Fab-based inhibitors reveal ubiquitin independent functions for HIV Vif neutralization of APOBEC3 restriction factors

Abstract

The lentiviral protein Viral Infectivity Factor (Vif) counteracts the antiviral effects of host APOBEC3 (A3) proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs) to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity.

Conformational dynamics of the HIV Vif protein complex

“…While it is possible that all APOBEC3 proteins could rigidify the VCBC complex to the same extent and in the same conformation, other intrinsically disordered proteins have been demonstrated to adopt different conformations with their multiple binding partners (58,59). Recent work by Binning et al also showed that Vif disrupts the APOBEC3 packaging into virions by a degradation-independent mechanism that occurs when CUL5 is prevented from binding to the VCBC complex (19 which all simulations were initiated. c) VCBC structure (Vif is shown in pink, CBF- in green, of VCBC complex bound to dT14 in 50 mM NaCl (red) corresponds to an experimental molecular mass of 135 kDa, which is consistent with the theoretical molecular weight for a monomer of 66 kDa, indicating that the complex is a dimer.…”

“…The VCBC complex, consisting of consensus Vif (19), CBF- (residues 1-165), EloB (residues 1-118), and EloC (residues 17-112), was co-expressed in E. coli BL21 DE3 Star cells as described (19). Briefly, cells were grown to mid-log phase at 37 C followed by induction with To express the VCBC complex with 13 C isotope labels on the methyl groups (ILVMA) for NMR spectroscopy, the BL21 DE3 Star cells were grown in M9 minimal media with trace metals (36).…”

“…A solubility-optimized form of the A3F-C-terminal domain with 11 mutations (A3Fctd-11x) was expressed in E. coli BL21 DE3 Star cells as described (19,37). The sample was purified by nickel chromatography and the His-tag was cleaved with TEV then passed again over the nickel column to remove the tag.…”

“…The envelope calculated by SAXS was more elongated than the VCBC complex bound to CUL5 in the crystal structure, indicating that the VCBC complex alone can sample more extended conformations. A recent electron microscopy (EM) study of VCBC bound to antibody antigen-binding fragments (Fabs) also indicates flexibility in the VCBC complex, likely caused by the hinge at the linker region between the two Vif domains(19). The magnitude of conformation heterogeneity of VCBC we observe by MD and NMR would likely make EM of the VCBC complex alone intractable, but the binding of the Fabs stabilizes the complex enough that Binning et al were able to classify the EM micrographs into two structurally similar EM maps(19).…”

“…A recent electron microscopy (EM) study of VCBC bound to antibody antigen-binding fragments (Fabs) also indicates flexibility in the VCBC complex, likely caused by the hinge at the linker region between the two Vif domains(19). The magnitude of conformation heterogeneity of VCBC we observe by MD and NMR would likely make EM of the VCBC complex alone intractable, but the binding of the Fabs stabilizes the complex enough that Binning et al were able to classify the EM micrographs into two structurally similar EM maps(19). Overall, data from experiments and our simulations indicate that while the HIV-1 Vif protein can fold and form a stable complex with only EloB, EloC and CBF-β, additional binding partners further restrict Vif's conformational landscape.Previous MD simulations on a human CRL complex by Liu and Nussinov revealed conformational changes within the linker between the two domains of the substrate binding protein that correlated with changes in distance between the E2 ligase and substrate, which are critical for ubiquitin transfer…”