Vif hijacks CBF-β to degrade APOBEC3G and promote HIV-1 infection

Abstract

Restriction factors, such as the retroviral complementary DNA deaminase APOBEC3G, are cellular proteins that dominantly block virus replication. The AIDS virus, human immunodeficiency virus type 1 (HIV-1), produces the accessory factor Vif, which counteracts the host's antiviral defence by hijacking a ubiquitin ligase complex, containing CUL5, ELOC, ELOB and a RING-box protein, and targeting APOBEC3G for degradation. Here we reveal, using an affinity tag/purification mass spectrometry approach, that Vif additionally recruits the transcription cofactor CBF-β to this ubiquitin ligase complex. CBF-β, which normally functions in concert with RUNX DNA binding proteins, allows the reconstitution of a recombinant six-protein assembly that elicits specific polyubiquitination activity with APOBEC3G, but not the related deaminase APOBEC3A. Using RNA knockdown and genetic complementation studies, we also demonstrate that CBF-β is required for Vif-mediated degradation of APOBEC3G and therefore for preserving HIV-1 infectivity. Finally, simian immunodeficiency virus (SIV) Vif also binds to and requires CBF-β to degrade rhesus macaque APOBEC3G, indicating functional conservation. Methods of disrupting the CBF-β-Vif interaction might enable HIV-1 restriction and provide a supplement to current antiviral therapies that primarily target viral proteins.

Figure 1. AP–MS experiments identify CBF-β as a Vif-dependent component of the Vif–CUL5 ubiquitin ligase complex

a, Flow-chart of the proteomic analysis performed during the study. b, Affinity-tagged versions of Vif, Vpu and Vpr were purified using 3×Flag from HEK293 and Jurkat cells, subjected to SDS–PAGE and stained with silver. Visible bands corresponding to interactions that are known for each accessory factor are labelled. Note Vif and CBF-β run at a similar place on the gel. Tagged versions of Vpr and Vpu were used as specificity controls. c, A network representation of Vif–host protein–protein interactions from both HEK293 (blue) and Jurkat T cells (red) after subjecting the data derived from the AP–MS analysis to the MiST scoring system. The intensity of the node colours corresponds to the quantitative MiST score. Blue edges represent interactions derived during this work; black edges are previously described interactions between host factors; dashed edges correspond to previously described Vif–host interactions present in the database VirusMint. d, The double purification approach, which allows for the identification of stable, stoichiometric protein complexes. e, Double purifications were performed in triplicate using 3×Flag-tagged CUL5, A3G or CBF-β with 2×Strep-tagged Vif in HEK293 cells. Proteins that were identified in all three double purifications, after trypsin digestion and analysis by mass spectrometry, are represented. The coverage corresponds to the percentage of protein identified by tryptic peptides. f, Immunoblots showing that Vif recruits CBF-β to theCUL5/ELOBC/RBX2 ubiquitin ligase complex. HA-tagged ELOB or CUL5 were immunoprecipitated in the absence or presence of increasing amounts of Vif, and endogenous CBF-β was monitored by immunoblot. g, HIV and SIV Vif co-immunoprecipitate CBF-β and ELOC. GFP and HIV Nef were analysed in parallel as specificity controls.

Figure 2. CBF-β is a stoichiometric component of the Vif E3 ubiquitin ligase

a, Size exclusion chromatography of recombinant purifiedCUL5/RBX2 (blue) overlaid with CUL5/RBX2 mixed with 1.5 equivalents of purified Vif substrate adaptor containing Vif, ELOBC and CBF-β (red). b, Coomassie-stained SDS–PAGE of fractions labelled 1–3 in a indicating the Vif substrate adaptor and a six-protein assembly (CRL5–Vif–CBF-β) co-purify as stable monodisperse species. c, A3G, but not A3A, directly binds the tetrameric Vif substrate adaptor in pull-down experiments in vitro. d, CRL5–Vif–CBF-β is an E3 ligase that promotes polyubiquitination of A3G, but not A3A (detected using an anti-c-Myc antibody to the C-terminal tag on the deaminases). Ub, ubiquitin. e, CRL5–Vif–CBF-β and UBE2R1 catalyse formation of K48-linked chains on A3G. Immunoblots showing substrate in ubiquitination reactions containing UBE2R1 as E2, no ubiquitin, Me-ubiquitin, K48R-ubiquitin, K48-only ubiquitin or wild-type ubiquitin. Reactions with Me-ubiquitin indicate at least two distinct sites are modified on A3G; K48R recapitulates the pattern observed with Me-ubiquitin, whereas both wild type and K48R-only ubiquitin result in extensive polyubiquitin chains.

Figure 3. CBF-β and Vif collaborate to degrade APOBEC3G and enable HIV-1 infectivity

a, CBF-β-depleted HEK293T cells have lower steady-state Vif levels, which recover upon treatment with 2.5 μM MG132. b, Infectivity of replication-competent, Vif-proficient HIV-1 in the presence and absence of CBF-β and A3G (n = 3; mean, s.d.). Immunoblots are shown for the indicated proteins in virus-producing cells and viral particles. c, Infectivity of HIV-GFP produced using HEK293T-shCBF-β or HEK293T-shControl clones transfected with the single-cycle virus cocktail, A3G, Vif and CBF-β as indicated (n = 3; mean, s.d.). The corresponding immunoblots are shown below. d, Infectivity of a Vif-deficient HIV-1 molecular clone produced in the presence or absence of human or rhesus A3G-HA, HIV or SIV Vif–Myc, and CBF-β as indicated (n = 3; mean, s.d.). Immunoblots are shown for the indicated proteins in virus-producing cells and viral particles with two exposures of the anti-Myc (Vif) blot shown to clarify the SIV Vif signal (the longer exposure also shows endogenous c-Myc).

Figure 4. Model for Vif–CBF-β E3 ligase formation and APOBEC3G polyubiquitination and degradation

Vif is depicted hijacking cellular CBF-β to the E3 ubiquitin ligase complex required for A3G polyubiquitination and degradation. Vif may recruit newly translated CBF-β (not shown) and/or hijack existing CBF-β from RUNX transcription complexes.