Andrés Finzi

Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01.

Designer Anti-HIV Developing a protective HIV vaccine remains a top global health priority. One strategy to identify potential vaccine candidates is to isolate broadly neutralizing antibodies from infected individuals and then attempt to elicit the same antibody response through vaccination (see the Perspective by Burton and Weiss). Wu et al. (p. 856, published online 8 July) now report the identification of three broadly neutralizing antibodies, isolated from an HIV-1–infected individual, that exhibited great breadth and potency of neutralization and were specific for the co-receptor CD4-binding site of the glycoprotein 120 (gp120), part of the viral Env spike. Zhou et al. (p. 811, published online 8 July) analyzed the crystal structure for one of these antibodies, VRC01, in complex with an HIV-1 gp120. VRC01 focuses its binding onto a conformationally invariant domain that is the site of initial CD4 attachment, which allows the antibody to overcome the glycan and conformational masking that diminishes the neutralization potency of most CD4-binding-site antibodies. The epitopes recognized by these antibodies suggest potential immunogens that can inform vaccine design. A human antibody achieves broad neutralization by binding the viral site of recognition for the primary host receptor, CD4. During HIV-1 infection, antibodies are generated against the region of the viral gp120 envelope glycoprotein that binds CD4, the primary receptor for HIV-1. Among these antibodies, VRC01 achieves broad neutralization of diverse viral strains. We determined the crystal structure of VRC01 in complex with a human immunodeficiency virus HIV-1 gp120 core. VRC01 partially mimics CD4 interaction with gp120. A shift from the CD4-defined orientation, however, focuses VRC01 onto the vulnerable site of initial CD4 attachment, allowing it to overcome the glycan and conformational masking that diminishes the neutralization potency of most CD4-binding-site antibodies. To achieve this recognition, VRC01 contacts gp120 mainly through immunoglobulin V-gene regions substantially altered from their genomic precursors. Partial receptor mimicry and extensive affinity maturation thus facilitate neutralization of HIV-1 by natural human antibodies.

Topological Layers in the HIV-1 gp120 Inner Domain Regulate gp41 Interaction and CD4-Triggered Conformational Transitions

The entry of human immunodeficiency virus (HIV-1) into cells is initiated by binding of the gp120 exterior envelope glycoprotein to the receptor, CD4. How does CD4 binding trigger conformational changes in gp120 that allow the gp41 transmembrane envelope glycoprotein to mediate viral-cell membrane fusion? The transition from the unliganded to the CD4-bound state is regulated by two potentially flexible topological layers (layers 1 and 2) in the gp120 inner domain. Both layers apparently contribute to the noncovalent association of unliganded gp120 with gp41. After CD4 makes initial contact with the gp120 outer domain, layer 1-layer 2 interactions strengthen gp120-CD4 binding by reducing the off rate. Layer 1-layer 2 interactions also destabilize the activated state induced on HIV-1 by treatment with soluble CD4. Thus, despite lack of contact with CD4, the gp120 inner-domain layers govern CD4 triggering by participating in conformational transitions within gp120 and regulating the interaction with gp41.

A B-Box 2 Surface Patch Important for TRIM5α Self-Association, Capsid Binding Avidity, and Retrovirus Restriction

ABSTRACT TRIM5α is a tripartite motif (TRIM) protein that consists of RING, B-box 2, coiled-coil, and B30.2(SPRY) domains. The TRIM5αrh protein from rhesus monkeys recognizes the human immunodeficiency virus type 1 (HIV-1) capsid as it enters the host cell and blocks virus infection prior to reverse transcription. HIV-1-restricting ability can be eliminated by disruption of the B-box 2 domain. Changes in the TRIM5αrh B-box 2 domain have been associated with alterations in TRIM5αrh turnover, the formation of cytoplasmic bodies and higher-order oligomerization. We present here the nuclear magnetic resonance structure of the TRIM5 B-box 2 domain and identify an unusual hydrophobic patch (cluster 1) on the domain surface. Alteration of cluster 1 or the flanking arginine 121 resulted in various degrees of inactivation of HIV-1 restriction, in some cases depending on compensatory changes in other nearby charged residues. For this panel of TRIM5αrh B-box 2 mutants, inhibition of HIV-1 infection was strongly correlated with higher-order self-association and binding affinity for capsid complexes but not with TRIM5αrh half-life or the formation of cytoplasmic bodies. Thus, promoting cooperative TRIM5αrh interactions with the HIV-1 capsid represents a major mechanism whereby the B-box 2 domain potentiates HIV-1 restriction.

Molecular architecture of the uncleaved HIV-1 envelope glycoprotein trimer.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, a membrane-fusing machine, mediates virus entry into host cells and is the sole virus-specific target for neutralizing antibodies. Binding the receptors, CD4 and CCR5/CXCR4, triggers Env conformational changes from the metastable unliganded state to the fusion-active state. We used cryo-electron microscopy to obtain a 6-Å structure of the membrane-bound, heavily glycosylated HIV-1 Env trimer in its uncleaved and unliganded state. The spatial organization of secondary structure elements reveals that the unliganded conformations of both glycoprotein (gp)120 and gp41 subunits differ from those induced by receptor binding. The gp120 trimer association domains, which contribute to interprotomer contacts in the unliganded Env trimer, undergo rearrangement upon CD4 binding. In the unliganded Env, intersubunit interactions maintain the gp41 ectodomain helical bundles in a “spring-loaded” conformation distinct from the extended helical coils of the fusion-active state. Quaternary structure regulates the virus-neutralizing potency of antibodies targeting the conserved CD4-binding site on gp120. The Env trimer architecture provides mechanistic insights into the metastability of the unliganded state, receptor-induced conformational changes, and quaternary structure-based strategies for immune evasion.

A V3 Loop-Dependent gp120 Element Disrupted by CD4 Binding Stabilizes the Human Immunodeficiency Virus Envelope Glycoprotein Trimer

ABSTRACT Human immunodeficiency virus (HIV-1) entry into cells is mediated by a trimeric complex consisting of noncovalently associated gp120 (exterior) and gp41 (transmembrane) envelope glycoproteins. The binding of gp120 to receptors on the target cell alters the gp120-gp41 relationship and activates the membrane-fusing capacity of gp41. Interaction of gp120 with the primary receptor, CD4, results in the exposure of the gp120 third variable (V3) loop, which contributes to binding the CCR5 or CXCR4 chemokine receptors. We show here that insertions in the V3 stem or polar substitutions in a conserved hydrophobic patch near the V3 tip result in decreased gp120-gp41 association (in the unliganded state) and decreased chemokine receptor binding (in the CD4-bound state). Subunit association and syncytium-forming ability of the envelope glycoproteins from primary HIV-1 isolates were disrupted more by V3 changes than those of laboratory-adapted HIV-1 envelope glycoproteins. Changes in the gp120 β2, β19, β20, and β21 strands, which evidence suggests are proximal to the V3 loop in unliganded gp120, also resulted in decreased gp120-gp41 association. Thus, a gp120 element composed of the V3 loop and adjacent beta strands contributes to quaternary interactions that stabilize the unliganded trimer. CD4 binding dismantles this element, altering the gp120-gp41 relationship and rendering the hydrophobic patch in the V3 tip available for chemokine receptor binding.

Macrophage Infection via Selective Capture of HIV-1-Infected CD4+ T Cells

Summary Macrophages contribute to HIV-1 pathogenesis by forming a viral reservoir and mediating neurological disorders. Cell-free HIV-1 infection of macrophages is inefficient, in part due to low plasma membrane expression of viral entry receptors. We find that macrophages selectively capture and engulf HIV-1-infected CD4+ T cells leading to efficient macrophage infection. Infected T cells, both healthy and dead or dying, were taken up through viral envelope glycoprotein-receptor-independent interactions, implying a mechanism distinct from conventional virological synapse formation. Macrophages infected by this cell-to-cell route were highly permissive for both CCR5-using macrophage-tropic and otherwise weakly macrophage-tropic transmitted/founder viruses but restrictive for nonmacrophage-tropic CXCR4-using virus. These results have implications for establishment of the macrophage reservoir and HIV-1 dissemination in vivo.

Role of CD4 Receptor Down-regulation During HIV-1 Infection

Human immunodeficiency virus has evolved several redundant mechanisms to remove its receptor, the CD4 molecule, from the cell surface. Indeed, HIV-1 encodes three proteins, Nef, Vpu and Env, that have a profound effect on CD4 trafficking and catabolism. Given this functional convergence, it is believed that cell surface CD4 regulation constitutes an important determinant of viral replication and pathogenesis in vivo. This review highlights recent progress made in our understanding of the molecular mechanisms underlying the down-regulation of the CD4 receptor by HIV-1 and describes our current comprehension of the role of CD4 down-regulation during HIV-1 infection.

Resistance of Transmitted Founder HIV-1 to IFITM-Mediated Restriction

Summary Interferon-induced transmembrane proteins (IFITMs) restrict the entry of diverse enveloped viruses through incompletely understood mechanisms. While IFITMs are reported to inhibit HIV-1, their in vivo relevance is unclear. We show that IFITM sensitivity of HIV-1 strains is determined by the co-receptor usage of the viral envelope glycoproteins as well as IFITM subcellular localization within the target cell. Importantly, we find that transmitted founder HIV-1, which establishes de novo infections, is uniquely resistant to the antiviral activity of IFITMs. However, viral sensitivity to IFITMs, particularly IFITM2 and IFITM3, increases over the first 6 months of infection, primarily as a result of neutralizing antibody escape mutations. Additionally, the ability to evade IFITM restriction contributes to the different interferon sensitivities of transmitted founder and chronic viruses. Together, these data indicate that IFITMs constitute an important barrier to HIV-1 transmission and that escape from adaptive immune responses exposes the virus to antiviral restriction.

Conformational Characterization of Aberrant Disulfide-linked HIV-1 gp120 Dimers Secreted from Overexpressing Cells

The envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) mediate viral entry and are also the primary target of neutralizing antibodies. The gp160 envelope glycoprotein precursor undergoes proteolytic cleavage in the Golgi complex to produce the gp120 exterior glycoprotein and the gp41 transmembrane glycoprotein, which remain associated non-covalently in the trimeric Env complex. Monomeric soluble gp120 has been used extensively to investigate conformational states, structure, antigenicity and immunogenicity of the HIV-1 Env glycoproteins. Expression of gp120 alone (without gp41) leads to the accumulation not only of monomeric gp120 but also an aberrant dimeric form. The gp120 dimers were sensitive to reducing agents. The formation of gp120 dimers was disrupted by a single amino acid change in the inner domain, and was reduced by removal of the V1/V2 variable loops or the N and C termini. Epitopes on the gp120 inner domain and the chemokine receptor-binding surface were altered or occluded by gp120 dimerization. Awareness of the existence and properties of gp120 dimers should assist interpretation of studies of this key viral protein.

Transitions to and from the CD4-Bound Conformation Are Modulated by a Single-Residue Change in the Human Immunodeficiency Virus Type 1 gp120 Inner Domain

ABSTRACT Binding to the primary receptor CD4 induces conformational changes in the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein that allow binding to the coreceptor (CCR5 or CXCR4) and ultimately trigger viral membrane-cell membrane fusion mediated by the gp41 transmembrane envelope glycoprotein. Here we report the derivation of an HIV-1 gp120 variant, H66N, that confers envelope glycoprotein resistance to temperature extremes. The H66N change decreases the spontaneous sampling of the CD4-bound conformation by the HIV-1 envelope glycoproteins, thus diminishing CD4-independent infection. The H66N change also stabilizes the HIV-1 envelope glycoprotein complex once the CD4-bound state is achieved, decreasing the probability of CD4-induced inactivation and revealing the enhancing effects of soluble CD4 binding on HIV-1 infection. In the CD4-bound conformation, the highly conserved histidine 66 is located between the receptor-binding and gp41-interactive surfaces of gp120. Thus, a single amino acid change in this strategically positioned gp120 inner domain residue influences the propensity of the HIV-1 envelope glycoproteins to negotiate conformational transitions to and from the CD4-bound state.