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. 2018 Jul 26;174(3):659-671.e14.
doi: 10.1016/j.cell.2018.07.004.

HIV-1 Nefs Are Cargo-Sensitive AP-1 Trimerization Switches in Tetherin Downregulation

Free PMC article

HIV-1 Nefs Are Cargo-Sensitive AP-1 Trimerization Switches in Tetherin Downregulation

Kyle L Morris et al. Cell. .
Free PMC article


The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, HIV-1 Nef, and the cytosolic tail of the restriction factor tetherin suggested a mechanism for inactivating tetherin by Golgi retention. The 4.3 Å structure of a mutant Nef-induced dimer of AP-1 showed how the closed trimer is regulated by the dileucine loop of Nef. HDX-MS and mutational analysis were used to show how cargo dynamics leads to alternative Arf1 trimerization, directing Nef targets to be either retained at the trans-Golgi or sorted to lysosomes. Phosphorylation of the NL4-3 M-Nef was shown to regulate AP-1 trimerization, explaining how O-Nefs lacking this phosphosite counteract tetherin but most M-Nefs do not. These observations show how the higher-order organization of a vesicular coat can be allosterically modulated to direct cargoes to distinct fates.

Keywords: HIV; HIV-Nef; adaptor protein; clathrin; cryo-EM; trafficking.

Conflict of interest statement

Declaration of Interests

The authors declare no competing interests.


Figure 1.
Figure 1.. Atomic model of the AP1:Arf1:tetherin-Nef closed trimer.
(A) The closed trimeric state consists of three copies of γΑrf1 assembling three AP-1 cores with one copy of HIV-Nef at each AP-1 dimer interface. The subunit reconstruction is shown in the context of the transparent whole trimer reconstruction. The AP-1 core and γArf1 subunits are sharpened at −50 Å2, Nef and βArf1 subunits are unsharpened and the transparent whole closed trimer low pass filtered to 18 Å is shown for clarity. (B) The trimer subunit contains the AP-1 core, two copies of Arf1 (γArf1 and βArf1) and one copy of Tetherin-HIV-Nef. A 2D class, corresponding volume slice and projection are shown for reference. (C) A subunit schematic for the closed trimer showing interaction interfaces and the break from perfect three-fold symmetry of the complex allowing movement of the cargo Nef trans AP-1 bridge. (D) A model for the closed trimer docked at the trans golgi network. See also Figure S1-3.
Figure 2.
Figure 2.. Nef bridging via the dileucine loop stabilizes the closed trimer.
(A) An AP-1:µ1:Tetherin:Nef sandwich stabilises the Nef core to AP-1:µ1. (B) The canonical Nef acidic patch binds directly to AP-1:µ1. (C) The ExxxLL motif of Nef binds to AP-1σ1 (D) These binding regions of Nef are found on the unstructured loops L1 and L4 but are stabilized in the trimeric assembly. (E) Consequently, the trimer architecture is closed by HIV-Nef at the AP1 trimeric dimer interface. (F) Mutating the dileucine loop, tetherin-NefLL164–165AA, responsible for binding AP-1:σ1 results in closed trimer destabilization and dimer formation as indicated by SEC. (G) The dileucine mutant adopts a dimeric oligomeric state as confirmed by cryo-EM. A 2D class, corresponding volume slice and projection are shown for reference. (H) The unstable trimer created by the knockout of the AP-1:σ1:LL interaction leads to a rearrangement into dimers driven by new βArf1:AP-1:γ interactions. See also Figure S1-3 and 4.
Figure 3.
Figure 3.. γArfl and βArf1 control AP-1 oligomerization in a cargo dependent manner.
(A) The binding loops of the closed AP-1 trimer are mediated through γArf1 loop 9 - loop 3 contacts. (B) Conversely the trimerisation architecture observed in COPI assemblies exhibits a rearrangement to utilize loop 9 - loop 5 contacts. (C) The counterclockwise turn of the Arf1 assemblies allows these new contacts to be made, yet both still utilize loop 9. In the COPI-like Arf1 L9–5 trimeric architecture, assembly via βArf1 is sterically plausible. (D) In the presence of tetherin-Nef cargo, Arf1 dependent trimerization of AP-1 is dependent on γArf1 but not βArf1. (E) In the presence of MHC- I-Nef cargo, AP-1 may trimerize via both γArf1 or βArf1. Negative stain EM 2D class averages and SEC support that an assembly in the presence of MHC-I-Nef cargo thus may be assembled via βArf1 trimerization. (F) The knockout of the γArf1:AP-1:γ interface completely destabilizes tetherin trimers to monomer, but does not disrupt MHC- I assembly in the same manner. (G) The knockout of the βArf1:AP-1:β1 does not affect the tetherin trimer assembly. In the case of MHC-I trimers, assembly via γArf1 leads to a closed trimer like state. Critically, MHC-I trimers can be seen assemble via both γArf1 and βArf1 oligomerization. See also Figure S4.
Figure 4.
Figure 4.. Cargo modulates Nef dynamics.
(A) Mapping of the changes in deuteration levels of Nef between tetherin-Nef:AP1:µ1-CTD and MHC-Nef:AP-1:µ1-CTD. The HDX-MS Difference at 10s is overlaid onto the cryoEM structure of the AP- 1:Arf1:tetherin-Nef trimer. (B) Mass spectra of the peptide from Nef (66–80) in tetherin- Nef (orange) or MHC-Nef (blue) upon AP-1:µ 1-CTD binding. A Gaussian fit is used to represent the distribution of peak heights of the ion peaks across the m/z values, and overlay with mass spectra (straight line). (C) The stronger binding of Nef to AP-1:µ 1 in the presence of MHC-I cargo may prevent Nef from reaching its AP-1:σ1 binding site. See also Figure S5.
Figure 5.
Figure 5.. Phosphorylation of NL4–3 Nef by CK1 disrupts the interaction between AP-1 σ1 and Nef dileucine motif.
(A) Amino acid alignment of Nef proteins in the D/ExxxLL motif region. The dileucine motifs that are conserved in all Nef strains are colored in blue. The residues aligned with NL4–3 Nef Ser169 predicted CK1 phosphorylation site (Hornbeck et al., 2015) are highlighted in gray. (B) Collision- induced dissociation (CID) fragmentation spectrum of the phosphorylated peptide in tetherin-Nef treated by CK1. Ser169 in NL4–3 Nef is phosphorylated. (C) GST pull down result suggest that Nef phosphorylation by CK1 largely reduces Nef dileucine motif binding to AP-1. AP-1 coreµCTD-GST is selected to represent the interaction with the Nef dileucine motif. The phosphorylation of Nef largely reduces the binding to AP-1coreµCTD- GST on the beads. (D) Gel filtration and SDS gel analysis showing the contents of trimer peak. The phosphorylation of NL4–3 Nef disrupts the closed trimer assembly. (E) The effect of Nef differing in the Ser phosphorylation site on infectious virus release in the presence of tetherin. Infectious virus yield from 293T cells co-transfected with an HIV-1 NL4–3 ΔVpuΔNef construct and vectors expressing the indicated nef alleles or human tetherin was determined by infection of TZM-bl cells (right). Data show mean percentages (± SEM) relative to those detected in the absence of tetherin (100%) obtained in five independent experiments. Results obtained for NL4–3 Vpu are shown for comparison. The bar diagrams show the increase in virus production in the presence of Nef compared to the vector control. Stars refer to the difference from the EGFP control panel. *p < 0.05, **p < 0.01.
Figure 6.
Figure 6.. Model for allosteric regulation of Nef target fate.
Tetherin cargo loaded AP- 1:Arf1 trimers are retained at the trans golgi network in a trimeric architecture not competent to assemble into higher species and thus recruit clathrin. MHC-I cargo loaded AP-1:Arf1 trimers may assemble through allosterically induced architectural and assembly changes that permits higher order assembly, clathrin recruitment and trafficking to the lysosome. See also Figure S6.

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