Christopher Aiken

Nef induces CD4 endocytosis: Requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain

CD4 is crucial for antigen-driven helper T cell signaling and is used as receptor by the human immunodeficiency virus (HIV). The HIV early protein Nef causes a loss of CD4 from cell surfaces through a previously undefined posttranscriptional mechanism. Here, we demonstrate that Nef acts by inducing CD4 endocytosis, resulting in its degradation in lysosomes. CD4 down-regulation is strongly enhanced by the association of Nef with cell membranes through myristoylation. The study of chimeric molecules reveals that 20 membrane-proximal residues of the CD4 cytoplasmic domain are sufficient to confer Nef sensitivity. Within this region, a dileucine motif, reminiscent of an endocytosis and lysosomal targeting signal found in the CD3 gamma and delta chains, is crucial for CD4 response to Nef.

Mature HIV-1 capsid structure by cryo-electron microscopy and all-atom molecular dynamics.

Retroviral capsid proteins are conserved structurally but assemble into different morphologies. The mature human immunodeficiency virus-1 (HIV-1) capsid is best described by a ‘fullerene cone’ model, in which hexamers of the capsid protein are linked to form a hexagonal surface lattice that is closed by incorporating 12 capsid-protein pentamers. HIV-1 capsid protein contains an amino-terminal domain (NTD) comprising seven α-helices and a β-hairpin, a carboxy-terminal domain (CTD) comprising four α-helices, and a flexible linker with a 310-helix connecting the two structural domains. Structures of the capsid-protein assembly units have been determined by X-ray crystallography; however, structural information regarding the assembled capsid and the contacts between the assembly units is incomplete. Here we report the cryo-electron microscopy structure of a tubular HIV-1 capsid-protein assembly at 8 Å resolution and the three-dimensional structure of a native HIV-1 core by cryo-electron tomography. The structure of the tubular assembly shows, at the three-fold interface, a three-helix bundle with critical hydrophobic interactions. Mutagenesis studies confirm that hydrophobic residues in the centre of the three-helix bundle are crucial for capsid assembly and stability, and for viral infectivity. The cryo-electron-microscopy structures enable modelling by large-scale molecular dynamics simulation, resulting in all-atom models for the hexamer-of-hexamer and pentamer-of-hexamer elements as well as for the entire capsid. Incorporation of pentamers results in closer trimer contacts and induces acute surface curvature. The complete atomic HIV-1 capsid model provides a platform for further studies of capsid function and for targeted pharmacological intervention.

Formation of a Human Immunodeficiency Virus Type 1 Core of Optimal Stability Is Crucial for Viral Replication

ABSTRACT Virions of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses contain conical cores consisting of a protein shell composed of the viral capsid protein (CA) surrounding an internal viral ribonucleoprotein complex. Although genetic studies have implicated CA in both early and late stages of the virus replication cycle, the mechanism of core disassembly following penetration of target cells remains undefined. Using quantitative assays for analyzing HIV-1 core stability in vitro, we identified point mutations in CA that either reduce or increase the stability of the HIV-1 core without impairing conical core formation in virions. Alterations in core stability resulted in severely attenuated HIV-1 replication and impaired reverse transcription in target cells with only minimal effects on viral DNA synthesis in permeabilized virions in vitro. We conclude that formation of a viral core of optimal stability is a prerequisite for efficient HIV-1 infection and suggest that disassembly of the HIV-1 core is a regulated step in infection that may be an attractive target for pharmacologic intervention.

HIV-1 infection of nondividing cells: C-terminal tyrosine phosphorylation of the viral matrix protein is a key regulator

The HIV-1 matrix (MA) protein contains two subcellular localization signals with opposing effects. A myristoylated N-terminus governs particle assembly at the plasma membrane, and a nucleophilic motif facilitates import of the viral preintegration complex into the nucleus of nondividing cells. Here, we show that myristoylation acts as the MA dominant targeting signal in HIV-1 producer cells. During virus assembly, a subset of MA is phosphorylated on the C-terminal tyrosine by a virion-associated cellular protein kinase. Tyrosine-phosphorylated MA is then preferentially transported to the nucleus of target cells. An MA tyrosine mutant virus grows normally in dividing cells, but is blocked for nuclear import in terminally differentiated macrophages. MA tyrosine phosphorylation thus reveals the karyophilic properties of this protein within the HIV-1 preintegration complex, thereby playing a critical role for infection of nondividing cells.

Escape from the Dominant HLA-B27-Restricted Cytotoxic T-Lymphocyte Response in Gag Is Associated with a Dramatic Reduction in Human Immunodeficiency Virus Type 1 Replication

ABSTRACT Human leukocyte antigen (HLA)-B27-positive subjects are uncommon in their ability to control infection with human immunodeficiency virus type 1 (HIV-1). However, late viral escape from a narrowly directed immunodominant Gag-specific CD8+ T-lymphocyte (CTL) response has been linked to AIDS progression in these individuals. Identifying the mechanism of the immune-mediated control may provide critical insights into HIV-1 vaccine development. Here, we illustrate that the CTL escape mutation R264K in the HLA-B27-restricted KK10 epitope in the capsid resulted in a significant defect in viral replication in vitro. The R264K variant was impaired in generating late reverse transcription products, indicating that replication was blocked at a postentry step. Notably, the R264K mutation was associated in vivo with the development of a rare secondary mutation, S173A, which restored viral replication in vitro. Furthermore, infectivity of the R264K variant was rescued by the addition of cyclosporine A or infection of a cyclophilin A-deficient cell line. These data demonstrate a severe functional defect imposed by the R264K mutation during an early step in viral replication that is likely due to the inability of this variant to replicate efficiently in the presence of normal levels of cyclophilin A. We conclude that the impact of the R264K substitution on capsid structure constrains viral escape and enables long-term maintenance of the dominant CTL response against B27-KK10, providing an explanation for the protective effect of HLA-B27 during HIV infection.

Assembly Properties of the Human Immunodeficiency Virus Type 1 CA Protein

ABSTRACT During retroviral maturation, the CA protein oligomerizes to form a closed capsid that surrounds the viral genome. We have previously identified a series of deleterious surface mutations within human immunodeficiency virus type 1 (HIV-1) CA that alter infectivity, replication, and assembly in vivo. For this study, 27 recombinant CA proteins harboring 34 different mutations were tested for the ability to assemble into helical cylinders in vitro. These cylinders are composed of CA hexamers and are structural models for the mature viral capsid. Mutations that diminished CA assembly clustered within helices 1 and 2 in the N-terminal domain of CA and within the crystallographically defined dimer interface in the CA C-terminal domain. These mutations demonstrate the importance of these regions for CA cylinder production and, by analogy, mature capsid assembly. One CA mutant (R18A) assembled into cylinders, cones, and spheres. We suggest that these capsid shapes occur because the R18A mutation alters the frequency at which pentamers are incorporated into the hexagonal lattice. The fact that a single CA protein can simultaneously form all three known retroviral capsid morphologies supports the idea that these structures are organized on similar lattices and differ only in the distribution of 12 pentamers that allow them to close. In further support of this model, we demonstrate that the considerable morphological variation seen for conical HIV-1 capsids can be recapitulated in idealized capsid models by altering the distribution of pentamers.

Structural convergence between Cryo-EM and NMR reveals intersubunit interactions critical for HIV-1 capsid function.

Mature HIV-1 particles contain conical-shaped capsids that enclose the viral RNA genome and perform essential functions in the virus life cycle. Previous structural analysis of two- and three-dimensional arrays of the capsid protein (CA) hexamer revealed three interfaces. Here, we present a cryoEM study of a tubular assembly of CA and a high-resolution NMR structure of the CA C-terminal domain (CTD) dimer. In the solution dimer structure, the monomers exhibit different relative orientations compared to previous X-ray structures. The solution structure fits well into the EM density map, suggesting that the dimer interface is retained in the assembled CA. We also identified a CTD-CTD interface at the local three-fold axis in the cryoEM map and confirmed its functional importance by mutagenesis. In the tubular assembly, CA intermolecular interfaces vary slightly, accommodating the asymmetry present in tubes. This provides the necessary plasticity to allow for controlled virus capsid dis/assembly.

Coupling of Human Immunodeficiency Virus Type 1 Fusion to Virion Maturation: a Novel Role of the gp41 Cytoplasmic Tail

ABSTRACT Retrovirus particles are not infectious until they undergo proteolytic maturation to form a functional core. Here we report a link between human immunodeficiency virus type 1 (HIV-1) core maturation and the ability of the virus to fuse with target cells. Using a recently developed reporter assay of HIV-1 virus-cell fusion, we show that immature HIV-1 particles are 5- to 10-fold less active for fusion with target cells than are mature virions. The fusion of mature and immature virions was rendered equivalent by truncating the gp41 cytoplasmic domain or by pseudotyping viruses with the glycoprotein of vesicular stomatitis virus. An analysis of a panel of mutants containing mutated cleavage sites indicated that HIV-1 fusion competence is activated by the cleavage of Gag at any site between the MA and NC segments and not as an indirect consequence of an altered core structure. These results suggest a mechanism by which binding of the gp41 cytoplasmic tail to Gag within immature HIV-1 particles inhibits Env conformational changes on the surface of the virion that are required for membrane fusion. This “inside-out” regulation of HIV-1 fusion could play an important role in the virus life cycle by preventing the entry of immature, noninfectious particles.

Evidence for a Stable Interaction of gp41 with Pr55 Gag in Immature Human Immunodeficiency Virus Type 1 Particles

ABSTRACT Assembly of infectious human immunodeficiency virus type 1 (HIV-1) virions requires incorporation of the viral envelope glycoproteins gp41 and gp120. Several lines of evidence have suggested that the cytoplasmic tail of the transmembrane glycoprotein, gp41, associates with Pr55Gag in infected cells to facilitate the incorporation of HIV-1 envelope proteins into budding virions. However, direct evidence for an interaction between gp41 and Pr55Gagin HIV-1 particles has not been reported. To determine whether gp41 is associated with Pr55Gag in HIV-1 particles, viral cores were isolated from immature HIV-1 virions by sedimentation through detergent. The cores contained a major fraction of the gp41 that was present on untreated virions. Association of gp41 with cores required the presence of the gp41 cytoplasmic tail. In HIV-1 particles containing a functional protease, a mutation that prevents cleavage of Pr55Gag at the matrix-capsid junction was sufficient for the detergent-resistant association of gp41 with the isolated cores. In addition to gp41, a major fraction of virion-associated gp120 was also detected on immature HIV-1 cores. Isolation of cores under conditions known to disrupt lipid rafts resulted in the removal of a raft-associated protein incorporated into virions but not the HIV-1 envelope proteins. These results provide biochemical evidence for a stable interaction between Pr55Gag and the cytoplasmic tail of gp41 in immature HIV-1 particles. Moreover, findings in this study suggest that the interaction of Pr55Gag with gp41 may regulate the function of the envelope proteins during HIV-1 maturation.

The HIV-1 Nef protein acts as a connector with sorting pathways in the Golgi and at the plasma membrane

The HIV Nef protein down-regulates the cell surface expression of CD4 and of MHC I at least in part through accelerated endocytosis. To investigate further the mechanism of this effect, we created chimeric integral membrane proteins comprising the extracellular and transmembrane regions of CD4 or CD8 and Nef as the cytoplasmic domain. These fusion molecules could down-modulate CD4 in trans in a dileucine-dependent manner. Furthermore, in spite of lacking receptor-derived internalization signals, the Nef-containing chimeras underwent both Golgi retention and rapid endocytosis via clathrin-coated pits. Taken together, these data suggest that Nef down-regulates CD4 and probably MHC I by physically connecting these receptors with sorting pathways in the Golgi and at the plasma membrane.