Research Article

HIV-1 Nef hijacks clathrin coats by stabilizing AP-1:Arf1 polygons

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Science  23 Oct 2015:
Vol. 350, Issue 6259, aac5137
DOI: 10.1126/science.aac5137

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HIV proteins exploit clathrin coats

Clathrin-coated vesicles are involved in the sorting of membrane and cargo at the trans-Golgi network. Clathrin coats exploit adaptor proteins, including AP-1 and AP-2, in selecting their cargoes, assisted by the small membrane-trafficking associated GTPase Arf1. Shen et al. were interested in how the HIV-1 Nef protein affected this process. They leveraged a structure solved in the presence of Nef to show that the AP-1:clathrin coat is far more intricately organized than previously thought. Furthermore, Arf1 played a central structural role in these coated vesicles. It seems that HIV-1 Nef hijacks the clathrin pathway to its own ends through very sophisticated structural perturbations.

Science, this issue p. 10.1126/science.aac5137

Structured Abstract

INTRODUCTION

Clathrin-coated vesicles mediate endocytosis and sorting from the trans-Golgi network (TGN) and endosomes to lysosomes. Adaptor protein (AP) complexes such as AP-1 connect membrane proteins to clathrin. AP-1 needs to be “unlocked” by activators in order to bind cargo and clathrin. The small guanosine triphosphatase Arf1 unlocks AP-1 at the TGN by coupling AP-1:Arf1 dimerization to conformational changes. Major histocompatibility class I (MHC-I) proteins and the viral restriction factor tetherin are normally present on the cell surface. The viruses HIV-1, HIV-2, and simian immunodeficiency virus (SIV) use their Nef proteins to hijack clathrin and AP-1 and thus redirect MHC-I and tetherin to lysosomes.

RATIONALE

The down-regulatory functions of Nef are important for HIV-1 infectivity. Previous structural studies of Nef and AP complexes revealed that Nef only binds to unlocked APs. We sought to determine whether Nef could potentiate the physiological unlocking mechanisms in down-regulation of tetherin and MHC-I. We used Förster resonance energy transfer (FRET) to determine the conformation of AP-1 complexes bound to Arf1, Nef, and cargo. To understand the mechanism by which Nef hijacks AP-1, we determined the structures of trimeric AP-1:Arf1:tetherin-Nef complexes by cryo–electron microscopy (cryo-EM) in active and inactive conformations. We predicted that the active AP-1:Arf1 trimer could form hexagonal lattices, which we visualized directly. To study the function of the hexagons, we reconstituted clathrin cage formation in vitro and showed that mutations in the lattice contacts blocked Arf1- and Nef-promoted cage formation.

RESULTS

In the presence of a tetherin-Nef fusion protein, AP-1:Arf1 complexes became trimeric. FRET analysis revealed that individual AP-1 complexes in the trimer were in the unlocked state. With the use of cryo-EM, we identified two kinds of trimers: closed and open. The closed trimer yielded a 7 Å reconstruction, which allowed docking of known atomic models of unlocked AP-1, Arf1, and Nef. The trimer is centered on a trimeric Arf1 interface. Although AP-1 was unlocked, the closed trimer hides the membrane binding sites and is thus inactive. The open trimer was more mobile, and the structure was resolved to 17 Å. The open trimer preserved the Arf1 trimeric interface while exposing membrane binding sites. Docking the open trimer with the known AP-1:Arf1 dimer yielded a hexagonal model that matched the dimensions of clathrin. The hexagons were visualized in AP-1:Arf1:MHC-I–Nef mixtures. Efficient clathrin cage assembly at neutral pH required Nef and intact Arf1 dimer and trimer interfaces.

CONCLUSION

Although we set out to explain how HIV-1 Nef hijacked the AP-1 complex, we also found that the inner layer of the AP-1 clathrin coat is far more intricately organized than anticipated. AP-1 and its Arf1 binding sites are conserved throughout eukaryotes; thus, the organization of the inner layer is ancient. HIV-1 has taken advantage of this complexity to subvert membrane traffic. The degree to which HIV-1 Nef can drive AP-1 hexagon formation seems to be coupled to its cargo preferences, as Nef recruits MHC-I more effectively than tetherin. Our findings elucidate the structured organization of the inner layer of clathrin coats that is conserved across eukaryotes, as well as the means by which HIV-1 uses Nef to subvert this structure.

HIV-1 Nef, Arf1, and the hexagonal inner layer of an AP-1 clathrin coat.

(Top) Cryo-EM reconstructions of closed (far left) and open (center left) trimers of tetherin–HIV-1 Nef fusion protein, Arf1, and the AP-1 core. Combining the open trimer and known dimer structure leads to a hexagonal model (center right) that matches the dimensions of the clathrin coat (right). (Bottom) Concept for Nef-activated assembly of the AP-1–clathrin coat.

Abstract

The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and adaptive immune defenses by hijacking the adaptor protein 1 (AP-1) and AP-2 clathrin adaptors. We found that HIV-1 Nef and the guanosine triphosphatase Arf1 induced trimerization and activation of AP-1. Here we report the cryo–electron microscopy structures of the Nef- and Arf1-bound AP-1 trimer in the active and inactive states. A central nucleus of three Arf1 molecules organizes the trimers. We combined the open trimer with a known dimer structure and thus predicted a hexagonal assembly with inner and outer faces that bind the membranes and clathrin, respectively. Hexagons were directly visualized and the model validated by reconstituting clathrin cage assembly. Arf1 and Nef thus play interconnected roles in allosteric activation, cargo recruitment, and coat assembly, revealing an unexpectedly intricate organization of the inner AP-1 layer of the clathrin coat.

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