GPR182 is an endothelium-specific atypical chemokine receptor that maintains hematopoietic stem cell homeostasis

G protein–coupled receptor 182 (GPR182) has been shown to be expressed in endothelial cells; however, its ligand and physiological role has remained elusive. We found GPR182 to be expressed in microvascular and lymphatic endothelial cells of most organs and to bind with nanomolar affinity the chemokines CXCL10, CXCL12, and CXCL13. In contrast to conventional chemokine receptors, binding of chemokines to GPR182 did not induce typical downstream signaling processes, including G_q- and G_i-mediated signaling or β-arrestin recruitment. GPR182 showed relatively high constitutive activity in regard to β-arrestin recruitment and rapidly internalized in a ligand-independent manner. In constitutive GPR182-deficient mice, as well as after induced endothelium-specific loss of GPR182, we found significant increases in the plasma levels of CXCL10, CXCL12, and CXCL13. Global and induced endothelium-specific GPR182-deficient mice showed a significant decrease in hematopoietic stem cells in the bone marrow as well as increased colony-forming units of hematopoietic progenitors in the blood and the spleen. Our data show that GPR182 is a new atypical chemokine receptor for CXCL10, CXCL12, and CXCL13, which is involved in the regulation of hematopoietic stem cell homeostasis.

G protein–coupled receptors (GPCRs) represent the largest group of transmembrane receptors encoded in the genome, and they are the largest group of proteins targeted by approved drugs (1, 2). GPCRs are very versatile and can bind ligands of different physicochemical properties, including ions, lipids, biogenic amines, peptides, or proteins, such as chemokines (3). Primarily by activation of heterotrimeric G proteins, GPCRs regulate multiple functions in basically all cells of mammalian organisms (4). Despite their large physiological and pharmacological relevance, the endogenous ligands, activating mechanisms and physiological functions of more than 100 GPCRs, are still not known and these receptors are therefore referred to as “orphan” receptors (3, 5). G protein–coupled receptor 182 (GPR182) is an orphan receptor, although it has been suggested to bind adrenomedullin (6), but this observation could not be confirmed (7). GPR182 was initially described to be widely expressed in various organs (8). More-detailed analyses in developing zebrafish and in mice revealed that Gpr182 is preferentially expressed in the vascular endothelium (9, 10). Widespread expression in endothelial cells of adult mice was shown using a mouse line expressing β-galactosidase under the control of the Gpr182-promoter (11), and expression of GPR182 in sinusoidal endothelial cells was reported based on immunohistochemical analysis (12). Whereas the role of GPR182 in endothelial cells is unknown, GPR182 expression was also reported in intestinal stem cells, where the receptor was shown to negatively regulate proliferation during regeneration and adenoma formation (11).Chemokine receptors are a family of 22 GPCRs that respond to 52 chemokines (13). Upon activation, they induce G protein–mediated intracellular signaling processes which, in many cases, regulate the migration of leukocytes (14). However, more recent work has shown that the function of chemokines goes beyond the regulation of leukocyte migration and can also affect other cell functions and cell types (13, 15, 16). In addition, and in contrast to other groups of GPCRs, the chemokine receptor family contains several members, which bind chemokines but are unable to signal through G proteins. These so-called “atypical chemokine receptors” (ACKRs) can indirectly regulate the interactions between chemokines and conventional chemokine receptors by controlling chemokine localization, distribution, and abundance (13, 16, 17). As most conventional chemokine receptors, ACKRs typically bind subgroups of chemokines. For instance, ACKR1 binds various chemokines and transports them across endothelial cells or, when expressed on erythrocytes, buffers chemokine levels in the blood (18). ACKR2 functions as a scavenger receptor by binding several C-C motif chemokine ligand (CCL) chemokines and plays various roles in the immune system (19). ACKR3 only binds C-X-C motif chemokine ligand 11 (CXCL11) and CXCL12 and controls the CXCL12–CXCR4 signaling axis by direct interaction with CXCR4 and by scavenging CXCL12 (20, 21). ACKR4 binds the conventional chemokine receptor ligands CCL19, CCL21, CCL25, and CXCL19 (18, 19).Here, we show that GPR182 functions as an atypical chemokine receptor for CXCL10, CXCL12, and CXCL13 and that it is involved in preventing hematopoietic stem cell egress from bone marrow.     

CXCR4/CXCL12-mediated entrapment of axons at the injury site compromises optic nerve regeneration

Regenerative failure in the mammalian optic nerve is generally attributed to axotomy-induced retinal ganglion cell (RGC) death, an insufficient intrinsic regenerative capacity, and an extrinsic inhibitory environment. Here, we show that a chemoattractive CXCL12/CXCR4-dependent mechanism prevents the extension of growth-stimulated axons into the distal nerve. The chemokine CXCL12 is chemoattractive toward axonal growth cones in an inhibitory environment, and these effects are entirely abolished by the specific knockout of its receptor, CXCR4 (CXCR4^−/−), in cultured regenerating RGCs. Notably, 8% of naïve RGCs express CXCL12 and transport the chemokine along their axons in the nerve. Thus, axotomy causes its release at the injury site. However, most osteopontin-positive α-RGCs, the main neuronal population that survives optic nerve injury, express CXCR4 instead. Thus, CXCL12-mediated attraction prevents growth-stimulated axons from regenerating distally in the nerve, indicated by axons returning to the lesion site. Accordingly, specific depletion of CXCR4 in RGC reduces aberrant axonal growth and enables long-distance regeneration. Likewise, CXCL12 knockout in RGCs fully mimics these CXCR4^−/− effects. Thus, active CXCL12/CXCR4-mediated entrapment of regenerating axons to the injury site contributes to regenerative failure in the optic nerve.

Retinal ganglion cells (RGCs) convey the visual input from the eye through the optic nerve and optic tract into the brain’s target regions. As typical neurons of the central nervous system (CNS), mammalian RGCs lose most of their capability to regrow injured axons after birth (1, 2), leading to an irreversible functional loss after optic nerve damage. To date, regenerative failure has been mainly attributed to three leading causes: 1) axotomy-induced apoptosis of RGCs, 2) the low intrinsic capacity to regrow axons, and 3) the external inhibitory environment with CNS myelin and glial scar proteins (3, 4).One widely used approach to delay axotomy-induced RGC degeneration and activate the intrinsic regenerative capacity of injured axons is inflammatory stimulation (IS) in the eye induced by a lens injury, intravitreal Pam₃Cys, or zymosan injection (5–7). IS leads to the expression and release of CNTF, LIF, and IL-6 from retinal astrocytes and Müller cells (8–10), which directly interact with RGCs and activate neuroprotective/regenerative signaling such as the JAK/STAT3 pathway (8, 9, 11, 12). IS, therefore, enables moderate axon regeneration beyond the lesion site of the optic nerve. Although combinatorial strategies, together with measures overcoming the inhibitory CNS environment synergistically, further improve IS-mediated optic nerve regeneration (13–17), the overall outcome remains mostly unsatisfactory. Thus, additional unknown mechanisms besides neurodegeneration, low intrinsic capacity, and the inhibitory environment might contribute to optic nerve regeneration failure.The chemokine receptor CXCR4, a seven-transmembrane G protein–coupled receptor, is expressed in embryonic and adult neurons (18–20). We have recently shown that this receptor is also expressed in the somata and axons of adult rat RGCs (18). Next to its role as a coreceptor for HIV entry and cancer-cell migration/proliferation (21, 22), CXCR4 is reportedly involved in neurogenesis and axonal pathfinding during the embryonal development of RGCs (20, 23, 24). CXCR4 regulates different signaling pathways upon binding its ligand CXCL12 (also known as stromal cell–derived factor 1, SDF-1), which is part of the chemokine family of chemotactic cytokines in the immune system involved in the attraction of lymphocytes (25, 26). CXCL12 is also reportedly expressed by some CNS neurons, astrocytes, and microglia (19, 27–30). As the CXCR4/CXCL12 axis is highly conserved between different species (31) and involved in axonal pathfinding during embryonal development of RGCs (20, 32), we speculated that CXCR4 expression in adult RGCs might also play a role in the regenerative processes of mature axons.The current study shows that growth-stimulated axons of RGCs are actively attracted and entrapped at the lesion site of the optic nerve by a CXCL12/CXCR4-dependent mechanism. CXCL12 is expressed in a subpopulation of RGCs and axonally transported, implying its release at the injury site. A different RGC subpopulation expressed CXCR4, causing axons in the distal nerve to return to the injury site. Specific depletion of CXCR4 or CXCL12 in RGCs abolished aberrant growth. It enabled long-distance regeneration in the optic nerve, with some axons reaching the optic chiasm 3 wk after injury. Thus, active CXCL12/CXCR4-mediated entrapment markedly compromises axon extension into the distal optic nerve and contributes to regenerative failure in the optic nerve.     

Chemical mutagenesis of a GPCR ligand: Detoxifying “inflammo-attraction” to direct therapeutic stem cell migration

A transplanted stem cell’s engagement with a pathologic niche is the first step in its restoring homeostasis to that site. Inflammatory chemokines are constitutively produced in such a niche; their binding to receptors on the stem cell helps direct that cell’s “pathotropism.” Neural stem cells (NSCs), which express CXCR4, migrate to sites of CNS injury or degeneration in part because astrocytes and vasculature produce the inflammatory chemokine CXCL12. Binding of CXCL12 to CXCR4 (a G protein-coupled receptor, GPCR) triggers repair processes within the NSC. Although a tool directing NSCs to where needed has been long-sought, one would not inject this chemokine in vivo because undesirable inflammation also follows CXCL12–CXCR4 coupling. Alternatively, we chemically “mutated” CXCL12, creating a CXCR4 agonist that contained a strong pure binding motif linked to a signaling motif devoid of sequences responsible for synthetic functions. This synthetic dual-moity CXCR4 agonist not only elicited more extensive and persistent human NSC migration and distribution than did native CXCL 12, but induced no host inflammation (or other adverse effects); rather, there was predominantly reparative gene expression. When co-administered with transplanted human induced pluripotent stem cell-derived hNSCs in a mouse model of a prototypical neurodegenerative disease, the agonist enhanced migration, dissemination, and integration of donor-derived cells into the diseased cerebral cortex (including as electrophysiologically-active cortical neurons) where their secreted cross-corrective enzyme mediated a therapeutic impact unachieved by cells alone. Such a “designer” cytokine receptor-agonist peptide illustrates that treatments can be controlled and optimized by exploiting fundamental stem cell properties (e.g., “inflammo-attraction”).

A transplanted stem cell’s engagement with a pathologic niche is the first step in cell-mediated restoration of homeostasis to that region, whether by cell replacement, protection, gene delivery, milieu alteration, toxin neutralization, or remodeling (1–4). Not surprisingly, the more host terrain covered by the stem cells, the greater their impact. We and others found that a propensity for neural stem cells (NSCs) to home in vivo to acutely injured or actively degenerating central nervous system (CNS) regions—a property called “pathotropism” (1–12), now viewed as central to stem cell biology—is undergirded, at least in part, by the presence of chemokine receptors on the NSC surface, enabling them to follow concentration gradients of inflammatory cytokines constitutively elaborated by pathogenic processes and expressed by reactive astrocytes and injured vascular endothelium within the pathologic niche (5–9). This engagement of NSC receptors was first described for the prototypical chemokine receptor CXCR4 (C-X-C chemokine receptor type 4; also known as fusin or cluster of differentiation-184 [CD184]) and its unique natural cognate agonist ligand, the inflammatory chemokine CXCL12 (C-X-C motif chemokine ligand-12; also known as stromal cell-derived factor 1α [SDF-1α]) (5), but has since been described for many chemokine receptor-agonist pairings (6–9). Chemokine receptors belong to a superfamily that is characterized by seven transmembrane GDP-binding protein-coupled receptors (GPCRs) (13–21). In addition to their role in mediating inflammatory reactions and immune responses (22, 23), these receptors and their agonists are components of the regulatory axes for hematopoiesis and organogenesis in other systems (21, 24). Therefore, it is not surprising that binding of CXCL12 to CXCR4 mediates not only an inflammatory response, but also triggers within the NSC a series of intracellular processes associated with migration (as well as proliferation, differentiation, survival, and, during early brain development, proper neuronal lamination) (10).A tool directing therapeutic NSCs to where they are needed has long been sought in regenerative medicine (11, 12). While it was appealing to contemplate electively directing reparative NSCs to any desired area by emulating this chemoattractive property through the targeted injection of exogenous recombinant inflammatory cytokines, it ultimately seemed inadvisable to risk increasing toxicity in brains already characterized by excessive and usually inimical inflammation from neurotraumatic or neurodegenerative processes. However, the notion of engaging the homing function of these NSC-borne receptors without triggering that receptor’s undesirable downstream inflammatory signaling [particularly given that the NSCs themselves can exert a therapeutic antiinflammatory action in the diseased region (1, 2)] seemed a promising heretofore unexplored “workaround.”There had already been an impetus to examine the structure–function relationships of CXCR4, known to be the entry route into cells for HIV-1, in order to create CXCR4 antagonists that block viral infection (25–30). Antagonists of CXCR4 were also devised to forestall hematopoietic stem cells from homing to the bone marrow, hence prolonging their presence in the peripheral blood (31) to treat blood dyscrasias. An agonist, however, particularly one with discrete and selective actions, had not been contemplated. In other words, if CXCL12 could be stripped of its undesirable actions while preserving its tropic activity, an ideal chemoattractant would be derived.Based on the concept that CXCR4’s functions are conveyed by two distinct molecular “pockets”—one mediating binding (i.e., allowing a ligand to engage CXCR4) and the other mediating signaling (i.e., enabling a ligand, after binding, to trigger CXCR4-mediated intracellular cascades that promote not only inflammation but also migration) (13–18)—we performed chemical mutagenesis that should optimize binding while narrowing the spectrum of signaling. We created a simplified de novo peptide agonist of CXCR4 that contained a strong pure binding motif derived from CXCR4’s strongest ligand, viral macrophage inflammatory protein-II (vMIP-II) and linked it to a truncated signaling motif (only 8 amino acid residues) derived from the N terminus of native CXCL12 (19, 20). This synthetic dual-moiety CXCR4 agonist, which is devoid of a large portion of CXCL12’s native sequence (presumably responsible for undesired functions such as inflammation) not only elicited (with great specificity) more extensive and long-lasting human NSC (hNSC) migration and distribution than native CXCL12 (overcoming migratory barriers), but induced no host inflammation (or other adverse effects). Furthermore, because all of the amino acids in the binding motif were in a D-chirality, rendering the peptide resistant to enzymatic degradation, persistence of this benign synthetic agonist in vivo was prolonged. The hNSC’s gene ontology expression profile was predominantly reparative in contrast to inflammatory as promoted by native CXCL12. When coadministered with transplanted human induced pluripotent stem cell (hiPSC)-derived hNSCs (hiPSC derivatives are now known to have muted migration) in a mouse model of a prototypical neurodegenerative disease [the lethal neuropathic lysosomal storage disorder (LSD) Sandhoff disease (29), where hiPSC-hNSC migration is particularly limited], the synthetic agonist enhanced migration, dissemination, and integration of donor-derived cells into the diseased cortex (including as electrophysiologically active cortical neurons), where their secreted cross-corrective enzyme could mediate a histological and functional therapeutic impact in a manner unachieved by transplanting hiPSC-derived cells alone.In introducing such a “designer” cytokine receptor agonist, we hope to offer proof-of-concept that stem cell-mediated treatments can be controlled and optimized by exploiting fundamental stem cell properties (e.g., “inflammo-attraction”) to alter a niche and augment specific actions. Additionally, when agonists are strategically designed, the various functions of chemokine receptors (and likely other GCPRs) may be divorced. We demonstrate that such a strategy might be used safely and effectively to direct cells to needed regions and broaden their chimerism. We discuss the future implications and uses within the life sciences of such a chemical engineering approach.     

Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists

Although class A G protein?coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer?dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.

G protein?coupled receptors (GPCRs) constitute the largest class of cell surface receptors and are the main targets of clinically approved drugs (1). While GPCRs have classically been thought to exist and function as simple monomers, substantial data show that many of them may form dimers and higher-order oligomers, which might be relevant for their function (2). In particular, it is now well-appreciated that class C GPCRs form obligatory dimers (3) and that intermolecular rearrangements of such dimers play a key role for receptor activation (4, 5). Even though members of the much larger family of class A GPCRs have been shown to transduce signals as monomeric entities (6), emerging evidence demonstrates that they can form oligomers both in vitro and in vivo (7, 8). Several recent studies using advanced fluorescence microscopy methods suggested a dynamic nature of dimerization for a number of class A GPCRs (9–12). Thus, the stability, dynamics and functional relevance of individual class A GPCR dimers remains debatable.The CXC chemokine receptor 4 (CXCR4) is a prime example of a class A GPCR where the quaternary organization may be functionally and pharmacologically important. While CXCR4 regulates physiological processes mainly associated with cell migration and development, dysregulation of CXCR4 expression and function plays an important role in cancer progression, as well as viral and immune diseases (13). Following the discovery of two ligand binding pockets (major and minor) of CXCR4, several drugs targeting this receptor have been studied (14). With one marketed and others in clinical trials, CXCR4 drugs receive substantial interest for numerous diseases (15).Multiple studies using fluorescence-based methods described CXCR4 oligomerization ranging from expression-dependent complex formation to stable and ligand-independent CXCR4 homodimers and homooligomers (16, 17). Agonist binding was proposed to induce conformational changes between the CXCR4 protomers (18) and even to induce higher-order complexes (19).These agonist-induced CXCR4 nanoclusters were suggested to define cellular functions of CXCR4, and mutations that alter the cluster organization also inhibit receptor signaling in vivo (19). CXCR4 dimerization was also described in malignant cells (20). Moreover, CXCR4 crystal structures with three different ligands displayed dimeric units with similar interfaces (21, 22). Although a 1:1 stoichiometry between CXCR4 and its main endogenous chemokine ligand, CXCL12, was shown to be sufficient for signal transduction (23), others reported homomeric CXCR4 complexes as functionally distinct units (24). Altogether, there is no clear picture of how and to what extent CXCR4 oligomerizes and how oligomerization is modulated. However, such an understanding would be needed as a basis to define druggable sites as well as ligands for this clinically important receptor.Here, we combine advanced microscopy methods—single-molecule microscopy and fluorescence fluctuation spectroscopy—to explore the quaternary organization of CXCR4 in living cells, at different expression levels and both under basal conditions and in the presence of diverse, chemically distinct CXCR4 ligands. We observe a transient formation of CXCR4 homodimers, which is dynamic, depends on expression levels, and is specifically disrupted by inverse agonists that bind to the minor subpocket of the receptor. Our data suggest a possible link between receptor dimerization and the basally active state of CXCR4.     

Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy

Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases and induces optic nerve regeneration in animal models. Paradoxically, however, although CNTF gene therapy promotes extensive regeneration, recombinant CNTF (rCNTF) has little effect. Because intraocular viral vectors induce inflammation, and because CNTF is an immune modulator, we investigated whether CNTF gene therapy acts indirectly through other immune mediators. The beneficial effects of CNTF gene therapy remained unchanged after deleting CNTF receptor alpha (CNTFRα) in retinal ganglion cells (RGCs), the projection neurons of the retina, but were diminished by depleting neutrophils or by genetically suppressing monocyte infiltration. CNTF gene therapy increased expression of C-C motif chemokine ligand 5 (CCL5) in immune cells and retinal glia, and recombinant CCL5 induced extensive axon regeneration. Conversely, CRISPR-mediated knockdown of the cognate receptor (CCR5) in RGCs or treating wild-type mice with a CCR5 antagonist repressed the effects of CNTF gene therapy. Thus, CCL5 is a previously unrecognized, potent activator of optic nerve regeneration and mediates many of the effects of CNTF gene therapy.

Like most pathways in the mature central nervous system (CNS), the optic nerve cannot regenerate once damaged due in part to cell-extrinsic suppressors of axon growth (1, 2) and the low intrinsic growth capacity of adult retinal ganglion cells (RGCs), the projection neurons of the eye (3–5). Consequently, traumatic or ischemic optic nerve injury or degenerative diseases such as glaucoma lead to irreversible visual losses. Experimentally, some degree of regeneration can be induced by intraocular inflammation or growth factors expressed by inflammatory cells (6–10), altering the cell-intrinsic growth potential of RGCs (3–5), enhancing physiological activity (11, 12), chelating free zinc (13, 14), and other manipulations (15–19). However, the extent of regeneration achieved to date remains modest, underlining the need for more effective therapies.Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for glaucoma and other ocular diseases (20–23). Activation of the downstream signal transduction cascade requires CNTF to bind to CNTF receptor-α (CNTFRα) (24), which leads to recruitment of glycoprotein 130 (gp130) and leukemia inhibitory factor receptor-β (LIFRβ) to form a tripartite receptor complex (25). CNTFRα anchors to the plasma membrane through a glycosylphosphatidylinositol linkage (26) and can be released and become soluble through phospholipase C-mediated cleavage (27). CNTF has been reported to activate STAT3 phosphorylation in retinal neurons, including RGCs, and to promote survival, but it is unknown whether these effects are mediated by direct action of CNTF on RGCs via CNTFRα (28). Our previous studies showed that CNTF promotes axon outgrowth from neonate RGCs in culture (29) but fails to do so in cultured mature RGCs (8) or in vivo (6). Although some studies report that recombinant CNTF (rCNTF) can promote optic nerve regeneration (20, 30, 31), others find little or no effect unless SOCS3 (suppressor of cytokine signaling-3), an inhibitor of the Jak-STAT pathway, is deleted in RGCs (5, 6, 32). In contrast, multiple studies show that adeno-associated virus (AAV)-mediated expression of CNTF in RGCs induces strong regeneration (33–40). The basis for the discrepant effects of rCNTF and CNTF gene therapy is unknown but is of considerable interest in view of the many promising clinical and preclinical outcomes obtained with CNTF to date.Because intravitreal virus injections induce inflammation (41), we investigated the possibility that CNTF, a known immune modulator (42–44), might act by elevating expression of other immune-derived factors. We report here that the beneficial effects of CNTF gene therapy in fact require immune system activation and elevation of C-C motif chemokine ligand 5 (CCL5). Depletion of neutrophils, global knockout (KO) or RGC-selective deletion of the CCL5 receptor CCR5, or a CCR5 antagonist all suppress the effects of CNTF gene therapy, whereas recombinant CCL5 (rCCL5) promotes axon regeneration and increases RGC survival. These studies point to CCL5 as a potent monotherapy for optic nerve regeneration and to the possibility that other applications of CNTF and other forms of gene therapy might similarly act indirectly through other factors.     

HIV envelope tail truncation confers resistance to SERINC5 restriction

SERINC5 is a potent lentiviral restriction factor that gets incorporated into nascent virions and inhibits viral fusion and infectivity. The envelope glycoprotein (Env) is a key determinant for SERINC restriction, but many aspects of this relationship remain incompletely understood, and the mechanism of SERINC5 restriction remains unresolved. Here, we have used mutants of HIV-1 and HIV-2 to show that truncation of the Env cytoplasmic tail (ΔCT) confers complete resistance of both viruses to SERINC5 and SERINC3 restriction. Critically, fusion of HIV-1 ΔCT virus was not inhibited by SERINC5 incorporation into virions, providing a mechanism to explain how EnvCT truncation allows escape from restriction. Neutralization and inhibitor assays showed ΔCT viruses have an altered Env conformation and fusion kinetics, suggesting that EnvCT truncation dysregulates the processivity of entry, in turn allowing Env to escape targeting by SERINC5. Furthermore, HIV-1 and HIV-2 ΔCT viruses were also resistant to IFITMs, another entry-targeting family of restriction factors. Notably, while the EnvCT is essential for Env incorporation into HIV-1 virions and spreading infection in T cells, HIV-2 does not require the EnvCT. Here, we reveal a mechanism by which human lentiviruses can evade two potent Env-targeting restriction factors but show key differences in the capacity of HIV-1 and HIV-2 to exploit this. Taken together, this study provides insights into the interplay between HIV and entry-targeting restriction factors, revealing viral plasticity toward mechanisms of escape and a key role for the long lentiviral EnvCT in regulating these processes.

The evolutionary arms race between human immunodeficiency virus type-1 (HIV-1) and host has allowed for efficient viral replication and transmission. Host cells are naturally hostile environments for invading pathogens and have evolved to express an arsenal of antiviral proteins, collectively termed restriction factors that target different steps of the viral life cycle to block replication. In response, viruses including pandemic HIV-1 and related lentiviruses have evolved to evade or directly antagonize host restriction factors, often by encoding viral accessory proteins (reviewed in refs. 1 and 2).The most recently described family of lentiviral restriction factors are the Serine Incorporator proteins (SERINCs) (3, 4). HIV-1 is most potently restricted by SERINC5 and to a lesser extent by SERINC3 (3, 4). In addition to human and simian lentiviruses, SERINC5 has broad antiviral activity against other retroviruses, including equine infectious anemia virus (5, 6) and murine leukemia virus (3, 4). SERINC5 is composed of 10 transmembrane domains and is primarily localized at the plasma membrane (7) where it gets incorporated into budding virions and inhibits viral entry (3, 4, 8). The lentiviral accessory protein Nef antagonizes SERINC5, excluding it from incorporation into viral particles (3, 4, 8) by removing SERINC5 from the plasma membrane via AP-2–dependent endocytosis (9). This relocalizes SERINC5 to the endosomal pathway, particularly into Rab5⁺ early endosomes, Rab7⁺ late endosomes, and eventually LAMP1+ compartments, for subsequent lysosomal degradation (9).Although the exact mechanism of how SERINC5 restricts lentiviruses remains incompletely understood, it is clear that the envelope glycoprotein (Env) is crucial in determining sensitivity to SERINC5 (8, 10–12). Env is a heterotrimeric structure composed of gp120 (receptor-binding subunit) and gp41, which contains an extracellular domain that includes the fusion peptide, a transmembrane domain, and a long but largely enigmatic 150-amino–acid cytoplasmic tail (reviewed in ref. 13). HIV-1 enters target cells by fusing with the host cell plasma membrane after the gp120 subunit engages entry receptors CD4 and coreceptor, either CXCR4 or CCR5. Biochemical studies suggest SERINC5 acts to inhibit small fusion pore formation, thereby reducing the efficiency of this fusion process (8), but whether SERINC5 directly interacts with Env to inhibit fusion, or whether indirect effects mediate restriction remains an open question (8, 11, 12). It has been proposed that SERINC5 extracellular loops 3 and 5 potentially interact with the membrane-proximal external region (MPER) of Env gp41 to mediate restriction (7). By contrast, a recent study investigating the effect of SERINC5 on Env clustering on virions indicated that SERINC5 clusters do not colocalize with Env clusters in the viral membrane (11), questioning the contribution of direct interactions to the restriction mechanism.The HIV-1 Env cytoplasmic tail (EnvCT) is crucial for Env trafficking to sites of virus assembly at the plasma membrane and for incorporation into virions, most importantly during viral replication in CD4+ T cells that are the main targets for HIV-1 in vivo (13–15). The membrane-proximal N terminus of the EnvCT contains a YxxL endocytic motif that binds the clathrin adaptor AP-2 and a C-terminal dileucine motif (16–19) that both act to limit the amount of Env present on the surface of infected cells. While the EnvCT clearly plays an important role in HIV-1 replication and virion morphogenesis, many aspects of the biology of this region of Env remain unclear. Most notably, why lentiviruses like HIV-1 require such a long EnvCT compared to related retroviruses that do not. Interestingly, the majority of the EnvCT is believed to be embedded within the plasma membrane (20) similar to SERINCs (7), where the EnvCT has been shown to influence Env mobility in membranes (21, 22). However, whether this critical region of Env interacts with SERINC5 and what role the EnvCT plays in SERINC5-mediated restriction remains unknown.Here, we examined the role of the EnvCT in determining sensitivity to SERINC5-mediated restriction with the goal of better understanding the complex relationship between Env and SERINC5. Using replication-competent viral mutants, we show that EnvCT truncation (ΔCT virus) renders both HIV-1 and HIV-2 resistant to SERINC5 and SERINC3 inhibition of infection, despite SERINC being incorporated into viral particles. Further analysis revealed that EnvCT truncation allowed HIV-1 to overcome the SERINC5-mediated block to viral fusion. Truncation of the EnvCT altered Env conformation and functionality, including fusogenicity, thermostability, and entry kinetics, all of which likely contribute to SERINC5 evasion. Furthermore, EnvCT truncation also rendered HIV-1 and HIV-2 viruses resistant to IFITM1, another entry-targeting restriction factor, suggesting an overlap between IFITM and SERINC restriction mechanism and viral evasion. Notably, while HIV-1 cannot replicate in T cells without the EnvCT, HIV-2 viruses are able to replicate efficiently, highlighting important differences in the requirement for the long EnvCT between pandemic HIV-1 and nonpandemic HIV-2. Our results suggest a model in which truncation of the cytoplasmic tail dysregulates Env conformation and functionality, allowing evasion from entry-targeting restriction factors, providing insights into the biology of the EnvCT, specifically its role in innate immune evasion and lentiviral replication.     

CD4 receptor diversity represents an ancient protection mechanism against primate lentiviruses

Infection with human and simian immunodeficiency viruses (HIV/SIV) requires binding of the viral envelope glycoprotein (Env) to the host protein CD4 on the surface of immune cells. Although invariant in humans, the Env binding domain of the chimpanzee CD4 is highly polymorphic, with nine coding variants circulating in wild populations. Here, we show that within-species CD4 diversity is not unique to chimpanzees but found in many African primate species. Characterizing the outermost (D1) domain of the CD4 protein in over 500 monkeys and apes, we found polymorphic residues in 24 of 29 primate species, with as many as 11 different coding variants identified within a single species. D1 domain amino acid replacements affected SIV Env-mediated cell entry in a single-round infection assay, restricting infection in a strain- and allele-specific fashion. Several identical CD4 polymorphisms, including the addition of N-linked glycosylation sites, were found in primate species from different genera, providing striking examples of parallel evolution. Moreover, seven different guenons (Cercopithecus spp.) shared multiple distinct D1 domain variants, pointing to long-term trans-specific polymorphism. These data indicate that the HIV/SIV Env binding region of the primate CD4 protein is highly variable, both within and between species, and suggest that this diversity has been maintained by balancing selection for millions of years, at least in part to confer protection against primate lentiviruses. Although long-term SIV-infected species have evolved specific mechanisms to avoid disease progression, primate lentiviruses are intrinsically pathogenic and have left their mark on the host genome.

Simian immunodeficiency viruses (SIVs) comprise a large group of lentiviruses that infect over 45 African primate species, including numerous guenons (Cercopithecus spp.), African green monkeys (Chlorocebus spp.), mandrills and drills (Mandrillus spp.), mangabeys (Cercocebus spp.), colobus monkeys (Colobus spp., Piliocolobus spp.), as well as chimpanzees (Pan troglodytes) and western gorillas (Gorilla gorilla) (1). Although the prevalence rates and geographic distribution of these infections vary widely, most SIVs are host-specific (i.e., their genomes form species-specific clusters in phylogenetic trees) (2–5). This has enabled the identification of instances when SIVs have crossed species barriers, including from apes and monkeys to humans (6). Phylogenetic analyses have shown that both pandemic and nonpandemic forms of HIV type 1 (HIV-1) resulted from the cross-species transmission of SIVs infecting central chimpanzees (P. troglodytes troglodytes) and western lowland gorillas (G. gorilla gorilla), while the various groups of HIV type 2 (HIV-2) emerged following the transfer of SIVsmm strains naturally infecting sooty mangabeys (Cercocebus atys) (6–9).SIVs have also jumped between nonhuman primate species, generating new SIV lineages. Cross-species transmission and recombination between ancestors of viruses today infecting greater spot-nosed (Cercopithecus nictitans), mustached (Cercopithecus cephus) and mona (Cercopithecus mona) monkeys (SIVgsn/SIVmus/SIVmon), and SIVrcm infecting red-capped mangabeys (Cercocebus torquatus) gave rise to SIVcpz in chimpanzees (10), and onward transmission of this virus to western lowland gorillas generated SIVgor (11). Additional cross-species transmissions and recombination events have generated mosaic SIV lineages in green monkeys (Chlorocebus sabaeus) and mandrills (Mandrillus sphinx) (12, 13). Finally, repeated introductions of diverse SIVs into the same primate species have resulted in cocirculating lineages, such as SIVkcol1 and SIVkcol2 in Kibale black-and-white colobus (Colobus guereza), and SIVmus-1, SIVmus-2, and SIVmus-3 in mustached monkeys (14, 15). Thus, primate lentiviruses have a high propensity to cross species barriers and have done so on numerous occasions throughout their evolutionary history.Lentiviruses have existed for tens of millions of years as evidenced by the finding of endogenous viruses in the genomes of species from four orders of mammals, including lemurs (16, 17), colugos (18), rabbits (19, 20), and weasels (21, 22). Some SIVs, such as those infecting green monkeys (Chlorocebus spp.) (23, 24) and the lhoesti group of guenons (Allochrocebus spp.) (25, 26) are at least several million years old because they appear to have coevolved with their respective hosts since these diverged from a common ancestor. Although an upper limit of 6 to 10 million y has been suggested for SIVs based on the fact that they have so far been found only in African, but not Asian, lineages of Old World monkeys (6), certain features of antiviral defense genes suggest that monkeys may have been exposed to lentiviruses long before this (27). Cellular restriction factors, such as APOBEC3G and TRIM5, are exquisitely antiviral and are counteracted by dedicated SIV accessary proteins. These restriction factors have evolved under strong positive selection at sites specifically involved in the interaction with lentiviruses, in both African and Asian monkeys (28, 29). However, if SIV indeed infected the common ancestor of African and Asian monkeys, this would imply numerous subsequent infection losses from multiple host lineages. Thus, it remains unclear when lentiviruses first infected primates.Among lentiviruses, those infecting primates are unique in their use of the CD4 receptor for entry into target cells. The viral envelope glycoprotein (Env) interacts with CD4 and subsequently undergoes conformational changes to expose the coreceptor binding site, which is required for viral–cell membrane fusion (30). CD4 is an immunoglobulin-like integral membrane protein that is expressed on multiple immune cells and stabilizes the interaction of the T cell receptor (TCR) with major histocompatibility complex class II (MHC II) molecules (31, 32). The most outward domain of CD4 (the D1 domain) binds a nonpolymorphic region on MHC II, which enhances TCR signaling (32). Importantly, the D1 domain is also the region that is bound by the HIV/SIV Env glycoprotein (33, 34). In HIV-infected humans and SIVmac-infected macaques, continuous high level viral replication leads to CD4⁺ T cell depletion, systemic immune activation, T cell exhaustion, and the development of AIDS (35, 36). Naturally occurring SIVs can also cause immunodeficiency and disease, as shown for chimpanzees and mandrills (37–40), indicating that these viruses are intrinsically pathogenic (41–43). However, a number of primate species with presumed longstanding SIV infections, such as African green monkeys, sooty mangabeys, and Ugandan red colobus monkeys (Piliocolobus tephrosceles), have evolved unique mechanisms that prevent disease progression despite continuous high viral replication (44–48). While the time required to evolve these adaptations is unknown, such protective mechanisms are absent from hosts that acquire new SIV infections.Unlike restriction factors, which prevent or limit viral replication, CD4 is a dependency factor (i.e., a host protein that is required for successful infection). Since there are many examples of host receptors coevolving with pathogens (49–51), it has been assumed that pressures exerted by pathogenic SIVs are responsible for the rapid diversification of primate CD4 (52–54). However, direct evidence for this hypothesis has been lacking. Examining the functional consequences of CD4 diversity in chimpanzees, we recently found that naturally occurring amino acid replacements in the D1 domain were able to inhibit SIVcpz infection, both in vitro and in vivo (55). Protective mechanisms included charged residues at the CD4–Env interface and steric hindrance between CD4- and Env-encoded glycans, which were effective not only against SIVcpz but also other SIVs that chimpanzees frequently encounter. These results suggested that CD4 diversity protects wild chimpanzee populations from SIV infection, possibly by conferring a heterozygote advantage (55). Since humans lack polymorphisms and glycans in the D1 domain, we asked whether CD4 diversification was a unique adaptation of chimpanzees. Sequencing the D1 domain in members of 36 African primate species, we identified a remarkable degree of CD4 diversity, both within and between species. The observed polymorphisms altered the cell entry of a panel of diverse SIV Envs, with the level of restriction depending on the particular allele and virus strain analyzed. Thus, the diversification of the primate CD4 receptor appears to have resulted from an ancient arms race between primate lentiviruses and their hosts.     

Drebrin regulates cytoskeleton dynamics in migrating neurons through interaction with CXCR4

Stromal cell-derived factor-1 (SDF-1) and chemokine receptor type 4 (CXCR4) are regulators of neuronal migration (e.g., GnRH neurons, cortical neurons, and hippocampal granule cells). However, how SDF-1/CXCR4 alters cytoskeletal components remains unclear. Developmentally regulated brain protein (drebrin) stabilizes actin polymerization, interacts with microtubule plus ends, and has been proposed to directly interact with CXCR4 in T cells. The current study examined, in mice, whether CXCR4 under SDF-1 stimulation interacts with drebrin to facilitate neuronal migration. Bioinformatic prediction of protein–protein interaction highlighted binding sites between drebrin and crystallized CXCR4. In migrating GnRH neurons, drebrin, CXCR4, and the microtubule plus-end binding protein EB1 were localized close to the cell membrane. Coimmunoprecipitation (co-IP) confirmed a direct interaction between drebrin and CXCR4 using wild-type E14.5 whole head and a GnRH cell line. Analysis of drebrin knockout (DBN1 KO) mice showed delayed migration of GnRH cells into the brain. A decrease in hippocampal granule cells was also detected, and co-IP confirmed a direct interaction between drebrin and CXCR4 in PN4 hippocampi. Migration assays on primary neurons established that inhibiting drebrin (either pharmacologically or using cells from DBN1 KO mice) prevented the effects of SDF-1 on neuronal movement. Bioinformatic prediction then identified binding sites between drebrin and the microtubule plus end protein, EB1, and super-resolution microscopy revealed decreased EB1 and drebrin coexpression after drebrin inhibition. Together, these data show a mechanism by which a chemokine, via a membrane receptor, communicates with the intracellular cytoskeleton in migrating neurons during central nervous system development.

Proper neuronal migration is essential for neural circuits formation (1) and requires coordination of extracellular signals and intracellular processes that results in cytoskeletal changes. Several modes of neuronal migration have been characterized, but all must transduce extracellular signals into appropriate movement. For bipolar migrating neurons (cerebellar granule cells (2, 3), cortical pyramidal cells (4), and neuroendocrine gonadotropin releasing hormone (GnRH) cells (5), the leading process extends toward a target, and the pulling forces of microtubule bundles anchored to the thin layer of cortical actin via end-binding proteins moves the nucleus forward. This process is sequentially repeated as the neuron migrates toward its final location (6–8). Although the basic dynamics of cell movement are being delineated, how a neuron ends up in the correct location, integrating extracellular guidance cues into cytoskeletal modifications, is still unclear.The stromal cell–derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) chemotactic axis is directly involved in migration of hippocampal (9, 10), cortical (4, 11), cerebellar (2, 12–14), and GnRH cells (15–17). Research in T lymphocytes documented SDF-1–induced cytoskeletal alterations, where rapid actin dynamics occur during formation of immune synapses (18). Developmentally regulated brain protein (drebrin), an actin filament side-binding protein that stabilizes the double-strained F-actin structure, was colocalized with the CXCR4 receptor cytoplasmic domain in these immune synapses. A physical interaction between CXCR4 and drebrin via coimmunoprecipitation (co-IP) was shown, suggesting that after SDF-1 activation, CXCR4 directly regulated cytoskeletal components by binding to drebrin. The function of drebrin in migrating neurons has only been analyzed in the rostral migratory stream, cerebellar granule neurons, and oculomotor neurons in mice. In these areas, Drebrin short hairpin RNA reduced the migration distance and velocity of migrating neurons (19–21).In contrast to drebrin, multiple studies have documented changes in neuronal migration after SDF-1/CXCR4 perturbations. SDF-1/CXCR4 has been shown to act as a chemoattractant for cerebellar granule cell progenitor migration and Purkinje neuron migration (2, 12), while in the developing cerebral cortex, it regulates interneuron migration (4, 11). In the hippocampus, meningeal/Cajal–Retzuis cells secrete SDF-1 (22). Here, the SDF-1 gradient is important for radial migration in the dentate gyrus (DG), specifically granule cells. CXCR4 mutant mice have a defect in granule cell position, while ectopic expression of SDF-1 disrupted DG granule cell migration (9). In addition, blocking SDF-1/CXCR4 signaling resulted in precocious differentiation, delayed migration, and ectopic granule cell progenitors (23). SDF-1/CXCR4 has also been well studied in the developing GnRH system. Neuroendocrine GnRH neurons migrate from the olfactory pit into the forebrain (5). The primary source of SDF-1 for GnRH neuronal migration are cells located close to the nasal midline cartilage, just beneath the cribriform plate (17). These cells create a gradient of SDF-1 that guides the GnRH neurons to the nasal/forebrain junction. Silencing of CXCR4 results in fewer GnRH neurons reaching the nasal forebrain junction (15, 16), while application of SDF-1 augments GnRH saltatory movement (17), a moving pattern similar to radial migrating neurons (24). Although CXCR4 on the plasma membrane has been shown to be internalized upon ligand binding (23), how SDF-1/CXCR4 regulates cytoskeleton dynamics in neurons is still unclear.The present study examined the interaction of drebrin and CXCR4 in migrating neurons. Bioinformatic analysis predicted a protein–protein interaction between drebrin and CXCR4. Co-IP confirmed a direct interaction between drebrin and CXCR4 in GnRH cells and in hippocampal areas. Analysis of drebrin knockout (DBN1 KO) mice showed delayed migration of GnRH cells into the brain and disrupted organization of granule cells in the hippocampus. Migration assays on primary GnRH cells, as well as modified Boyden chamber assays on primary DG granule cells, established that inhibiting drebrin (either pharmacologically or using cells from DBN1 KO mice) prevented the effects of SDF-1/CXCR4 on neuronal movement. Bioinformatic prediction then was used to identify potential binding sites between drebrin and the microtubule plus end protein EB1, and super-resolution microscopy revealed decreased EB1 and drebrin coexpression after drebrin or CXCR4 inhibition. Together, these data show a mechanism by which a chemokine, via a membrane receptor, communicates with the intracellular cytoskeleton in migrating neurons during early central nervous system (CNS) development.     

Mutational fitness landscapes reveal genetic and structural improvement pathways for a vaccine-elicited HIV-1 broadly neutralizing antibody

Vaccine-based elicitation of broadly neutralizing antibodies holds great promise for preventing HIV-1 transmission. However, the key biophysical markers of improved antibody recognition remain uncertain in the diverse landscape of potential antibody mutation pathways, and a more complete understanding of anti–HIV-1 fusion peptide (FP) antibody development will accelerate rational vaccine designs. Here we survey the mutational landscape of the vaccine-elicited anti-FP antibody, vFP16.02, to determine the genetic, structural, and functional features associated with antibody improvement or fitness. Using site-saturation mutagenesis and yeast display functional screening, we found that 1% of possible single mutations improved HIV-1 envelope trimer (Env) affinity, but generally comprised rare somatic hypermutations that may not arise frequently in vivo. We observed that many single mutations in the vFP16.02 Fab could enhance affinity >1,000-fold against soluble FP, although affinity improvements against the HIV-1 trimer were more measured and rare. The most potent variants enhanced affinity to both soluble FP and Env, had mutations concentrated in antibody framework regions, and achieved up to 37% neutralization breadth compared to 28% neutralization of the template antibody. Altered heavy- and light-chain interface angles and conformational dynamics, as well as reduced Fab thermal stability, were associated with improved HIV-1 neutralization breadth and potency. We also observed parallel sets of mutations that enhanced viral neutralization through similar structural mechanisms. These data provide a quantitative understanding of the mutational landscape for vaccine-elicited FP-directed broadly neutralizing antibody and demonstrate that numerous antigen-distal framework mutations can improve antibody function by enhancing affinity simultaneously toward HIV-1 Env and FP.

The tremendous circulating sequence diversity of HIV-1 and its capacity to evade host immunity pose unique challenges for vaccine design (reviewed in refs. 1, 2). Broadly neutralizing antibodies (bNAbs) identified from HIV-1 patients target conserved vulnerable epitopes on the HIV-1 envelope protein (Env) to prevent HIV-1 infection, and HIV-1 bNAb elicitation has become a major goal for HIV-1 vaccine design (3, 4). Several HIV-1 vulnerable epitopes have been described (5) including the following: the V1V2 apex (6, 7), the CD4-binding site (8–10), the membrane-proximal external region (11, 12), the glycan-V3 region (also known as N332 glycan supersite) (10, 13), the highly glycosylated region at the center of the silent face on the gp120 subunit (14), and the fusion peptide (FP), which is required for viral entry (15–17). Several complementary approaches seek to develop immunogens that elicit broadly neutralizing HIV-1 antibodies, with promising results (4, 18, 19). However, vaccine-elicited HIV-1 antibodies are often either less potent or less broad than many of the bNAbs identified from human patients. There is a pressing need to better understand bNAb developmental pathways and outline the genetic and structural antibody features that can provide enhanced neutralization breadth and potency for HIV-1 vaccines.Clinical data show that broadly neutralizing serum antibodies develop naturally in around 20% of individuals with chronic HIV-1 infection (20, 21) and that a smaller number of individuals show highly potent neutralization (22, 23). bNAbs develop via the accumulation of somatic hypermutations (SHM) and affinity maturation following their initial B-cell selection and expansion. While HIV-1–infected individuals may have high titers against HIV-1 antigens, the rarity of bNAbs suggests that the mutations acquired during bNAb development are correspondingly rare and/or that the mutational pathways to effective bNAb development are explored only in the context of chronic HIV-1 antigen exposure over years of viral infection (24). Evidence that high viral load is correlated with higher HIV-1 serum breadth and potency also suggests that larger numbers of sampled antibody maturation pathways are correlated with broad HIV-1 neutralization (20). A major question for HIV-1 vaccine design is thus how to quickly and effectively induce B-cell maturation to bNAbs from a small number of controlled immunizations.HIV-1 bNAbs are often highly somatically mutated (25–28), and reverting these mutations to antibody germline sequences results in drastic reductions of neutralization breadth and potency (9, 29–31). Complementarity determining regions (CDRs) are known to be mutational hotspots and are often in direct contact with antigen to enable recognition. Antibody framework region (FR) mutations can also modulate neutralization breadth and potency by altering the structural orientation, particularly at heavy:light interface alignments, and by altering the intrinsic flexibility of the paratope (16, 32–35). Not all observed bNAb somatic mutations are required for high neutralization breadth and potency (36), and a better understanding of the critical mutations and antibody structural features (both naturally elicited and vaccine-induced) to improve neutralization breadth and potency would enhance our understanding of bNAb development and guide efficient HIV-1 vaccine strategies (16, 17, 37–41).Among various immunization approaches reported for eliciting broadly neutralizing antibodies (16–19, 38–42), one strategy targets the HIV-1 FP epitope and has elicited 59 and 31% HIV-1 neutralization breadth in rhesus macaques and mice, respectively, against a broad panel of 208 HIV-1 strains (16, 17). Priming the immune response with soluble FP followed by three Env trimer boosts elicited antibodies with FP-targeted trimer recognition and broad HIV-1 neutralization; however, the structural mechanisms that enable effective anti-FP antibody maturation and pathway selection are not fully understood. Several key unanswered questions include the following: 1) What are the structural features of FP recognition vs. trimer recognition that may appropriately guide antibody development?; 2) What critical parameters control virus neutralization of FP-targeting bNAbs?; and 3) How do beneficial mutations fit into the entire landscape of possible mutational pathways, and what fraction of possible mutations provide increased affinity, neutralization breadth, and potency?To address these questions, we characterized the genetic and functional fitness landscape of an anti-FP bNAb and used biophysical and structural techniques to follow the mechanisms of antibody improvement. We implemented these screening strategies using the anti-FP bNAb vFP16.02, a vaccine-elicited antibody that neutralizes ∼30% of HIV-1 viral isolates (16). We applied yeast display and fluorescence-activated cell sorting (FACS) coupled with next-generation sequencing (NGS) to identify single mutations that enhanced binding or fitness to multiple HIV-1 BG505 Env trimer variants (Fig. 1). We mapped possible single mutations by their functional impacts and identified a panel of mutations with enhanced binding affinity and neutralization. Structural analyses of a subset of these mutations provided insights into the mechanisms of enhanced neutralization. These data confirm that several parallel mutational pathways exist for HIV-1 bNAb improvement and underscore the importance of improved Env trimer affinity for enhancing neutralization potency and breadth in HIV-1 vaccine designs.Open in a separate windowFig. 1.Experimental workflow for comprehensive functional analysis of all possible single mutations in an HIV-1 bNAb. SSM libraries were designed for VH and VL regions of the anti–HIV-1 FP bNAb vFP16.02. SSM libraries were cloned into yeast Fab surface display libraries and screened by FACS to determine the functional impact of each possible single mutation. Single-mutation display libraries were sorted for their affinity against a BG505 DS-SOSIP HIV-1 Env trimer with two different FP sequences. Sorted yeast libraries were prepped for NGS to enable quantitative variant tracking across three screening rounds; bioinformatic analyses of NGS data were used to interpret the functional impact of each possible amino acid mutation. Selected variants were characterized for neutralization activity and affinity against soluble FP and against HIV-1 Env. Structural analyses were performed to understand the mechanistic basis of anti–HIV-1 FP bNAb improvement.     

CCR5/CD4/CXCR4 oligomerization prevents HIV-1 gp120IIIB binding to the cell surface

CCR5 and CXCR4, the respective cell surface coreceptors of R5 and X4 HIV-1 strains, both form heterodimers with CD4, the principal HIV-1 receptor. Using several resonance energy transfer techniques, we determined that CD4, CXCR4, and CCR5 formed heterotrimers, and that CCR5 coexpression altered the conformation of both CXCR4/CXCR4 homodimers and CD4/CXCR4 heterodimers. As a result, binding of the HIV-1 envelope protein gp120_IIIB to the CD4/CXCR4/CCR5 heterooligomer was negligible, and the gp120-induced cytoskeletal rearrangements necessary for HIV-1 entry were prevented. CCR5 reduced HIV-1 envelope-induced CD4/CXCR4-mediated cell-cell fusion. In nucleofected Jurkat CD4 cells and primary human CD4⁺ T cells, CCR5 expression led to a reduction in X4 HIV-1 infectivity. These findings can help to understand why X4 HIV-1 strains infection affect T-cell types differently during AIDS development and indicate that receptor oligomerization might be a target for previously unidentified therapeutic approaches for AIDS intervention.For HIV-1 to enter a target cell, the viral envelope glycoprotein gp120 must interact with a set of cell surface molecules that include the primary receptor, CD4 (1), and a chemokine receptor (CCR5 or CXCR4) that acts as a coreceptor (2, 3). These molecules form CD4/chemokine receptor complexes, as deduced from coprecipitation data for CXCR4 or CCR5 with CD4 (4–8).Most HIV-1 variants isolated from newly infected individuals use CCR5 and CD4 to enter host cells; these M-tropic R5 strains are predominant in acute and asymptomatic phases of HIV infection. CD4⁺ T helper type 1 (Th1) cells, which express high CCR5 levels (9, 10), are implicated in maintaining asymptomatic status (11, 12). The “viral shift” from R5 to T-tropic X4 HIV-1 strains correlates with AIDS progression (13, 14). X4 strains infect mainly CD4⁺ Th2 cells, which express little CCR5 and whose CXCR4 levels resemble those of Th1 cells (15, 16), which suggests that cell susceptibility to HIV-1 infection depends on the CD4/coreceptor ratio and on receptor levels during cell activation and/or differentiation (17). CXCR4 and CCR5 are present as homodimers and heterodimers at the plasma membrane (18–20). In addition, gp120-mediated CD4/CXCR4 and CD4/CCR5 association and clustering is reported (21–23). Nonetheless, little is known of how CCR5 expression influences the CD4/CXCR4 interaction, or of the molecular basis that underlies the differences in X4 strains infection relative to CCR5 levels at the cell surface.Here, we identify CD4/CXCR4/CCR5 oligomers at the cell membrane, even in the absence of ligands. CCR5 expression in these complexes modifies the heterodimeric CD4/CXCR4 conformation and blocks gp120_IIIB binding, without altering binding of the CXCR4 ligand CXCL12 and its subsequent signaling. gp120_IIIB-triggered LIMK1 activation, cofilin dephosphorylation, and the actin cytoskeleton rearrangement necessary for cell-cell fusion were impeded in CD4/CXCR4/CCR5-expressing cells. The data obtained using recombinant gp120_IIIB glycoprotein were confirmed by experiments showing that X4 HIV-1 infection of Jurkat and primary T cells is regulated by CCR5 expression.     

The high-affinity immunoglobulin receptor FcγRI potentiates HIV-1 neutralization via antibodies against the gp41 N-heptad repeat

The HIV-1 gp41 N-heptad repeat (NHR) region of the prehairpin intermediate, which is transiently exposed during HIV-1 viral membrane fusion, is a validated clinical target in humans and is inhibited by the Food and Drug Administration (FDA)-approved drug enfuvirtide. However, vaccine candidates targeting the NHR have yielded only modest neutralization activities in animals; this inhibition has been largely restricted to tier-1 viruses, which are most sensitive to neutralization by sera from HIV-1–infected individuals. Here, we show that the neutralization activity of the well-characterized NHR-targeting antibody D5 is potentiated >5,000-fold in TZM-bl cells expressing FcγRI compared with those without, resulting in neutralization of many tier-2 viruses (which are less susceptible to neutralization by sera from HIV-1–infected individuals and are the target of current antibody-based vaccine efforts). Further, antisera from guinea pigs immunized with the NHR-based vaccine candidate (ccIZN36)₃ neutralized tier-2 viruses from multiple clades in an FcγRI-dependent manner. As FcγRI is expressed on macrophages and dendritic cells, which are present at mucosal surfaces and are implicated in the early establishment of HIV-1 infection following sexual transmission, these results may be important in the development of a prophylactic HIV-1 vaccine.

Membrane fusion between HIV-1 and host cells is mediated by the viral envelope glycoprotein (Env), a trimer consisting of the gp120 and gp41 subunits. Upon interaction with cellular receptors, Env undergoes a dramatic conformational change and forms the prehairpin intermediate (PHI) (1–3), in which the fusion peptide region at the amino terminus of gp41 inserts into the cell membrane. In the PHI, the N-heptad repeat (NHR) region of gp41 is exposed and forms a stable, three-stranded α-helical coiled coil. Subsequently, the PHI resolves when the NHR and the C-heptad repeat (CHR) regions of gp41 associate to form a trimer-of-hairpins structure that brings the viral and cell membranes into proximity, facilitating membrane fusion (Fig. 1).Open in a separate windowFig. 1.HIV-1 membrane fusion. The surface protein of the HIV-1 envelope is composed of the gp120 and gp41 subunits. After Env binds to cell-surface receptors, gp41 inserts into the host cell membrane and undergoes a conformational change to form the prehairpin intermediate. The N-heptad repeat (orange) region of gp41 is exposed in the PHI and forms a three-stranded coiled coil. To complete viral fusion, the PHI resolves to a trimer-of-hairpins structure in which the C-heptad repeat (blue) adopts a helical conformation and binds the NHR region. Fusion inhibitors such as enfuvirtide bind the NHR, preventing viral fusion by inhibiting formation of the trimer of hairpins (1–3). The membrane-proximal external region (red) is located adjacent to the transmembrane (TM) region of gp41.The NHR region of the PHI is a validated therapeutic target in humans: the Food and Drug Administration (FDA)-approved drug enfuvirtide binds the NHR and inhibits viral entry into cells (4, 5). Various versions of the three-stranded coiled coil formed by the NHR have been created and used as vaccine candidates in animals (6–10). The neutralization potencies of these antisera, as well as those of anti-NHR monoclonal antibodies (mAbs) (11–15), are modest and mostly limited to HIV-1 isolates that are highly sensitive to antibody-mediated neutralization [commonly referred to as tier-1 viruses (16)]. These results have led to skepticism about the PHI as a vaccine target.Earlier studies showed that the neutralization activities of mAbs that bound another region of gp41, the membrane-proximal external region (MPER) (Fig. 1), were enhanced as much as 5,000-fold in cells expressing FcγRI (CD64) (17, 18), an integral membrane protein that binds the Fc portion of immunoglobulin G (IgG) molecules with high (nanomolar) affinity (19, 20). This effect was not attributed to phagocytosis and occurred when the cells were preincubated with antibody and washed before adding virus (17, 18). Since the MPER is a partially cryptic epitope that is not fully exposed until after Env engages with cellular receptors (21, 22), these results suggest that by binding the Fc region, FcγRI provides a local concentration advantage for MPER mAbs at the cell surface that enhances viral neutralization (17, 18). While not expressed on T cells, FcγRI is expressed on macrophages and dendritic cells (23), which are present at mucosal surfaces and are implicated in sexual HIV-1 transmission and the early establishment of HIV-1 infection (22–34).Here we investigated whether FcγRI expression also potentiates the neutralizing activity of antibodies targeting the NHR, since that region, like the MPER, is preferentially exposed during viral fusion. We found that D5, a well-characterized anti-NHR mAb (11, 12), inhibits HIV-1 infection ∼5,000-fold more potently in TZM-bl cells expressing FcγRI (TZM-bl/FcγRI cells) than in TZM-bl cells that do not. Further, while antisera from guinea pigs immunized with (ccIZN36)₃, an NHR-based vaccine candidate (7), displayed weak neutralizing activity in TZM-bl cells, they exhibited enhanced neutralization in TZM-bl/FcγRI cells, including against some tier-2 HIV-1 isolates that are more resistant to antibody-mediated neutralization (16) and that serve as benchmarks for antibody-based vaccine efforts. These results indicate that FcγRI can play an important role in neutralization by antibodies that target the PHI. Since these receptors are expressed on cells prevalent at mucosal surfaces thought to be important for sexual HIV-1 transmission, our results motivate vaccine strategies that harness this potentiating effect.     

Tropospheric carbonyl sulfide mass balance based on direct measurements of sulfur isotopes

Robust estimates for the rates and trends in terrestrial gross primary production (GPP; plant CO₂ uptake) are needed. Carbonyl sulfide (COS) is the major long-lived sulfur-bearing gas in the atmosphere and a promising proxy for GPP. Large uncertainties in estimating the relative magnitude of the COS sources and sinks limit this approach. Sulfur isotope measurements (³⁴S/³²S; δ³⁴S) have been suggested as a useful tool to constrain COS sources. Yet such measurements are currently scarce for the atmosphere and absent for the marine source and the plant sink, which are two main fluxes. Here we present sulfur isotopes measurements of marine and atmospheric COS, and of plant-uptake fractionation experiments. These measurements resulted in a complete data-based tropospheric COS isotopic mass balance, which allows improved partition of the sources. We found an isotopic (δ³⁴S ± SE) value of 13.9 ± 0.1‰ for the troposphere, with an isotopic seasonal cycle driven by plant uptake. This seasonality agrees with a fractionation of −1.9 ± 0.3‰ which we measured in plant-chamber experiments. Air samples with strong anthropogenic influence indicated an anthropogenic COS isotopic value of 8 ± 1‰. Samples of seawater-equilibrated-air indicate that the marine COS source has an isotopic value of 14.7 ± 1‰. Using our data-based mass balance, we constrained the relative contribution of the two main tropospheric COS sources resulting in 40 ± 17% for the anthropogenic source and 60 ± 20% for the oceanic source. This constraint is important for a better understanding of the global COS budget and its improved use for GPP determination.

The Earth system is going through rapid changes as the climate warms and CO₂ level rises. This rise in CO₂ is mitigated by plant uptake; hence, it is important to estimate global and regional photosynthesis rates and trends (1). Yet, robust tools for investigating these processes at a large scale are scarce (2). Recent studies suggest that carbonyl sulfide (COS) could provide an improved constraint on terrestrial photosynthesis (gross primary production, GPP) (2–12). COS is the major long-lived sulfur-bearing gas in the atmosphere and the main supplier of sulfur to the stratospheric sulfate aerosol layer (13), which exerts a cooling effect on the Earth’s surface and regulates stratospheric ozone chemistry (14).During terrestrial photosynthesis, COS diffuses into leaf stomata and is consumed by photosynthetic enzymes in a similar manner to CO₂ (3–5). Contrary to CO₂, COS undergoes rapid and irreversible hydrolysis mainly by the enzyme carbonic-anhydrase (6, 7). Thus, COS can be used as a proxy for the one-way flux of CO₂ removal from the atmosphere by terrestrial photosynthesis (2, 8–11). However, the large uncertainties in estimating the COS sources weaken this approach (10–12, 15). Tropospheric COS has two main sources: the oceans and anthropogenic emissions, and one main sink–terrestrial plant uptake (8, 10–13). Smaller sources include biomass burning, soil emissions, wetlands, volcanoes, and smaller sinks include OH destruction, stratospheric destruction, and soil uptake (12). The largest source of COS to the atmosphere is the ocean, both as direct COS emission, and as indirect carbon disulfide (CS₂) and dimethylsulfide (DMS) emissions that are rapidly oxidized to COS (10, 16–20). Recent studies suggest oceanic COS emissions are in the range of 200–4,000 GgS/y (19–22). The second major COS source is the anthropogenic source, which is dominated by indirect emissions derived from CS₂ oxidation, mainly from the use of CS₂ as an industrial solvent. Direct emissions of COS are mainly derived from coal and fuel combustion (17, 23, 24). Recent studies suggest that anthropogenic emissions are in the range of 150–585 GgS/y (23, 24). The terrestrial plant uptake is estimated to be in the range of 400–1,360 GgS/y (11). Measurements of sulfur isotope ratios (δ³⁴S) in COS may be used to track COS sources and thus reduce the uncertainties in their flux estimations (15, 25–27). However, the isotopic mass balance approach works best if the COS end members are directly measured and have a significantly different isotopic signature. Previous δ³⁴S measurements of atmospheric COS are scarce and there have been no direct measurements of two important components: the δ³⁴S of oceanic COS emissions, and the isotopic fractionation of COS during plant uptake (15, 25–27). In contrast to previous studies that used assessments for these isotopic values, our aim was to directly measure the isotopic values of these missing components, and to determine the tropospheric COS δ³⁴S variability over space and time.     

Genetically edited CD34+ cells derived from human iPS cells in vivo but not in vitro engraft and differentiate into HIV-resistant cells

Genetic editing of induced pluripotent stem (iPS) cells represents a promising avenue for an HIV cure. However, certain challenges remain before bringing this approach to the clinic. Among them, in vivo engraftment of cells genetically edited in vitro needs to be achieved. In this study, CD34⁺ cells derived in vitro from iPS cells genetically modified to carry the CCR5Δ32 mutant alleles did not engraft in humanized immunodeficient mice. However, the CD34⁺ cells isolated from teratomas generated in vivo from these genetically edited iPS cells engrafted in all experiments. These CD34⁺ cells also gave rise to peripheral blood mononuclear cells in the mice that, when inoculated with HIV in cell culture, were resistant to HIV R5-tropic isolates. This study indicates that teratomas can provide an environment that can help evaluate the engraftment potential of CD34⁺ cells derived from the genetically modified iPS cells in vitro. The results further confirm the possibility of using genetically engineered iPS cells to derive engraftable hematopoietic stem cells resistant to HIV as an approach toward an HIV cure.

A major objective of recent HIV research is to develop a “cure” for this virus infection that avoids lifelong adherence to antiretroviral therapy (ART). One of the approaches toward reaching this objective has been to genetically delete or mutate genes encoding for proteins that promote HIV infection and spread. An attractive candidate for this strategy is the Ccr5 gene, for which a genetic mutation causing a 32-bp deletion has been shown to be associated with natural protection from HIV infection and disease (1, 2). The Ccr5 gene encodes CCR5, a human cell-surface chemokine receptor that is a coreceptor for HIV attachment and infection of cells (3, 4). The Ccr5 allele with its 32-bp deletion results in a truncated isoform of the CCR5 receptor, CCR5Δ32, which is not expressed at the cell surface. Thus, entry of the virus into the cell is blocked (5).Induced pluripotent stem (iPS) cells (6), because of their capacity to differentiate into CD34⁺ hematopoietic stem cells (HSCs) (7), can reconstitute a full immune system (8, 9). These iPS cells are therefore a target of choice for genetic engineering. Our group and others have demonstrated that iPS cells generated from the peripheral blood mononuclear cells (PBMC) of both healthy individuals (10) and HIV-infected patients under ART (11) can have their wild-type allele of the Ccr5 gene genetically edited to carry the Ccr5 Δ32 mutation (12, 13). Notably, using CRISPR/Cas9 technology, the Ccr5 gene can be modified to have the naturally occurring Δ32 variant allele that has been associated with resistance to R5-tropic viruses. Moreover, while it is not present at the cell surface, the truncated CCR5Δ32 protein is still expressed and, as such, could have other important physiological roles (14–17).We have confirmed that the genetically modified Ccr5 Δ32 iPS cells can be differentiated into CD34⁺ HSCs in vitro (10, 18). Under appropriate cell culture conditions, they can give rise to various myeloid and lymphoid cell lineages (10, 11, 18). This result can also be observed with the formation of teratomas following the injection of large quantities of iPS cells into mice. Teratomas are multicellular tumors composed of many different cell types including HSCs. Notably, immune cells with the CCR5Δ32 mutation differentiated in vitro from the genetically modified iPS cell-derived HSCs and inoculated with HIV are resistant to R5-tropic virus infection (10, 18).These results have suggested that editing Ccr5 in iPS cells from HIV-infected subjects can be a promising strategy toward an HIV cure. The pluripotent stem cells can be induced from a small number of PBMC from the patients and genetically modified to become resistant to HIV infection (10, 11, 18). In this case, leukapheresis to obtain large amounts of these cells (19) is not required. The edited HSCs could then be transplanted back to the original patient without concern for immune cell rejection. Therefore, because these experiments were performed in cell culture, an important remaining question is whether in vitro-edited iPS cells can differentiate into HSCs that can be transplanted back into a recipient in vivo (20).To address this question, transplantation of the in vitro-derived CD34⁺ cells was attempted under various conditions in animal models of humanized or immunodeficient mice (21). In approaches to obtain sufficient numbers of CD34⁺ cells for transplantation, our ability to grow them in vitro offered an opportunity. However, although we could expand CD34⁺ cells substantially in culture (18), we observed that engraftment of these cell culture-derived CD34⁺ cells in humanized NSG-BLT mice did not occur. Thus, alternatively, to study the genetically edited cells in vivo, we explored the use of differentiated CD34⁺ cells in vivo via the generation of teratomas from iPS cells. We found that not only did these teratomas successfully yield human CD34⁺ cells, but importantly, these CD34⁺ cells could engraft in recipient immunodeficient NSG mice. This observation has been made by Nakauchi and colleagues (22) with different mouse strains. Finally, we confirmed that the PBMC formed in mice from these teratoma-derived genetically edited CD34⁺ cells are resistant to ex vivo R5-tropic HIV infection when they carry the mutant Δ32 Ccr5 allele.     

Scavenging of soluble and immobilized CCL21 by ACKR4 regulates peripheral dendritic cell emigration

Leukocyte homing driven by the chemokine CCL21 is pivotal for adaptive immunity because it controls dendritic cell (DC) and T cell migration through CCR7. ACKR4 scavenges CCL21 and has been shown to play an essential role in DC trafficking at the steady state and during immune responses to tumors and cutaneous inflammation. However, the mechanism by which ACKR4 regulates peripheral DC migration is unknown, and the extent to which it regulates CCL21 in steady-state skin and lymph nodes (LNs) is contested. Specifically, our previous findings that CCL21 levels are increased in LNs of ACKR4-deficient mice [I. Comerford et al., Blood 116, 4130–4140 (2010)] were refuted [M. H. Ulvmar et al., Nat. Immunol. 15, 623–630 (2014)], and no differences in CCL21 levels in steady-state skin of ACKR4-deficient mice were reported despite compromised CCR7-dependent DC egress in these animals [S. A. Bryce et al., J. Immunol. 196, 3341–3353 (2016)]. Here, we resolve these issues and reveal that two forms of CCL21, full-length immobilized and cleaved soluble CCL21, exist in steady-state barrier tissues, and both are regulated by ACKR4. Without ACKR4, extracellular CCL21 gradients in barrier sites are saturated and nonfunctional, DCs cannot home directly to lymphatic vessels, and excess soluble CCL21 from peripheral tissues pollutes downstream LNs. The results identify the mechanism by which ACKR4 controls DC migration in barrier tissues and reveal a complex mode of CCL21 regulation in vivo, which enhances understanding of functional chemokine gradient formation.

CCL21 is a chemokine that mediates recruitment of multiple leukocyte subsets through CCR7-mediated signaling during the steady state and inflammation. CCL21 plays crucial roles in priming adaptive immunity via governing egress of dendritic cells (DCs) from barrier tissues and T cell entry and positioning in secondary lymphoid organs (1–5). A well-characterized site of CCL21 gradient formation is the skin, where CCL21 is secreted by lymphatic endothelial cells (LECs) and immobilized on extracellular heparan sulfate moieties via interactions with the charged, elongated C-terminal tail of CCL21 (6–8). Here, immobilized CCL21 gradients are essential for interstitial DC trafficking toward lymphatic vessels (LVs) (8), after which CCL21 further contributes to LV attachment (9), infiltration (10), downstream luminal migration (11), and migration from the lymph node (LN) subcapsular sinus (SCS) to the paracortex (12). In-vitro studies have shown that the C-terminal tail of CCL21 can also be proteolytically cleaved by mature DCs to generate solubilized CCL21 (13), with signaling properties distinct from another soluble CCR7 ligand, CCL19 (14, 15). While CCL19 is dispensable for steady-state DC migration (16), important questions regarding the in vivo processing and function of cleaved CCL21 remain.Both forms of CCL21 are also ligands for the atypical chemokine receptor ACKR4 (17), which regulates chemokine bioavailability rather than directly mediating cell migration. ACKR4 expression has been identified in multiple barrier tissues (18–20) and lymphoid tissues (12, 21) where expression is largely restricted to stromal cell populations, with the exception of germinal-center B cells (22). Despite clear evidence of ACKR4 scavenging of CCL21 in vitro, the extent to which it regulates CCL21 in vivo is disputed. We have shown increased CCL21 in the LNs of Ackr4^−/− mice, which was associated with exacerbated Th17 responses in autoimmunity (23) and an ACKR4-dependent increase in CCL21 in tumors that promotes antitumor immunity (24). However, no differences in dermal CCL21 abundance were previously reported in steady-state Ackr4^−/− mice despite steady-state CCR7-dependent DC migratory defects being independent of CCL19 (19), and the contribution of ACKR4 in regulating LN CCL21 abundance has been disputed despite a clear role for ACKR4 in maintaining interfollicular CCL21 gradients in LN (12). These discrepancies have remained unresolved but point to previously unrecognized complexity in ACKR4-dependent regulation of CCL21 in both barrier and lymphoid tissues.     

Environmental noise degrades hippocampus-related learning and memory

The neural mechanisms underlying the impacts of noise on nonauditory function, particularly learning and memory, remain largely unknown. Here, we demonstrate that rats exposed postnatally (between postnatal days 9 and 56) to structured noise delivered at a sound pressure level of ∼65 dB displayed significantly degraded hippocampus-related learning and memory abilities. Noise exposure also suppressed the induction of hippocampal long-term potentiation (LTP). In parallel, the total or phosphorylated levels of certain LTP-related key signaling molecules in the synapses of the hippocampus were down-regulated. However, no significant changes in stress-related processes were found for the noise-exposed rats. These results in a rodent model indicate that even moderate-level noise with little effect on stress status can substantially impair hippocampus-related learning and memory by altering the plasticity of synaptic transmission. They support the importance of more thoroughly defining the unappreciated hazards of moderately loud noise in modern human environments.

The noise pollution accompanying industrialization is a risk factor to human health. Earlier studies have extensively examined the deleterious impacts of noise in the auditory systems of both humans and animal models (1–6), showing that noise exposure either early or late in life can induce progressive hearing loss, change neural coding along the auditory pathway, and alter auditory-related perception and behavior.The auditory system, however, contains direct and indirect pathways to other systems and structures of the brain that are necessary for functional integration. For example, earlier studies found that the hippocampus, the core area of the brain associated with learning and memory processes, receives neuronal inputs from the auditory system through the lemniscal and nonlemniscal pathways (7–11). It is thus conceivable that noise-evoked activities might be transmitted via these connections to the hippocampus, thereby affecting learning and memory. Indeed, animal studies have shown that exposure to loud noise (e.g., above a sound pressure level [SPL] of 95 dB) that induces temporary or permanent shifts in the auditory threshold disrupts hippocampal histology, decreases neurogenesis in the hippocampus, and impairs hippocampus-related learning and memory abilities (12–16). In addition, epidemiological studies have demonstrated that environment noise has substantially negative effects on children’s learning outcomes and cognitive abilities (17–19). While the usual explanations for the origins of these noise-induced effects on nonauditory functions have relied on stress-related processes (15, 16, 20–23), the underlying neural mechanisms remain largely unknown.In this study, we exposed rat pups to structured noise delivered at ∼65 dB SPL for a 7-wk period. Exposure to a moderate level of modulated broad-spectrum noise more realistically models the noise environments people encountered in industrial workplaces and other modern acoustic settings (2, 4, 24–26). We then evaluated the behavioral consequences of noise exposure on hippocampus-related learning and memory for these noise-exposed rats. In addition, we explored the mechanisms underlying possible postexposure changes in learning and memory via physiological and molecular assessments of the hippocampus.