How HIV Nef Proteins Hijack Membrane Traffic To Promote Infection

Abstract

The accessory protein Nef of human immunodeficiency virus (HIV) is a primary determinant of viral pathogenesis. Nef is abundantly expressed during infection and reroutes a variety of cell surface proteins to disrupt host immunity and promote the viral replication cycle. Nef counteracts host defenses by sequestering and/or degrading its targets via the endocytic and secretory pathways. Nef does this by physically engaging a number of host trafficking proteins. Substantial progress has been achieved in identifying the targets of Nef, and a structural and mechanistic understanding of Nef's ability to command the protein trafficking machinery has recently started to coalesce. Comparative analysis of HIV and simian immunodeficiency virus (SIV) Nef proteins in the context of recent structural advances sheds further light on both viral evolution and the mechanisms whereby trafficking is hijacked. This review describes how advances in cell and structural biology are uncovering in growing detail how Nef subverts the host immune system, facilitates virus release, and enhances viral infectivity.

Keywords: Nef; antiviral restriction factors; host-pathogen; human immunodeficiency virus; immune receptors; lentiviruses; protein structure; simian immunodeficiency virus; trafficking.

FIG 1

Nef downregulates host factors. Schematic representation of the clathrin machinery hijacked for CD4 (red), tetherin (green), MHC-I (blue), and SERINC3/5 (teal) downregulation by HIV-1 Nef. These host factors are synthesized in the ER and transported to the Golgi apparatus and then the TGN. From the TGN, these proteins are shuttled to the plasma membrane (PM) via the constitutive secretory pathway where they engage in viral restriction and immune signaling. For both SIV and HIV, Nef-induced downregulation of CD4 and SERINCs occurs at the PM by linking these host factors to AP-2. For the case of CD4, it is believed that a tripartite assembly of Nef-CD4-AP-2 is formed. For SERINCs, this is not known. The Nef-AP-2 assembly triggers clathrin recruitment and budding of CD4 and SERINC3/5 into clathrin-coated pits (CCPs) and eventual budding into clathrin-coated vesicles (CCVs). These CCVs are then routed into a lysosomal degradation pathway. SIVs utilize the same pathway to downregulate simian tetherin. Both SIV and HIV Nefs downregulate newly synthesized MHC-I molecules by rerouting them into the endolysosomal system. This involves the formation of a tripartite assembly of Nef-MHC-I-AP-1 at the TGN membrane, which sequesters MHC-I molecules into CCVs. These CCVs are targeted to MVBs that will eventually fuse with the lysosomes, leading to MHC-I degradation. O-type HIV-1 Nef also targets human tetherin at the TGN similarly to targeting MHC-1, but the result is a Golgi accumulation mechanism of downregulation where Nef traps tetherin at the TGN in an AP-1-dependent manner. Nef-induced downregulation at the TGN is dependent on the formation of Arf1-mediated open and closed trimer of AP-1 for engagement with MHC-I and tetherin, respectively.

FIG 2

Nef-dependent downregulation of host factors is dependent on clathrin adaptor proteins AP-2 and AP-1. (A) Locked AP-2 interacts with phosphatidylinositol 4,5-bisphosphate (PIP₂) at the plasma membrane (PM) where it unlocks to expose its tyrosine (YxxΦ) and dileucine (ExxxLL) cargo binding sites. Nef can interact with unlocked AP-2 and specific host factors at these sites to force the downregulation of the targeted host factor. The host factors targeted by Nef include CD4, CD8, CD3, CD28, SERINC3/5, and simian tetherin. Nef accomplishes this by inducing clathrin-mediated endocytosis (CME) and shuttling the host factor into the lysosomal degradation pathway. (B) Locked AP-1 interacts with Arf1 at the trans-Golgi network (TGN), where it unlocks and forms dimers. Upon binding cargo and Nef at its cargo binding sites, AP-1 can form either closed or open trimers, depending on the specific cargo and the type of Nef, which is centered at a trimeric Arf1 interface. Host factors targeted at the TGN by Nef include MHC-I and human (Hs) tetherin. Closed and open trimers represent the distinct Nef-induced downregulation mechanisms of TGN accumulation and lysosomal degradation, respectively. In the case of the Nef-induced lysosomal degradation mechanism, with additional engagement of unlocked AP-1 dimers, Nef-bound open trimers can form a large hexagonal lattice which matches the hexagonal lattice of clathrin. This lattice binds clathrin to form CCVs and shuttles the bound cargo into the lysosomal degradation pathway.

FIG 3

Nef sites of interaction responsible for host factor downregulation and engagement with host trafficking machinery. Ribbon diagram of a composite structure of HIV-1 NL4-3 (orange). Residues 5 to 24 and 55 to 79 are from PDB ID 4EMZ, and residues 80 to 203 are from PDB ID 4NEE. Composite structure was built by aligning the Nef core of the two PDBs and combining them into a single PDB in Chimera. Where no structural data are available, the disordered regions are represented as dashed lines. For clarity, Nef residues 35 to 65 are represented by a single disordered region though they overlap the ₅₇WL₅₈ and ₆₂EEEE₆₅ sites of interaction. Sites of host protein engagement are highlighted with various colors, and their interacting residues are modeled. Human is abbreviated Hs. Sites that directly bind host factors, resulting in their downregulation, are in red text. Direct AP-binding sites required for host factor downregulation are in purple text; the labels indicate which AP binds at the highlighted site, followed by which host factors are downregulated as a result. Sites implicated in cargo binding, scaffolding, and other non-AP interactions in host substrate downregulation are in blue text. Putative Nef engagement sites of nonclathrin trafficking machinery, direct or indirect, are indicated in green text; the interacting protein is listed, followed by the trafficking machinery affected by binding. The N terminus (NT) and C terminus (CT) of Nef are labeled for reference.

FIG 4

Structures of Nef bound to AP-2 or AP-1 and host factors targeted for downregulation. Central composite structures of AP-2 and AP-1 were composed by aligning PDB IDs 6CM9, 4EMZ, 4NEE, and 6OWT. In this composite structure, Nef is bound at both the tyrosine and dileucine binding sites. The sites where Nef binds host factors CD4 (PDB ID 5NUI), CD3ζ (PDB ID 3IK5), and simian tetherin (6OWT) are indicated for the dileucine binding site of AP-2. The AP-2 β2 subunit is indicated to highlight the formation of the N-terminal β-hairpin upon binding to simian tetherin. The sites where Nef binds host factors MHC-I (PDB ID 4EMZ) and human tetherin (6CM9) are indicated for the tyrosine binding site of AP-1. All positions of the host factors were determined by aligning the Nef core of the respective PDBs to the Nef cores of PDB ID 4NEE (AP-2 interacting partners) and PDB ID 4EMZ (AP-1-interacting partners).