Huiyi Miao

Identification of the platelet-derived chemokine CXCL4/PF-4 as a broad-spectrum HIV-1 inhibitor.

The natural history of HIV-1 infection is highly variable in different individuals, spanning from a rapidly progressive course to a long-term asymptomatic infection. A major determinant of the pace of disease progression is the in vivo level of HIV-1 replication, which is regulated by a complex network of cytokines and chemokines expressed by immune and inflammatory cells. The chemokine system is critically involved in the control of HIV-1 replication by virtue of the role played by specific chemokine receptors, most notably CCR5 and CXCR4, as cell-surface coreceptors for HIV-1 entry; hence, the chemokines that naturally bind such coreceptors act as endogenous inhibitors of HIV-1. Here, we show that the CXC chemokine CXCL4 (PF-4), the most abundant protein contained within the α-granules of platelets, is a broad-spectrum inhibitor of HIV-1 infection. Unlike other known HIV-suppressive chemokines, CXCL4 inhibits infection by the majority of primary HIV-1 isolates regardless of their coreceptor-usage phenotype or genetic subtype. Consistent with the lack of viral phenotype specificity, blockade of HIV-1 infection occurs at the level of virus attachment and entry via a unique mechanism that involves direct interaction of CXCL4 with the major viral envelope glycoprotein, gp120. The binding site for CXCL4 was mapped to a region of the gp120 outer domain proximal to the CD4-binding site. The identification of a platelet-derived chemokine as an endogenous antiviral factor may have relevance for the pathogenesis and treatment of HIV-1 infection.

Quaternary contact in the initial interaction of CD4 with the HIV-1 envelope trimer

Binding of the gp120 envelope (Env) glycoprotein to the CD4 receptor is the first step in the HIV-1 infectious cycle. Although the CD4-binding site has been extensively characterized, the initial receptor interaction has been difficult to study because of major CD4-induced structural rearrangements. Here we used cryogenic electron microscopy (cryo-EM) to visualize the initial contact of CD4 with the HIV-1 Env trimer at 6.8-Å resolution. A single CD4 molecule is embraced by a quaternary HIV-1–Env surface formed by coalescence of the previously defined CD4-contact region with a second CD4-binding site (CD4-BS2) in the inner domain of a neighboring gp120 protomer. Disruption of CD4-BS2 destabilized CD4-trimer interaction and abrogated HIV-1 infectivity by preventing the acquisition of coreceptor-binding competence. A corresponding reduction in HIV-1 infectivity occurred after the mutation of CD4 residues that interact with CD4-BS2. Our results document the critical role of quaternary interactions in the initial HIV-Env-receptor contact, with implications for treatment and vaccine design.

Treatment with IL-7 Prevents the Decline of Circulating CD4+ T Cells during the Acute Phase of SIV Infection in Rhesus Macaques

Although treatment with interleukin-7 (IL-7) was shown to transiently expand the naïve and memory T-cell pools in patients with chronic HIV-1 infection receiving antiretroviral therapy (ART), it is uncertain whether a full immunologic reconstitution can be achieved. Moreover, the effects of IL-7 have never been evaluated during acute HIV-1 (or SIV) infection, a critical phase of the disease in which the most dramatic depletion of CD4+ T cells is believed to occur. In the present study, recombinant, fully glycosylated simian IL-7 (50 µg/kg, s.c., once weekly for 7 weeks) was administered to 6 rhesus macaques throughout the acute phase of infection with a pathogenic SIV strain (mac251); 6 animals were infected at the same time and served as untreated controls. Treatment with IL-7 did not cause clinically detectable side effects and, despite the absence of concomitant ART, did not induce significant increases in the levels of SIV replication except at the earliest time point tested (day 4 post-infection). Strikingly, animals treated with IL-7 were protected from the dramatic decline of circulating naïve and memory CD4+ T cells that occurred in untreated animals. Treatment with IL-7 induced only transient T-cell proliferation, but it was associated with sustained increase in the expression of the anti-apoptotic protein Bcl-2 on both CD4+ and CD8+ T cells, persistent expansion of all circulating CD8+ T-cell subsets, and development of earlier and stronger SIV Tat-specific T-cell responses. However, the beneficial effects of IL-7 were not sustained after treatment interruption. These data demonstrate that IL-7 administration is effective in protecting the CD4+ T-cell pool during the acute phase of SIV infection in macaques, providing a rationale for the clinical evaluation of this cytokine in patients with acute HIV-1 infection.

Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2–V3 interaction and modulates neutralization sensitivity

Significance Despite intensive efforts, the structure of the native HIV-1 envelope trimer—the sole relevant target for vaccine design—has remained elusive. Our work identifies a key structural constraint that stabilizes the native envelope conformation and modulates its sensitivity to neutralization. We show that this constraint is established by previously unrecognized sulfated tyrosines within the second variable loop (V2) of the envelope glycoprotein subunit gp120, which mediate intramolecular interaction with the base of the third variable loop, V3. Strikingly, the V2 sulfotyrosines functionally mimic those present in the N terminus of the CCR5 coreceptor, which bind to the same V3 region. Our results shed light on the mechanisms adopted by HIV-1 to elude immunologic control and open new perspectives for vaccine design. Elicitation of broadly neutralizing antibodies is essential for the development of a protective vaccine against HIV-1. However, the native HIV-1 envelope adopts a protected conformation that conceals highly conserved sites of vulnerability from antibody recognition. Although high-definition structures of the monomeric core of the envelope glycoprotein subunit gp120 and, more recently, of a stabilized soluble gp140 trimer have been solved, fundamental aspects related to the conformation and function of the native envelope remain unresolved. Here, we show that the conserved central region of the second variable loop (V2) of gp120 contains sulfated tyrosines (Tys173 and Tys177) that in the CD4-unbound prefusion state mediate intramolecular interaction between V2 and the conserved base of the third variable loop (V3), functionally mimicking sulfated tyrosines in CCR5 and anti–coreceptor-binding-site antibodies such as 412d. Recombinant gp120 expressed in continuous cell lines displays low constitutive levels of V2 tyrosine sulfation, which can be enhanced markedly by overexpression of the tyrosyl sulfotransferase TPST2. In contrast, virion-associated gp120 produced by primary CD4+ T cells is inherently highly sulfated. Consistent with a functional role of the V2 sulfotyrosines, enhancement of tyrosine sulfation decreased binding and neutralization of HIV-1 BaL by monomeric soluble CD4, 412d, and anti-V3 antibodies and increased recognition by the trimer-preferring antibodies PG9, PG16, CH01, and PGT145. Conversely, inhibition of tyrosine sulfation increased sensitivity to soluble CD4, 412d, and anti-V3 antibodies and diminished recognition by trimer-preferring antibodies. These results identify the sulfotyrosine-mediated V2–V3 interaction as a critical constraint that stabilizes the native HIV-1 envelope trimer and modulates its sensitivity to neutralization.

The CD8-Derived Chemokine XCL1/Lymphotactin Is a Conformation-Dependent, Broad-Spectrum Inhibitor of HIV-1

CD8+ T cells play a key role in the in vivo control of HIV-1 replication via their cytolytic activity as well as their ability to secrete non-lytic soluble suppressive factors. Although the chemokines that naturally bind CCR5 (CCL3/MIP-1α, CCL4/MIP- 1β, CCL5/RANTES) are major components of the CD8-derived anti-HIV activity, evidence indicates the existence of additional, still undefined, CD8-derived HIV-suppressive factors. Here, we report the characterization of a novel anti-HIV chemokine, XCL1/lymphotactin, a member of the C-chemokine family that is produced primarily by activated CD8+ T cells and behaves as a metamorphic protein, interconverting between two structurally distinct conformations (classic and alternative). We found that XCL1 inhibits a broad spectrum of HIV-1 isolates, irrespective of their coreceptor-usage phenotype. Experiments with stabilized variants of XCL1 demonstrated that HIV-1 inhibition requires access to the alternative, all-β conformation, which interacts with proteoglycans but does not bind/activate the specific XCR1 receptor, while the classic XCL1 conformation is inactive. HIV-1 inhibition by XCL1 was shown to occur at an early stage of infection, via blockade of viral attachment and entry into host cells. Analogous to the recently described anti-HIV effect of the CXC chemokine CXCL4/PF4, XCL1-mediated inhibition is associated with direct interaction of the chemokine with the HIV-1 envelope. These results may open new perspectives for understanding the mechanisms of HIV-1 control and reveal new molecular targets for the design of effective therapeutic and preventive strategies against HIV-1.

Virion incorporation of integrin α4β7 facilitates HIV-1 infection and intestinal homing

Anti–integrin α4β7 therapy might directly interfere with the ability of HIV to home to intestinal reserviors. Taking HIV to the gut Antiretroviral therapy (ART) effectively limits HIV replication, but HIV+ individuals are medicated for life because ART withdrawal results in rebound of persistent virus. Developing therapies that keep viral loads low in the long term and prevent reinfection remains an important goal—one emerging approach is an antibody against integrin α4β7. Integrin α4β7 is a receptor that facilitates homing of CD4+ T cells to the gut, a key site for HIV persistence. ART-suppressed macaques that received antibodies against integrin α4β7 controlled the virus even after ART withdrawal. Here, Guzzo et al. demonstrate that integrin α4β7 is incorporated into the HIV envelope, suggesting that antibody treatment may directly interfere with the ability of HIV to target intestinal tissues. Their results change our perception of the role of integrin α4β7, a promising therapeutic target in HIV pathogenesis. The intestinal mucosa is a key anatomical site for HIV-1 replication and CD4+ T cell depletion. Accordingly, in vivo treatment with an antibody to the gut-homing integrin α4β7 was shown to reduce viral transmission, delay disease progression, and induce persistent virus control in macaques challenged with simian immunodeficiency virus (SIV). We show that integrin α4β7 is efficiently incorporated into the envelope of HIV-1 virions. Incorporated α4β7 is functionally active as it binds mucosal addressin cell adhesion molecule–1 (MAdCAM-1), promoting HIV-1 capture by and infection of MAdCAM-expressing cells, which in turn mediate trans-infection of bystander cells. Functional α4β7 is present in circulating virions from HIV-infected patients and SIV-infected macaques, with peak levels during the early stages of infection. In vivo homing experiments documented selective and specific uptake of α4β7+ HIV-1 virions by high endothelial venules in the intestinal mucosa. These results extend the paradigm of tissue homing to a retrovirus and are relevant for the pathogenesis, treatment, and prevention of HIV-1 infection.

Tyrosine-sulfated V2 peptides inhibit HIV-1 infection via coreceptor mimicry

Tyrosine sulfation is a post-translational modification that facilitates protein-protein interaction. Two sulfated tyrosines (Tys173 and Tys177) were recently identified within the second variable (V2) loop of the major HIV-1 envelope glycoprotein, gp120, and shown to contribute to stabilizing the intramolecular interaction between V2 and the third variable (V3) loop. Here, we report that tyrosine-sulfated peptides derived from V2 act as structural and functional mimics of the CCR5 N-terminus and potently block HIV-1 infection. Nuclear magnetic and surface plasmon resonance analyses indicate that a tyrosine-sulfated V2 peptide (pV2α-Tys) adopts a CCR5-like helical conformation and directly interacts with gp120 in a CD4-dependent fashion, competing with a CCR5 N-terminal peptide. Sulfated V2 mimics, but not their non-sulfated counterparts, inhibit HIV-1 entry and fusion by preventing coreceptor utilization, with the highly conserved C-terminal sulfotyrosine, Tys177, playing a dominant role. Unlike CCR5 N-terminal peptides, V2 mimics inhibit a broad range of HIV-1 strains irrespective of their coreceptor tropism, highlighting the overall structural conservation of the coreceptor-binding site in gp120. These results document the use of receptor mimicry by a retrovirus to occlude a key neutralization target site and provide leads for the design of therapeutic strategies against HIV-1.

Interdomain Stabilization Impairs CD4 Binding and Improves Immunogenicity of the HIV-1 Envelope Trimer

The HIV-1 envelope (Env) spike is a trimer of gp120/gp41 heterodimers that mediates viral entry. Binding to CD4 on the host cell membrane is the first essential step for infection but disrupts the native antigenic state of Env, posing a key obstacle to vaccine development. We locked the HIV-1 Env trimer in a pre-fusion configuration, resulting in impaired CD4 binding and enhanced binding to broadly neutralizing antibodies. This design was achieved via structure-guided introduction of neo-disulfide bonds bridging the gp120 inner and outer domains and was successfully applied to soluble trimers and native gp160 from different HIV-1 clades. Crystallization illustrated the structural basis for CD4-binding impairment. Immunization of rabbits with locked trimers from two different clades elicited neutralizing antibodies against tier-2 viruses with a repaired glycan shield regardless of treatment with a functional CD4 mimic. Thus, interdomain stabilization provides a widely applicable template for the design of Env-based HIV-1 vaccines.

Corrigendum: Quaternary contact in the initial interaction of CD4 with the HIV-1 envelope trimer

Nat. Struct. Mol. Biol. 24, 370–378 (2017); published online 20 February 2017; corrected after print 27 April 2017 In the version of this article initially published, funding information for B.C. and C.S.P. was missing NIH grant S10 OD019994-01. In addition, there was an incorrect comma in the introduction (after “glycoprotein” in the sentence “Upon binding to the primary cellular receptor, CD4, the external gp120 Env glycoprotein undergoes major conformational changes.