviral infection in non-permissive cell lines. The requirement for the vif gene in efficientHIV-1replication is tightly linked to the phenotype of the virus-producing cell. HIV-1lacking vif can efficiently replicate in permissive cell lines but not in non-permissivecells. CEM15, now usually known as human APOBEC3G(A3G) which is present onlyin nonpermissive cells confering a nonpermissive phenotype, was identified as ananti-HIV host factor in2002. It can inhibit early stages of the retrovirus life cycle.APOBEC3G functions by incorporation into various budding viruses (includingHIV-1/HIV-2, equine infectious anemia virus and simian immunodeficiency viruses etal) and subsequently triggers hypermutations by massive deamination within thenascent retroviral minus-strand cDNA, causing many missense and nonsense codonchanges.Vif antagonizes the antiviral activity of human cytidine deaminase APOBEC3proteins confering the non-permissive phenotype by tethering A3G to theVif-CBF-β-ElonginB-ElonginC-Cullin5-Rbx (Vif-CBF-β-EloB-EloC-Cul5-Rbx) E3complex to induce its proteasomal degradation. The functional domains that Vif utilizesto recruit the E3complex have been well studied: The N-terminal region of Vif containsthe main sites involved in binding of CBF-β and APOBEC3proteins; while itsC-terminal domain contains a so-called SOCS-box motif, which is responsible forbinding to the EloB-EloC complex, and a conserved HCCH zinc coordination site thatmediates selective binding to Cul5. Every component of the Vif-Cul5E3ligase is indispensable for degradation of APOBEC3proteins. Vif acts as a substrate adaptormolecule to bridge A3G with the Cul5E3ligase, and the newly found factor CBF-β isreported to be a unique regulator of Vif-Cul5E3ligase by promoting folding of Vif,EloB and EloC are also known as regulatory subunits, whereas Cul5functions as ascaffold protein. Though the biochemical data is rapidly accumulated, the structural dataon HIV-1Vif protein is insufficient so far. The crystal structure of the HIV-1VifBC-box-ElonginB-ElonginC complex has been determined in2008. More structuralinformation to investigate the relationship between the structure and the function of Vifis still waiting to be explored. CBF-β is a newly identified key regulator of Vif functionand more information is needed to further clarify its regulation mechanism.Our study focused on the structure of Vif-EloB-EloC and the mechanism of theinteractions among the components of Vif E3complex. We tried various ofVif-EloB-EloC truncations to attempt to further analyse Vif-EloB-EloC structure byX-ray. For the purpose of obtaining the most direct informations to guide thetherapeutic drug design, we comprehensively investigated the interactions of thecomponents of the Vif-Cul5E3ligase complex by using solubility analysis， RNAinterference, co-immunoprecipitation assay, cycloheximide stability assay, viralinfectivity assay and the critical motif mutation et al.In the structure analysis, by using a modified vector PMR1and a polycistronicco-expression plasmid pST39, We constructed and expressed Vif protein as well as aseries of soluble protein complex of EloC-EloB-Vif；EloC-EloB-Vif-Cul511-191；EloC-EloB-Vif-Cul51-159；EloC-EloB-Vif-Cul51-230；EloCΔN17-EloB-VifΔN97；EloCΔN17-EloBΔC20-VifΔN97in BL21E. coli cells. All of these proteins had beenisolated for growing crystals. The purities of Vif，EloCΔN17-EloB-VifΔN97complexand EloCΔN17-EloBΔC20-VifΔN97complex were even>95%. We obtained a singlecrystal of Vif which diffrated to1.6resolution, however, the structrue could not beresolved because there was not any known protein structural motif in Vif and becausethe failure of getting the selenomethionine-incorporated protein. Then we carried out mild trypsin treatment to the EloCΔN17-EloB-VifΔN97complex to remove thefractions with low compactness and high flexibility in the backbone. After the cleavagereaction, the cleaved complexes were isolated from the reaction mixture by a two-steppurification: Ion exchange-Gel filtration, for the purpose of getting compactly foldedglobulin complexes. This cleaved complex could be crystallized and the crystals diffractto2.40resolution. However, so far we are unable to determine the structure of thetrypsin-treated HisVifΔN97-EloB-EloCΔN17complex in this research because thediffraction data could not be indexed since the crystals are twin, consequently lacking ofthe data parameters. Refinements of the crystallization condition is in progress bychanging the crystallization temperature, concentration of precipitant, buffer pH, proteinsolution concentration, drop size, and additives in order to get single crystals.SDS-PAGE and Western blot analysis showed that after incubation with trypsin,HisVifΔN97had a decreased molecular weight about4-5kDa whereas ElonginB andElonginCΔN17had almost no changes. The Vif fragment in the cleaved complex wasthen identified by LC-MS/MS, the result indicated the peptide of Vif C-terminal123-168or more amino acid residues which in complex with ElonginB and ElonginCwas well folded or bound compactly to ElonginB-ElonginC therefore shielded frommild tryptic attack, revealing the folding core in Vif C-terminal. We also found that thecleaved complex showed a increase in thermal stability by using turbidity analysis,analyzing the folding of Vif C-terminal domain from a new perspective.About function analysis of the E3complex, in an attempt to comprehensivelyinvestigate the components functions of the Vif-CBF-β-EloB-EloC-Cul5E3ligase, weknocked down endogenous EloB expression by using siRNA. Major findings were asfollows:(1) We found it was consistent with the existing results obtained by Hwang thatthe levels of EloC were greatly reduced in EloB-silencing cells, confirmed again thatEloB formed an obligate heterodimer with EloC.(2) Knock-down of endogenous EloBexpression impaired Vif-induced A3G degradation and the ability of Vif to block theincorporation of A3G into the budding virus. Silencing of endogenous EloB could also impair the ability of Vif to neutralize the antiviral activity of A3G.(3) To explore themechanism used by EloB to affect Vif function, we examined the interactions of HIV-1Vif with its substrate A3G and its cognate partners, endogenous Cul5, EloB/EloC andCBF-β in the condition of EloB silencing. We found that EloB silencing (together withEloC) could reduce Vif binding to CBF-β and Cul5, whereas binding to A3G seemednot be affected.(4) We used a VifΔSLQ mutant which dramatically impairsVif-EloB-EloC binding and found that there was almost no CBF-β interaction withVif-SLQ.(5) We constructed a mutant with a deletion of the34amino acids in theC-terminal tail which was reported to interact with Vif, EBΔC34. The Vif-mediateddepletion of A3G was impaired by co-expression of EBΔC34, and this effect ofEBΔC34on Vif function was dose-dependent, the presence of EBΔC34also interferedwith the ability of Vif to counteract the A3G antiviral activity.(6) Since EloB wasreported to interact with the PPLP motif of Vif, we wondered that whether thisinteraction could also affect CBF-β binding to Vif, to test this hypothesis, We used aVifΔPPL mutant and found that VifΔPPL indeed appeared to be less able to bindCBF-β.(7) We found that it was EloB-EloC but not CBF-β could greatly enhance thefolding of full-length Vif in Escherichia coli, and solubilities of Vif and CBF-β weresignificantly increased only when they were co-expressed with both EloB and EloC. Inconclusion, our results demonstrate that the interactions between HIV-1Vif and humanEloB-EloC are important for CBF-β binding to Vif. Moreover, we found that free EloBcan stabilize Vif via its UbH domain, further expanding the diversity of functions ofUbL proteins.Our studies provide a material basis for resolving the molecular structure of theVif-EloB-EloC interaction interface，extend the current understanding of the functionand mechanism of the Vif-CBF-β-Cul5E3ligase and offer new possibilities for therapeutic applications.