CD4 mimetics sensitize HIV-1-infected cells to ADCC

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.Worldwide, it is estimated that more than 35 million people are living with HIV. In 2013 alone, around 2.1 million people became newly infected with HIV, and 1.5 million people died from AIDS (1). Measures to prevent HIV-1 transmission are desperately needed. Prevention of HIV-1 transmission and progression likely requires approaches that can specifically target and eliminate HIV-1-infected cells. Interestingly, there is increasing evidence supporting a role of antibody (Ab)-dependent cell-mediated cytotoxicity (ADCC) in controlling HIV-1 transmission and disease progression (2–8). Analysis of the correlates of protection in the RV144 vaccine trial suggested that increased ADCC activity was linked with decreased HIV-1 acquisition (9), and Abs with potent ADCC activity were isolated from some RV144 vaccinees (10). Recent studies reported that the viral accessory proteins Nef and Vpu protect HIV-1-infected cells from anti-HIV-1 envelope (Env)-mediated ADCC responses (11–14). Importantly, we and others reported that Env in the CD4-bound conformation was preferentially targeted by ADCC-mediating Abs and sera from HIV-1-infected individuals (11, 12, 15, 16), which represent a significant proportion of anti-Env Abs elicited during natural HIV infection (11, 17). However, the vast majority of circulating HIV-1 strains worldwide express functional Nef and Vpu proteins, which limit the exposure of CD4-induced (CD4i) Env epitopes at the surface of infected cells, likely preventing ADCC responses.Theoretically, agents promoting the CD4-bound Env conformation should expose CD4i epitopes that are readily recognized by ADCC-mediating Abs and sera from infected individuals (11, 12, 15, 16, 18), resulting in the sensitization of HIV-1-infected cells to ADCC. Importantly, modulating Env conformation at the surface of HIV-1-infected cells has become feasible as a result of the availability of small CD4-mimetic compounds (CD4mc). The prototypes of such compounds, NBD-556 and NBD-557, were discovered in a screen for inhibitors of gp120-CD4 interaction (19). These small-molecule ∼337-Da compounds and recent derivatives (DMJ-I-228, JP-III-48) bind in the Phe-43 cavity (20–22), a highly conserved ∼150-ų pocket in the gp120 glycoprotein located at the interface of the inner domain, outer domain, bridging sheet, and CD4 receptor (23). CD4mc block gp120-CD4 interaction and induce thermodynamic changes in gp120 similar to those observed during CD4 or soluble CD4 (sCD4) binding (24). Accordingly, these small molecules, as well as sCD4, can promote the transition of Env to the CD4-bound conformation, thus sensitizing HIV-1 particles to neutralization by otherwise nonneutralizing CD4i Abs (17, 25). Additional strategies using scaffolded miniproteins targeting critical gp120 elements required for CD4 interaction allowed the identification of CD4 mimetics with nanomolar affinity for gp120 (26). One of these variants, M48U1, displayed remarkably potent neutralization of three HIV-1 isolates (27). Its crystal structure in complex with HIV-1 gp120 was recently solved, showing that M48U1 engages the Phe-43 cavity in a manner similar to that of CD4 (28); thus, M48U1 might induce gp120 to adopt the CD4-bound conformation and expose CD4i epitopes. Previous studies exploring the antiviral properties of CD4mc were performed on viral particles (17, 25, 27). However, whether these compounds are able to engage the large amounts of Env present at the surface of infected cells and modulate Env conformation in a way that allows exposure of ADCC-mediating epitopes is currently not known. In this study, we show that CD4mc strongly sensitize HIV-1-infected primary CD4 T cells to ADCC mediated by sera, cervicovaginal fluids, and breast milk from HIV-1-infected individuals, as well as help eliminate infected, ex vivo-expanded primary CD4 T cells from HIV-1-infected individuals. Therefore, CD4mc possess three valuable complementary antiviral properties: direct inactivation of viral particles, sensitization of viral particles to neutralization by otherwise nonneutralizing Abs, and sensitization of HIV-1-infected cells to ADCC-mediated killing.     

A mouse model for HIV-1 entry

Passive transfer of neutralizing antibodies against HIV-1 can prevent infection in macaques and seems to delay HIV-1 rebound in humans. Anti-HIV antibodies are therefore of great interest for vaccine design. However, the basis for their in vivo activity has been difficult to evaluate systematically because of a paucity of small animal models for HIV infection. Here we report a genetically humanized mouse model that incorporates a luciferase reporter for rapid quantitation of HIV entry. An antibody’s ability to block viral entry in this in vivo model is a function of its bioavailability, direct neutralizing activity, and effector functions.HIV-1 (HIV), the causative agent of AIDS, represents a formidable global challenge, with the development of an effective vaccine being of paramount importance (1–4). Rapid progress in this area has been hindered in part by lack of a widely available small animal model for HIV entry. Currently available animal models include nonhuman primates and immunodeficient humanized mice, neither of which is readily available or amenable to genetic modifications (5, 6).Some viral pathogens exhibit a narrow host range, one of those being HIV. HIV’s entry into target cells is mediated by binding of its trimeric envelope spike (gp160) to human CD4 (hCD4) (7) and subsequently to a coreceptor such as human CXCR4 (8) or human CCR5 (hCCR5) (9–11). hCCR5 is of particular interest because it seems to be the primary coreceptor used for transmission (12, 13), as evidenced by the finding that homozygous deletion in the CCR5 allele confers resistance against HIV-1 acquisition (14, 15) and can also lead to long-term control of HIV after stem cell transplantation (16). Finally, HeLa cells and other HIV-resistant cells, including mouse cells, support viral entry when they are engineered to express hCD4/hCCR5/hCXCR4 (17–19).Here, we describe a hCCR5- and hCD4-expressing luciferase reporter mouse that can be used to measure HIV pseudovirus entry and antibody-mediated protection against initial infection in vivo.     

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

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 development of a protective vaccine remains a high priority for the global control of the HIV/AIDS epidemic (1). However, the unique biological features of HIV-1 make this task extremely challenging. The main obstacles include the ability of the virus to integrate into the host chromosomes, a remarkable degree of genetic variability, and the cryptic, antibody-shielded conformation adopted by the viral envelope in the native spikes that protrude from the virion surface (2). These spikes are composed of homotrimers of heterodimers of the envelope glycoprotein subunits gp120 and gp41 maintained in an energetically unfavorable, metastable conformation (3, 4). Upon binding to CD4 and a coreceptor such as CCR5 or CXCR4, gp120 undergoes dramatic conformational changes that lead to a low-energy state, creating permissive conditions for activation of the gp41 fusogenic mechanism (3). In the prefusion conformation, gp120 effectively conceals its highly conserved receptor- and coreceptor-binding sites from antibody recognition, imposing a high-entropy penalty for interaction with CD4 or antibodies to the coreceptor-binding site such as 17b; in contrast, in the open, low-energy conformation, gp120 interacts with CD4 and 17b with minimal thermodynamic changes (4, 5). This conformational masking of the vulnerable receptor- and coreceptor-binding sites is believed to be a primary mechanism of immune evasion by HIV-1 (4).The inherent conformational flexibility of gp120, along with the extensive N-linked glycosylation that covers most of the exposed surface of the glycoprotein, has severely hampered attempts to elucidate the native structure of the HIV-1 envelope spike. As a consequence, most of the available high-definition structures of gp120 have been obtained with deglycosylated, variable loop-truncated core monomers in complex with stabilizing ligands such as soluble CD4 (sCD4) (6–10). Important information regarding the overall conformation and ligand interactions of the trimeric spike at intermediate resolution has emerged from the use of increasingly refined cryo-electron microscopy (cryo-EM) technologies (11–17). Moreover, the crystal structure of a stabilized, soluble, cleaved gp140 trimer (BG505 SOSIP.664) at 4.7-Å resolution was reported recently (18). However, despite these advances, many critical aspects related to the structural mechanisms of HIV-1 immune vulnerability and evasion remain unresolved. In particular, the fine molecular details of the interaction between the second and third variable loops (V2 and V3, respectively) of gp120, which are believed to play a critical role in stabilizing the prefusion envelope structure (19–21), are elucidated only partially. Functionally, V2 and V3 cooperate in the formation of quaternary epitopes targeted by some of the most potent and broadly neutralizing mAbs hitherto identified (22, 23), and V2 effectively masks neutralization epitopes in V3 (24–27). Cryo-EM studies have provided evidence that in the prefusion conformation V2 and V3 are spatially contiguous and account for most of the density at the apex of the trimeric envelope spike (12–17). Although various fragments of V2 and V3 were crystallized separately using antibody-complexed synthetic peptides (28, 29), scaffolded chimeric constructs of the first and second variable loops (V1V2) (30, 31), or a V3-containing gp120 core monomer (8), the only study in which the two loops were visualized simultaneously is the recent report of the BG505 SOSIP.664 trimer crystal structure (18). In this artificially stabilized trimer, which displays several antigenic features of the native envelope (32), V2 and V3 appear to interact directly at the trimer apex with the V3 β-hairpin extensively buried under the V1V2 four-stranded Greek-key β-sheet (18).In the present study, we provide evidence that the conserved central region of the gp120 V2 loop contains previously unrecognized sulfated tyrosines that, in the CD4-unbound prefusion state, mediate intramolecular interaction between V2 and the CCR5-binding site at the base of V3. Our results suggest that the sulfotyrosine-bolstered interaction between V2 and V3 is a key structural constraint that stabilizes the native conformation of the HIV-1 envelope trimer.     

Targeting CXCL12/CXCR4 signaling with oncolytic virotherapy disrupts tumor vasculature and inhibits breast cancer metastases

Oncolytic viruses hold promise for the treatment of cancer, but their interaction with the tumor microenvironment needs to be elucidated for optimal tumor cell killing. Because the CXCR4 receptor for the stromal cell-derived factor-1 (SDF-1/CXCL12) chemokine is one of the key stimuli involved in signaling interactions between tumor cells and their stromal microenvironment, we used oncolytic virotherapy with a CXCR4 antagonist to target the CXCL12/CXCR4 signaling axis in a triple-negative 4T1 breast carcinoma in syngeneic mice. We show here that CXCR4 antagonist expression from an oncolytic vaccinia virus delivered intravenously to mice with orthotopic tumors attains higher intratumoral concentration than its soluble counterpart and exhibits increased efficacy over that mediated by oncolysis alone. A systemic delivery of the armed virus after resection of the primary tumor was efficacious in inhibiting the development of spontaneous metastasis and increased overall tumor-free survival. Inhibition of tumor growth with the armed virus was associated with destruction of tumor vasculature, reductions in expression of CXCL12 and VEGF, and decrease in intratumoral numbers of bone marrow-derived endothelial and myeloid cells. These changes led to induction of antitumor antibody responses and resistance to tumor rechallenge. Engineering an oncolytic virus armed with a CXCR4 antagonist represents an innovative strategy that targets multiple elements within the tumor microenvironment. As such, this approach could have a significant therapeutic impact against primary and metastatic breast cancer.The chemokine receptor CXCR4 and its cognate ligand CXCL12 form a pivotal axis for enabling metastasis by many solid tumor types, including breast carcinomas (1). Overexpression of CXCR4 in primary breast tumors is related to an aggressive phenotype and lymph-node metastases (2–4). Similarly, elevated CXCR4 expression in estrogen (ER) and progestin receptor (PR)-negative breast cancers, as well as ER2⁻/PR2⁻/HER-2⁻ triple-negative breast cancers, is closely associated with lymph-node metastasis and poor prognosis (5, 6). Binding of CXCL12 to CXCR4 promotes tumor progression by several mechanisms associated with the activation of a number of signaling pathways required for biological responses, including chemotaxis (7). First, CXCR4 is essential for metastatic spread to organs where CXCL12 is expressed (8, 9). Second, CXCL12 can stimulate survival and growth of neoplastic cells in a paracrine fashion (10–12), and promotes tumor angiogenesis by recruiting circulating endothelial progenitor cells (CEPs) to the tumor stroma (10, 13). CXCL12 also attracts protumor Gr1⁺CD11b⁺ myeloid cells and regulatory T cells (CD4⁺ subtype) into the tumor (14, 15), which impede innate and adaptive immune mechanisms of tumor destruction.In contrast to normal breast tissue, breast cancer cells typically express high levels of CXCR4 that can direct chemotaxis and invasive responses (1, 4). Therefore, modulation of the CXCL12/CXCR4 signaling pathway in breast cancer could impact multiple aspects of tumor progression. Several CXCR4 antagonists have shown antitumor activity in preclinical models and have been evaluated in clinical trials (8, 16, 17). However, given the abundant expression of CXCR4 by many cell types, including those of the central nervous, gastrointestinal, and immune systems (18), the side-effects of these antagonists need to be considered. Moreover, the impact of soluble CXCR4 antagonists on the mobilization of CXCR4-expressing hematopoietic stem and progenitor cells represents an additional concern, particularly when combined with chemotherapeutic agents, because of the potential for increased toxicity to the normal process of hematopoiesis (8, 19).To overcome some of these concerns related to the systemic delivery of soluble CXCR4 antagonists, we designed a tumor cell-targeted therapy that delivered the CXCR4 antagonist via an oncolytic vaccinia virus (OVV). To that end, a new antagonist was cloned into the genome of OVV, whose selective replication in cancer cells is associated with cellular EGFR/Ras signaling, thymidine kinase (TK) elevation, and type-I IFN resistance (20, 21). We have chosen an OVV as the delivery vector because the virus has evolved mechanisms for intravenous stability and spread to distant tissues, including resistance to antibody- and complement-mediated neutralization in the blood (22, 23). In addition, the highly destructive nature of a poxvirus infection results in the release of several cellular and viral danger signals, leading to generation of potent inflammatory responses that ultimately overcome tumor-mediated immune suppression to clear the virus (24, 25). Furthermore, complete tumor responses in preclinical models with vaccinia viruses containing deletions of the B18R secreted inhibitor of type-I IFN (26) and TK genes were accompanied by immune-mediated protection against tumor rechallenge (27). Oncolytic poxvirus therapy may therefore be considered as a method to achieve vaccination in situ, with the adaptive immune response being able to clear minimal residual disease and provide long-term protection against tumor relapse.As a template for the virally delivered CXCR4 antagonist, we used the CTCE-9908 dimer corresponding to the N-terminal region of CXCL12 chemokine (KGVSLSYR-K-RYSLSVGK). This CXCR4 antagonist, the safety of which has been demonstrated in a phase I/II trial in cancer patients (17), is capable of blocking the CXCL12/CXCR4 interaction (28) and delaying the development of metastasis in breast cancer mouse models (29, 30). In the virally delivered construct, the first eight amino acids of CTCE-9908 were expressed in the context of murine (mFc) or human (hFc) fragment of IgG with disulfide bonds in a hinge region for preservation of its dimeric structure (Fig. S1 A–C). Using a highly metastatic 4T1 tumor model, which emulates stage IV breast cancer in humans (31), we demonstrated that the virally delivered CXCR4-A-mFc antagonist was predominantly retained in the tumor and inhibited the growth of both primary and metastatic lesions. A systemic delivery of the armed virus after resection of the primary tumor further reduced the development of spontaneous metastasis and resulted in increased tumor-free survival. The OVV-CXCR4-A-mFc antitumor efficacy was associated with destruction of intratumoral microvessels, lower accumulation of CEPs and neutrophils/granulocytic-myeloid derived suppressor cells (G-MDSCs), as well as enhancement of the vaccinia-mediated activation of antitumor antibody responses.     

Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain; and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. Thus, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.It is well accepted that direct virus neutralization is an important element of antibody-mediated protection against HIV-1 (refs. 1–6 and reviewed in ref. 7). In contrast, less is known about the role of Fc-mediated effector function in the control of HIV-1, although four lines of evidence signal its importance. First, studies in HIV-1–infected people (8–14) and in macaques infected with simian immunodeficiency virus (15, 16) consistently show an inverse correlation between Fc-mediated effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC) (8, 9) or antibody-dependent cell-mediated viral inhibition (ADCVI), and viral loads or decreased disease progression (17). Second, vaccine-elicited protection both in nonhuman primates (18–21) and in a subset of human subjects in the Vax-004 trial (22) correlates with Fc-mediated effector function often observed in the absence of detectable neutralizing antibodies (18–21). Similarly, there was an inverse relationship between acquisition of HIV-1 and ADCC in the RV144 trial for a subset of subjects who had low to moderate IgA anti-gp120 titers (23). Third, breast milk IgG ADCC responses to gp120 but not to virus neutralization correlated with reduced perinatal transmission of HIV-1 (24). Fourth, passive immunization studies in nonhuman primates (25, 26) showed that abrogation of Fc-mediated effector function diminished the sterilizing protection afforded by the neutralizing mAb b12. These compelling studies show that neutralization alone significantly protects against a simian-human immunodeficiency virus challenge and that Fc-mediated effector function augments this effect. Taken together, these four lines of investigation strongly suggest that Fc-mediated effector function in addition to neutralization contributes to antibody-mediated protection against HIV-1. Thus, it is important to determine the precise relationships among antibody specificity, neutralization, and Fc-mediated effector function in protection against HIV-1.In this report, we probe these relationships using a panel of human mAbs that recognize transitional epitopes exposed during the earliest stage of viral entry, the interaction of gp120 with CD4. Our studies deliberately focus on antibody responses to epitopes that become exposed during viral entry because passive immunization studies indicate that an antibody has at most a 24-h window to block transmission (ref. 27; reviewed in ref. 28). Thus, transmission-blocking antibodies must block infection by direct neutralization of HIV-1, by Fc-mediated killing of nascently infected cells, or both. Although these two effector functions often are coincident for a given mAb specificity (29, 30), they can be dissociated because nonneutralizing epitopes on both gp120 (12, 31) and gp41 (32) can be ADCC targets. In this report, we probe the relationships among antibody specificity, ADCC, and neutralization using a panel of human mAbs that recognize transitional epitopes exposed on target cells during viral entry.     

Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages

HIV type 1 (HIV-1) infects CD4⁺ T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as “Trojan horses” carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages.HIV type 1 (HIV-1) infects CD4⁺ T lymphocytes, myeloid dendritic cells, and monocyte-macrophages, cell types sharing the expression of the primary viral receptor (R) CD4 on their surface together with one or more chemokine R, usually CCR5 and/or CXCR4 (1, 2). The discovery of combination antiretroviral therapy (cART) in the mid 1990s has significantly impacted the natural history of the infection by virtually suppressing, in optimal conditions, the capacity of the virus to infect target cells, thereby resulting in a prolonged, near-normal life expectation of infected individuals. Therapy suspension almost inevitably results in the resumption of virus replication and disease progression in most individuals, owing to the existence of viral reservoirs of likely heterogeneous cellular origin that are unaffected by prolonged cART (3, 4). Consequently, complete eradication of HIV-1 from infected individuals is not currently achievable without targeting these cART-insensitive sources of infection. Latently infected CD4⁺ T cells with both “central memory” and “transitional effector memory” phenotypes have been well characterized as reservoirs of replication-competent HIV-1 (4, 5); in contrast, the contribution of non–T-cell subsets to the overall viral reservoir in cART-treated individuals has remained more elusive (6–8).Concerning mononuclear phagocytes, whether bone marrow precursor cells and circulating monocytes are truly infected in vivo is a matter of debate, whereas a greater consensus exists on the role played by resident tissue macrophages of different organs. In particular, brain-associated macrophages or microglia, together with astrocytes, have been reported as the main targets and source of virus in the central nervous system (4, 5, 8, 9). In addition, infected mononuclear phagocytes are considered responsible for the slower second-phase decay of plasma viremia observed in patients starting cART (9).Unlike CD4⁺ T cells, mononuclear phagocytes are depleted neither in vivo (at least in terms of circulating monocytes) nor in vitro on HIV-1 infection. In addition, macrophage infection, both in vitro and in vivo, has been described as characterized by budding and accumulation of HIV-1 particles in virus-containing compartments (VCC) of debated origin (10–13). These peculiar features of macrophage infection led to the “Trojan horse” hypothesis of a pathogenic role of mononuclear phagocytes as capable of accumulating virions in subcellular compartments invisible to immune recognition (14, 15) and relatively insensitive to antiretrovirals (15, 16). It is hypothesized that VCC-associated virions are released either as a consequence of cell death or by functional stimulation of the infected cells. In this regard, we have previously observed that IFN-γ stimulation of the chronically infected promonocytic U1 cell line differentiated to macrophage-like cells by phorbol-12, myristate, 13-acetate (PMA) caused a profound redirection of the major site of virion production from the plasma membrane to VCC (17). This observation was later extended to other stimulants of PMA-differentiated U1 cells, including urokinase-type plasminogen activator (uPA) and CD11b/CD18 (Mac-1) integrin ligands (18, 19). Of interest, a similar phenotype has been reported in infected primary human monocyte-derived macrophages (MDM) by interference with endogenously released CCL2/monocyte chemotactic protein-1 (MCP-1) (20).Regarding the nature of VCC, Gould et al. (14) proposed that retroviruses exploit the exosome biogenesis pathway for both the formation and release of infectious particles as well as for the uptake of a R-independent, gp120 envelope (Env)-dependent infection. More recently, VCC are believed to represent intracellular sequestration of plasma membrane areas rich in a subset of tetraspanins (12). Likely because of their origin, VCC can be transiently (11) connected to the extracellular medium through microchannels or conduits (21, 22). Of interest, VCC-like compartments accessible by the extracellular medium preexist in uninfected macrophages and express similar markers, including CD9, CD81, CD63 (23), and, identified more recently, CD36 (24). Recently, it was demonstrated that on HIV-1 infection, Gag is recruited to these preexisting compartments in which virion assembly occurs, leading to their conversion into VCC (24, 25). Whether the release of virions is a regulated process, and the nature of the signal(s) that may induce virion discharge from VCC, remain largely unknown, however.In the present study, we investigated the hypothesis that signals from an inflamed microenvironment may affect HIV-1 accumulation and release from macrophage-associated VCC. We focused on extracellular ATP (eATP), a molecule passively released during necrotic cell death (26), but also actively released on cell stimulation via innate immunity R, as reviewed previously (27). We indeed observed that eATP induced a rapid release of HIV-1 virions accumulated in VCC of both primary human MDM acutely infected with HIV-1 and chronically infected U1 cells differentiated to macrophage-like cells (D-U1 cells) (28). eATP-induced release of HIV-1 virions was not associated with gross cytopathic effects and was blocked by imipramine, an antidepressant agent known to inhibit the membrane-associated acid sphingomyelinase (aSMase), an enzyme involved in the formation of membrane microvesicles (29, 30). Finally, we found that eATP-dependent discharge of virions from VCC was attributed mainly to engagement of the P2X7R on the surface of infected macrophages.     

Heteromerization of chemokine (C-X-C motif) receptor 4 with α1A/B-adrenergic receptors controls α1-adrenergic receptor function

Recent evidence suggests that chemokine (C-X-C motif) receptor 4 (CXCR4) contributes to the regulation of blood pressure through interactions with α₁-adrenergic receptors (ARs) in vascular smooth muscle. The underlying molecular mechanisms, however, are unknown. Using proximity ligation assays to visualize single-molecule interactions, we detected that α_1A/B-ARs associate with CXCR4 on the cell surface of rat and human vascular smooth muscle cells (VSMC). Furthermore, α_1A/B-AR could be coimmunoprecipitated with CXCR4 in a HeLa expression system and in human VSMC. A peptide derived from the second transmembrane helix of CXCR4 induced chemical shift changes in the NMR spectrum of CXCR4 in membranes, disturbed the association between α_1A/B-AR and CXCR4, and inhibited Ca²⁺ mobilization, myosin light chain (MLC) 2 phosphorylation, and contraction of VSMC upon α₁-AR activation. CXCR4 silencing reduced α_1A/B-AR:CXCR4 heteromeric complexes in VSMC and abolished phenylephrine-induced Ca²⁺ fluxes and MLC2 phosphorylation. Treatment of rats with CXCR4 agonists (CXCL12, ubiquitin) reduced the EC₅₀ of the phenylephrine-induced blood pressure response three- to fourfold. These observations suggest that disruption of the quaternary structure of α_1A/B-AR:CXCR4 heteromeric complexes by targeting transmembrane helix 2 of CXCR4 and depletion of the heteromeric receptor complexes by CXCR4 knockdown inhibit α₁-AR–mediated function in VSMC and that activation of CXCR4 enhances the potency of α₁-AR agonists. Our findings extend the current understanding of the molecular mechanisms regulating α₁-AR and provide an example of the importance of G protein-coupled receptor (GPCR) heteromerization for GPCR function. Compounds targeting the α_1A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulate blood pressure.Chemokine (C-X-C motif) receptor 4 (CXCR4) is a G protein-coupled receptor (GPCR) that is essential during development. Animals lacking CXCR4 are not viable and demonstrate defects of the hematopoietic and cardiovascular system (1). After birth, CXCR4 is expressed in many tissues, including the heart and vasculature, and fulfills multiple functions in the immune system, such as regulation of leukocyte trafficking, stem cell mobilization, and homing (2, 3). Moreover, CXCR4 is involved in various disease processes, such as HIV infection, cancer metastasis, and tissue repair (3–5).In addition to these established functions, recent observations suggest that CXCR4 also contributes to the regulation of hemodynamics and blood pressure. Treatment with the CXCR4 antagonists AMD3100 and AMD3465 reduced blood pressure in experimental models of pulmonary arterial and systemic hypertension (6, 7). We have shown previously that AMD3100 reduces hemodynamic stability and blood pressure during the cardiovascular stress response to traumatic and hemorrhagic shock, whereas selective activation of CXCR4 with the noncognate agonist ubiquitin improves hemodynamic stability and increases systemic blood pressure after traumatic, hemorrhagic, and endotoxic shock (8–13). Because in vivo pharmacological targeting of CXCR4 did not affect myocardial function, these findings suggested that effects of CXCR4 on hemodynamics and blood pressure are mediated via modulation of vascular function (9). Accordingly, we observed that CXCR4 activation enhances and sensitizes vasoconstriction of isolated mesenteric arteries and veins in response to α₁-adrenergic receptor (AR) activation with phenylephrine (PE) (9). As these effects were independent of the vascular endothelium, interactions between CXCR4 and α₁-AR in vascular smooth muscle likely constitute the physiological basis for these observations (9). The molecular mechanisms underlying interactions between CXCR4 and α₁-AR in vascular smooth muscle, however, remain unknown.Crosstalk between GPCRs is a widely recognized principle that expands the physiological repertoire of GPCR-mediated signaling events and functions (14–19). Receptor crosstalk can be attributed to a variety of molecular mechanisms, including receptor hetero-oligomerization (14–23). The formation of homo- and/or hetero-oligomeric complexes among GPCRs is thought to be important for many aspects of GPCR function (22–24).CXCR4 has been shown to associate with multiple chemokine receptors in various expression systems (3, 25–28). ARs are also known to be able to form heteromeric receptor complexes (29–35), and recent evidence suggests that AR may also be able to form heteromeric complexes with chemokine receptors (36–38). Thus, we studied whether α₁-AR and CXCR4 may interact on the cell surface of vascular smooth muscle cells through the formation of heteromeric receptor complexes.Here, we provide evidence that heteromeric receptor complexes between α_1A-AR and CXCR4 and between α_1B-AR and CXCR4 are constitutively expressed in rat and human vascular smooth muscle cells (VSMC). We show that disruption of the quaternary structure of the heteromeric receptor complex by targeting transmembrane helix (TM) 2 of CXCR4 and depletion of heteromeric receptor complexes by CXCR4 knockdown inhibit α₁-AR agonist-induced key signaling events and contraction of VSMC. Furthermore, we show that treatment with CXCR4 agonists increases the potency of the α₁-AR agonist PE to increase blood pressure in vivo. Our observations suggest that α₁-AR function in VSMC is controlled through the formation of heteromeric α_1A/B-AR:CXCR4 complexes.     

MLV glycosylated-Gag is an infectivity factor that rescues Nef-deficient HIV-1

Optimal infectivity of HIV-1 virions requires synthesis of the HIV-1 regulatory protein Nef in some producer cells but not others. A survey of 18 lymphoid cell lines found that Nef was dispensable in three, each of which harbored gammaretroviruses. Nef-dependent cell lines were rendered Nef-independent by a cell-free supernatant from the independent lines or by transfection of cloned murine leukemia virus (MLV). Analysis of MLV deletion mutations identified glycosylated gag (glycogag) as the factor that rescues Nef-defective HIV-1 virions. Glycogag was also demonstrated to be required for the infectivity of MLV virions produced in lymphoid cells. Direct comparison of Nef and glycogag revealed identical dependence for activity on Env-pseudotype and producer cell type. The two proteins colocalize within cells, and both increase the yield of viral cDNA in target cells. The functional similarity of Nef and glycogag is a compelling example of convergent evolution in which two structurally unrelated proteins provide a function necessary for virion infectivity in lymphoid cells.Nef is a myristoylated protein encoded by HIV-1, HIV-2, and SIV, crucial for virus replication in vivo and rapid AIDS progression (1–3). It performs a remarkable array of activities by exploiting many of its surfaces to interact with several cellular molecules. By interacting with proteins implicated in intracellular trafficking, it modulates cell surface expression of numerous molecules, including the receptor CD4 (4, 5) and MHC-I (6). Alleles derived from most SIV isolates also down-regulate the TCR/CD3 complex (7–9). In addition, Nef alters the activation threshold of lymphocytes (10–12) by interacting with protein kinases (13–16) and modulates apoptotic signals (10, 17).Nef also has a positive effect on the infectivity of virions (18, 19), a function which remains mechanistically unexplained. Although the ability to down-regulate CD4 can contribute to the effect of Nef on infectivity (20), this activity is visible by using CD4-negative producer cells and was shown to be independent from other Nef effects (18, 21–23), it requires its expression in virus-producing cells and is manifested at an early step of the infection process of target cells (18, 22, 24–26). Nef might play a crucial role during penetration of retroviral cores into the cytoplasm (27). Accordingly, HIV-1 pseudotyped by vesicular stomatitis virus G (VSV-G), which relies on endosomal uptake and, therefore, might enhance cytoplasmic delivery, does not require Nef (28, 29). Although found in virus particles, recent data indicate that Nef itself might not function as a virion protein (30, 31), suggesting that it could induce a yet unknown modification of the particle. The effect of Nef on infectivity was shown to depend on dynamin 2 and clathrin activities in producer cells (32) and on a di-leucine motif critical for the interaction with the clathrin adaptor complexes AP2 (33, 34). The biogenesis and/or trafficking of intracellular vesicles in virus producing cells might therefore play a crucial role in modulating virion infectivity.Most gammaretroviruses encode an accessory protein (gPr80 or glycogag) from unspliced RNA via an alternative CUG initiation codon upstream and in-frame with the standard Gag polyprotein (35–39). As a result, a leader sequence [88 amino acids in MLV provirus genome (MoMLV)] is added to the N terminus of conventional Gag. The protein encoded has a type II transmembrane topology, with the conventional Gag residues being extracellular or located in the lumen of the ER, and glycosylated. Mature gPr80 is proteolytically cleaved, and only half of the conventional Gag sequence remains attached to the integral transmembrane protein (40). Although not strictly required for virus replication in vitro, gPr80 is crucial in vivo for sustained virus replication and disease progression (41–47). The mechanisms engaged by gPr80 to promote virus replication remain largely unknown. However, a recent report has revealed that gPr80 affects release of both MLV and HIV-1 by facilitating budding from lipid rafts (48).In this study, a screen of 18 producer cell lines identified three lymphoid cell lines capable of generating HIV-1 that does not require Nef for maximal infectivity. Glycosylated gag expressed from gammaretrovirus genomes harbored by these cell lines was found to functionally replace the infectivity function of Nef, unveiling a role for glycogag as an infectivity factor.     

Pepducin targeting the C-X-C chemokine receptor type 4 acts as a biased agonist favoring activation of the inhibitory G protein

Short lipidated peptide sequences derived from various intracellular loop regions of G protein-coupled receptors (GPCRs) are named pepducins and act as allosteric modulators of a number of GPCRs. Recently, a pepducin selectively targeting the C-X-C chemokine receptor type 4 (CXCR4) was found to be an allosteric agonist, active in both cell-based assays and in vivo. However, the precise mechanism of action of this class of ligands remains poorly understood. In particular, given the diversity of signaling effectors that can be engaged by a given receptor, it is not clear whether pepducins can show biased signaling leading to functional selectivity. To explore the ligand-biased potential of pepducins, we assessed the effect of the CXCR4 selective pepducin, ATI-2341, on the ability of the receptor to engage the inhibitory G proteins (Gi1, Gi2 and Gi3), G13, and β-arrestins. Using bioluminescence resonance energy transfer-based biosensors, we found that, in contrast to the natural CXCR4 ligand, stromal cell-derived factor-1α, which promotes the engagement of the three Gi subtypes, G13 and the two β-arrestins, ATI-2341 leads to the engagement of the Gi subtypes but not G13 or the β-arrestins. Calculation of the transduction ratio for each pathway revealed a strong negative bias of ATI-2341 toward G13 and β-arrestins, revealing functional selectivity for the Gi pathways. The negative bias toward β-arrestins results from the reduced ability of the pepducin to promote GPCR kinase-mediated phosphorylation of the receptor. In addition to revealing ligand-biased signaling of pepducins, these findings shed some light on the mechanism of action of a unique class of allosteric regulators.Pepducins represent a class of molecules that regulate the activity of G protein-coupled receptors (GPCRs). These lipid-modified peptides are derived from the amino acid sequences of one of the four intracellular loops of a target GPCR (1). Although the precise mode of action is not completely understood, it is believed that pepducins bind to their target receptors and allosterically modulate their signaling activity (2, 3). Pepducins have been identified for several GPCRs, including the protease-activated receptors PAR1 (1, 4–10), PAR2 (1, 11–13), and PAR4 (6, 9), the formyl peptide receptor-2 (14), the melanocortin type 4 receptor (1), the sphingosine 1-phosphate receptor 3 (15), and the C-X-C chemokine receptor type 1, 2 (CXCR1, CXCR2) (16), and 4 (CXCR4) (2, 17, 18). They have been found to act as allosteric agonists as well as negative or positive allosteric modulators. However, in most cases, their activity was assessed for only one or a few signaling pathways engaged by the receptors.One of the receptors for which pepducins were developed is CXCR4. This receptor, expressed in many tissues including hematopoietic and circulating cells, is a coreceptor for the entry of HIV (19, 20) and controls many physiological functions, including cell migration, mobilization, and retention of polymorphonuclear neutrophils (PMNs) and hematopoietic stem cells, as well as progenitor cells (HSPCs) in the bone marrow niche (21–23). It has also been found to play an important role in tumor progression, angiogenesis, and metastasis of a variety of cancers (24–26). A CXCR4 antagonist, AMD-3100 (Mozobil), is used to mobilize HSPCs from the bone marrow for transplantation of leukemic patients (27).A pepducin derived from the first intracellular loop of CXCR4 (ATI-2341) was found to be an allosteric agonist on the chemotactic response elicited on a human T-lymphoblastic leukemia cell line (CCRF-CEM cells) that endogenously expresses CXCR4 (18). This activity of ATI-2341 was confirmed in fresh human PMNs as well as in vivo for their ability to promote the mobilization of PMNs and HSPCs in the peripheral circulation of both mice and monkeys (18). Of note, unlike the clinically used CXCR4 antagonist AMD-3100 and stromal cell-derived factor-1α (SDF-1; also known as CXCL12) that promote the mobilization of lymphocytes in addition to PMNs and HSPCs (18, 28–30), ATI-2341 was without effect on the mobilization of lymphocytes (18), indicating that ATI-2341 may display functional selectivity.When assessing the effect of ATI-2341 on the signaling activity of CXCR4, the pepducin was found to be an allosteric agonist, activating the inhibitory heterotrimeric G protein (Gi) to promote inhibition of cAMP production and induce calcium mobilization (18, 31). In recent years, many GPCRs have been shown to engage in promiscuous signaling activities involving more than one G protein subtype as well as G protein-independent signaling. More importantly, it was found that different ligands can selectively couple to a subset of the signaling pathways that can be engaged by a receptor. In some cases, a given ligand can even have opposite efficacies on two different signaling pathways: a concept known as “ligand-biased signaling” or “functional selectivity” (32–36). In addition to its coupling to Gi, CXCR4 has also been found to signal through the engagement and activation of G13 (37, 38) and β-arrestin2 (39, 40), both pathways being proposed to contribute to the chemotactic responses. These observations raise the question of whether the CXCR4-selective pepducin, ATI-2341, is an allosteric agonist on all of the signaling pathways identified for the chemokine receptor or whether it could show bias toward selective signaling pathways and thus be functionally selective.To determine whether pepducins can display functional selectivity on CXCR4 signaling at the molecular level, we took advantage of bioluminescence resonance energy transfer (BRET)-based assays that allow the direct monitoring of the engagement and activation of proximal signaling effectors. More specifically, we compared the ability of ATI-2341 and the natural agonist of the receptor, SDF-1, to promote the engagement/activation of three Gi family members (Gi₁, Gi₂, and Gi₃), G13, β-arrestin1, and β-arrestin2. We found that, whereas SDF-1 promotes the engagement of all of the signaling effectors, ATI-2341 selectively led to the functional engagement of the Gi family members and had no effect on G13 or the two β-arrestins. The lack of recruitment of β-arrestins results from the poor recruitment of G protein-coupled receptor kinases (GRKs) to the receptor because, in contrast to SDF-1 that stimulates the phosphorylation of CXCR4 by protein kinase C (PKC), GRK2/3, and GRK6, ATI-2341 promotes effective PKC-dependent phosphorylation of the receptor but minimal GRK2/3 recruitment, as well as minimal GRK6-dependent phosphorylation.Taken together, our results demonstrate that the pepducin ATI-2341 is a functionally selective allosteric regulator of CXCR4 that activates Gi-dependent pathways without modulating G13 and β-arrestin pathways. These data indicate that ATI-2341 could have physiological actions that may differ from the natural ligand SDF-1 and the clinically used AMD-3100, raising the intriguing possibility that ATI-2341 may have distinct clinical properties. These findings also shed some light on the mechanism of action of pepducins and indicates that, similar to orthosteric ligands, these allosteric regulators can be functionally selective.     

Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions

Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.Cell division is a fundamental biological process that is tightly regulated by evolutionarily conserved signaling pathways (1, 2). The initial decision to start cell division, the fidelity of subsequent DNA replication, and the final formation of daughter cells is monitored and regulated by these essential pathways (2–6). The cyclin-dependent kinases (CDKs) are the central players that orchestrate this orderly progression through the cell cycle (1, 2, 6, 7). The enzymatic activity of CDKs is regulated by complex mechanisms that include posttranslational modifications and expression of activating and inhibitory proteins (1, 2, 6, 7). The spatial and temporal changes in the activity of these CDK complexes are thought to generate the distinct substrate specificities that lead to sequential and unidirectional progression of the cell cycle (1, 8, 9).Cell-cycle deregulation is a universal feature of human cancer and a long-sought-after target for anticancer therapy (1, 10–13). Frequent genetic or epigenetic changes in mitogenic pathways, CDKs, cyclins, or CDK inhibitors are observed in various human cancers (1, 4, 11). In particular, the G1/S-regulating CDK4/6–cyclin D–inhibitors of CDK4 (INK4)–retinoblastoma (Rb) protein pathway frequently is disrupted in cancer cells (11, 14). These observations provided an impetus to develop CDK inhibitors as anticancer drugs. However, the earlier class of CDK inhibitors had limited specificity, inadequate clinical activity, poor pharmacokinetic properties, and unacceptable toxicity profiles (10, 11, 14, 15). These disappointing initial efforts now have been followed by the development of the specific CDK4/6 inhibitors palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219), which have demonstrated manageable toxicities, improved pharmacokinetic properties, and impressive antitumor activity, especially in certain forms of breast cancer (14, 16). Successful early clinical trials with these three CDK4/6 inhibitors have generated cautious enthusiasm that these drugs may emerge as a new class of anticancer agents (14, 17). Palbociclib recently was approved by Food and Drug Administration for the treatment of metastatic breast cancer and became the first CDK4/6 inhibitor approved for anticancer therapy (18).In addition to its potential as an anticancer strategy, CDK4/6 inhibition in normal tissues could be exploited therapeutically for wide-ranging clinical conditions. For example, radiation-induced myelosuppression, caused by cell death of proliferating hematopoietic stem/progenitor cells, can be rescued by palbociclib (19, 20). Furthermore, cytotoxic anticancer agents cause significant toxicities to normal proliferating cells, which possibly could be mitigated by the concomitant use of CDK4/6 inhibitors (20, 21). More broadly, cell-cycle inhibition could have beneficial effects in disorders in which maladaptive proliferation of normal cells contributes to the disease pathology, as observed in vascular proliferative diseases, hyperproliferative skin diseases, and autoimmune disorders (22, 23). In support of this possibility, palbociclib treatment recently was reported to ameliorate disease progression in animal models of rheumatoid arthritis through cell-cycle inhibition of synovial fibroblasts (24).Abnormal cellular proliferation also is a hallmark of various kidney diseases (25), and cell-cycle inhibition has been shown to ameliorate significantly the pathogenesis of polycystic kidney disease (26), nephritis (27), and acute kidney injury (AKI) (28). Remarkably, during AKI, the normally quiescent renal tubular cells reenter the cell cycle (29–34), and blocking cell-cycle progression can reduce renal injury (28). Here, we provide evidence that the CDK4/6 pathway is activated early during AKI and demonstrate significant protective effects of CDK4/6 inhibitors in animal models of cisplatin-induced AKI. In addition, we found that the CDK4/6 inhibitors palbociclib and LEE011 are potent inhibitors of organic cation transporter 2 (OCT2), a cisplatin uptake transporter highly expressed in renal tubular cells (35–37). Our findings provide a rationale for the clinical development of palbociclib and LEE011 for the prevention and treatment of AKI.     

Distinct patterns of hepcidin and iron regulation during HIV-1, HBV,and HCV infections

During HIV type-1 (HIV-1), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections, altered iron balance correlates with morbidity. The liver-produced hormone hepcidin dictates systemic iron homeostasis. We measured hepcidin, iron parameters, cytokines, and inflammatory markers in three cohorts: plasma donors who developed acute HIV-1, HBV, or HCV viremia during the course of donations; HIV-1–positive individuals progressing from early to chronic infection; and chronically HIV-1–infected individuals (receiving antiretroviral therapy or untreated). Hepcidin increased and plasma iron decreased during acute HIV-1 infection, as viremia was initially detected. In patients transitioning from early to chronic HIV-1 infection, hepcidin in the first 60 d of infection positively correlated with the later plasma viral load set-point. Hepcidin remained elevated in individuals with untreated chronic HIV-1 infection and in subjects on ART. In contrast to HIV-1, there was no evidence of hepcidin up-regulation or hypoferremia during the primary viremic phases of HCV or HBV infection; serum iron marginally increased during acute HBV infection. In conclusion, hepcidin induction is part of the pathogenically important systemic inflammatory cascade triggered during HIV-1 infection and may contribute to the establishment and maintenance of viral set-point, which is a strong predictor of progression to AIDS and death. However, distinct patterns of hepcidin and iron regulation occur during different viral infections that have particular tissue tropisms and elicit different systemic inflammatory responses. The hypoferremia of acute infection is therefore a pathogen-specific, not universal, phenomenon.Disturbances in iron homeostasis commonly manifest in inflammatory and infectious diseases (1). The liver-produced hormone hepcidin regulates levels and compartmentalization of iron by inhibiting the iron exporter ferroportin (2), which is highly expressed by macrophages and duodenal enterocytes (3). Hepcidin excludes iron from serum by sequestering it in macrophages and preventing dietary uptake. In addition to its homeostatic regulation by iron, hepcidin is an acute-phase peptide induced by interleukin (IL)-6, IL-22, and type I interferon (IFN) (4–6). Persistent exclusion of iron from serum caused by hepcidin contributes to iron-restricted erythropoiesis and the anemia of chronic inflammation.Anemia is common during chronic HIV type-1 (HIV-1) infection (7) and is predictive of HIV-associated morbidity and mortality independently of established prognostic indicators such as CD4 count (8–11). The etiology of HIV-related anemia is complex (reviewed in ref. 10) but likely involves iron-restricted erythropoiesis (12). Iron sequestration within bone marrow macrophages, suggestive of hepcidin activity, inversely correlates with secondary infections and mortality in HIV-1 infection (13), and altered iron status (independently of anemia) correlates with HIV-associated mortality and morbidity, even after accounting for confounders including CD4 count (14–18). Hepcidin inversely correlates with CD4 counts in individuals with advanced HIV-1 (19). However, hepcidin levels in the crucial acute phase of HIV-1 infection, which dictate later events in the disease (20), are unexplored. In this early period, vast numbers of mucosal CD4+ T cells are lost (21), immune homeostasis is irreversibly perturbed, and proinflammatory cytokines are systemically elevated (22, 23).Unusually for a systemic inflammatory state, hepcidin is suppressed during chronic hepatitis C virus (HCV) infection, contributing to pathogenic liver iron loading (24). Hepcidin can also be suppressed during hepatitis B virus (HBV)-associated cirrhosis (25), although this may not be as marked as in HCV infection (26). Nothing is known about how hepcidin behaves during acute HCV or HBV infection, in particular whether the unusual low hepcidin observed in the chronic states may occur earlier.The aims of this study were to investigate the behavior of hepcidin during acute and chronic HIV-1 infection and second to compare and contrast hepcidin kinetics during acute HIV-1, HCV, and HBV infections.     

The HIV-1 reservoir in eight patients on long-term suppressive antiretroviral therapy is stable with few genetic changes over time

The source and dynamics of persistent HIV-1 during long-term combinational antiretroviral therapy (cART) are critical to understanding the barriers to curing HIV-1 infection. To address this issue, we isolated and genetically characterized HIV-1 DNA from naïve and memory T cells from peripheral blood and gut-associated lymphoid tissue (GALT) from eight patients after 4–12 y of suppressive cART. Our detailed analysis of these eight patients indicates that persistent HIV-1 in peripheral blood and GALT is found primarily in memory CD4⁺ T cells [CD45RO⁺/CD27(^+/−)]. The HIV-1 infection frequency of CD4⁺ T cells from peripheral blood and GALT was higher in patients who initiated treatment during chronic compared with acute/early infection, indicating that early initiation of therapy results in lower HIV-1 reservoir size in blood and gut. Phylogenetic analysis revealed an HIV-1 genetic change between RNA sequences isolated before initiation of cART and intracellular HIV-1 sequences from the T-cell subsets after 4–12 y of suppressive cART in four of the eight patients. However, evolutionary rate analyses estimated no greater than three nucleotide substitutions per gene region analyzed during all of the 4–12 y of suppressive therapy. We also identified a clearly replication-incompetent viral sequence in multiple memory T cells in one patient, strongly supporting asynchronous cell replication of a cell containing integrated HIV-1 DNA as the source. This study indicates that persistence of a remarkably stable population of infected memory cells will be the primary barrier to a cure, and, with little evidence of viral replication, this population could be maintained by homeostatic cell proliferation or other processes.Combinational, antiretroviral therapy (cART) effectively suppresses but does not eradicate HIV-1 infection (1). Persistent low-level HIV-1 can still be detected in plasma (2–7) and cellular reservoirs (8–10) even after several years of suppressive cART, and cessation of current treatments invariably results in resumption of viral replication. Resting-memory CD4⁺ T cells are a well-defined reservoir of HIV-1, and the reservoir is established when an activated CD4⁺ T cell becomes infected by HIV-1 but transitions to a resting state (9) or perhaps when resting cells are infected directly (11–13). Central and transitional memory T cells have recently been identified as major contributors to the HIV-1 reservoir in the memory T-cell population (14). Naïve T cells have also been demonstrated to contain HIV-1 DNA in patients on suppressive therapy, although at a lower infection frequency than the memory T-cell population (15). In addition, many other cell types, including monocyte/macrophages, have been proposed to play a role in HIV-1 persistence (reviewed in ref. 16). These long-lived HIV-1–infected cells have been detected in peripheral blood. Several studies, however, suggest that the reservoir is largely established and maintained in lymphoid tissues, and that the infected cells circulating in blood may not be representative of the population of infected cells in tissue. For example, the majority of lymphocytes are sequestered in the gastrointestinal tract, and gut-associated lymphoid tissue (GALT) has been shown to be a major viral reservoir in patients on suppressive antiretroviral therapy (17–22).In addition to the persistence of long-lived, latently infected cells, low-level viral replication has been proposed as a mechanism that maintains HIV-1 during cART. If complete viral replication cycles persist, despite suppressive antiretroviral therapy, this would lead to de novo cellular infection and a constant replenishment of the viral reservoir. Investigations into whether HIV-1 replication continues during suppressive therapy have been carried out with peripheral blood and GALT samples but have led to potentially contradictory results. Some studies have found an absence of genetic evolution in viral reservoirs (23–29) and no reduction of plasma RNA during intensification of cART (30, 31), suggesting that cART is effective in preventing viral replication in these anatomical sites. In contrast, increased numbers of 2-long terminal repeat circles in peripheral blood mononuclear cells and decreased amounts of unspliced HIV-1 RNA in CD4⁺ T cells isolated from the terminal ileum have been reported during raltegravir intensification, supporting the concept that some viral replication can occur despite suppressive cART (32, 33). Thus, the role of on-going replenishment via cycles of replication as a cause of persistence is not fully understood.To investigate the source and dynamics of HIV-1 reservoirs in peripheral blood and GALT, we sorted and genetically characterized intracellular HIV-1 from subsets of memory T cells, naïve T cells, and myeloid cells from these two compartments from eight patients who had been on suppressive therapy with undetectable viral loads (<40–75 copies/mL) for 4–12 y: five who initiated therapy during acute/early infection and three who initiated therapy during chronic infection. Our aim was to investigate the nature of the infected cell population during cART and explore the role of HIV-1 replication, as reflected by nucleotide sequence substitutions in maintaining this reservoir. Our study revealed that both memory T cells and naïve T cells harbor HIV-1 DNA after long-term suppressive therapy, and the infection frequency of these T cells was higher in patients treated during chronic infection compared with patients treated during early infection. In-depth phylogenetic analysis revealed little or no change in viral structure or divergence over time within the viral sequences isolated from the different T-cell populations compared with sequences isolated from plasma collected just before initiation of cART, indicating lack of on-going replication during long-term suppressive therapy.