James A. Hoxie

CD4-independent infection by HIV-2 is mediated by Fusin/CXCR4

Several members of the chemokine receptor family have been shown to function in association with CD4 to permit HIV-1 entry and infection. However, the mechanism by which these molecules serve as CD4-associated cofactors is unclear. In the present report, we show that one member of this family, termed Fusin/ CXCR4, is able to function as an alternative receptor for some isolates of HIV-2 in the absence of CD4. This conclusion is supported by the finding that (1) CD4-independent infection by these viruses is inhibited by an anti-Fusin monoclonal antibody, (2) Fusin expression renders human and nonhuman CD4-negative cell lines sensitive to HIV-2-induced syncytium induction and/or infection, and (3) Fusin is selectively down-regulated from the cell surface following HIV-2 infection. The finding that one chemokine receptor can function as a primary viral receptor strongly suggests that the HIV envelope glycoprotein contains a binding site for these proteins and that differences in the affinity and/or the availability of this site can extend the host range of these viruses to include a number of CD4-negative cell types.

Interactions of Mast Cell Tryptase with Thrombin Receptors and PAR-2

Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-protein-coupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca2+. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca2+. These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach.

Neuronal apoptosis induced by HIV-1 gp120 and the chemokine SDF-1α is mediated by the chemokine receptor CXCR4

CXCR4, a seven transmembrane domain G-protein-coupled receptor for the Cys-X-Cys class of chemokines, is one of several chemokine receptors that can act as a co-receptor with CD4 for the human immunodeficiency virus (HIV-1) glycoprotein gp120 [1-3]. CXCR4 can mediate the entry of HIV-1 strains that specifically infect T cells, such as the IIB strain (see [4] for review). Recent reports indicate that gp120 can signal through CXCR4 [5] and it has been suggested that signal transduction, mediated by the viral envelope, might influence viral-associated cytopathicity or apoptosis [6]. Neuronal apoptosis is a feature of HIV-1 infection in the brain [7,8], although the exact mechanism is unknown. Here, we address the possible role of CXCR4 in inducing apoptosis using cells of the hNT human neuronal cell line; these cells resemble immature post-mitotic cholinergic neurons and have a number of neuronal characteristics [9-15]. We have previously shown that gp120 from the HIV-1 IIIB strain binds with high affinity to CXCR4 expressed on hNT neurons [15]. We now find that both IIIB gp120 and the Cys-X-Cys chemokine SDF-1 alpha can directly induce apoptosis in hNT neurons in the absence of CD4 and in a dose-dependent manner. To our knowledge, this is the first report of a chemokine and an HIV-1 envelope glycoprotein eliciting apoptotic responses through a chemokine receptor.

CD4-independent association between HIV-1 gp120 and CXCR4: functional chemokine receptors are expressed in human neurons

BACKGROUND Chemokines are a family of proteins that chemoattract and activate immune cells by interacting with specific receptors on the surface of their targets. We have shown previously that chemokine receptors including the interleukin-8 receptor B (CXCR2) and the Duffy blood group antigen are expressed on subsets of neurons in various regions of the adult nervous system. RESULTS Using a combination of immunohistochemical staining and receptor binding studies, we show that hNT cells, which are differentiated human neurons derived from the cell line NTera2, express functional chemokine receptors of the C-X-X and C-C types. These chemokine receptors include CXCR2, CXCR4, CCR1 and CCR5. We demonstrate high-affinity binding of both types of chemokines to hNT neurons and dose-dependent chemotactic responses to these chemokines in differentiated, but no t undifferentiated, NTera 2 cells. In addition, we show that the envelop glycoprotein from the T-cell-tropic human immunodeficiency virus 1 (HIV-1) strain IIIB is a CD4-independent, dose-dependent inhibitor of the binding of stromal cell-derived factor 1 to its receptor, CXCR4. CONCLUSIONS These data support recent findings that members of the chemokine family, including CCR5 and LESTR/Fusin (CXCR4), function as coreceptors in combination with CD4 for HIV-1 invasion. This is the first report of functional expression of chemokine receptors on human neurons. Furthermore, our studies provide for direct CD4-independent association of the viral envelope protein of the HIV-1 strain III with the chemokine receptor CXCR4.

Envelope Glycoprotein Incorporation, Not Shedding of Surface Envelope Glycoprotein (gp120/SU), Is the Primary Determinant of SU Content of Purified Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) particles typically contain small amounts of the surface envelope protein (SU), and this is widely believed to be due to shedding of SU from mature virions. We purified proteins from HIV-1 and SIV isolates using procedures which allow quantitative measurements of viral protein content and determination of the ratios of gag- and env-encoded proteins in virions. All of the HIV-1 and most of the SIV isolates examined contained low levels of envelope proteins, with Gag:Env ratios of approximately 60:1. Based on an estimate of 1,200 to 2,500 Gag molecules per virion, this corresponds to an average of between 21 and 42 SU molecules, or between 7 and 14 trimers, per particle. In contrast, some SIV isolates contained levels of SU at least 10-fold greater than SU from HIV-1 isolates. Quantification of relative amounts of SU and transmembrane envelope protein (TM) provides a means to assess the impact of SU shedding on virion SU content, since such shedding would be expected to result in a molar excess of TM over SU on virions that had shed SU. With one exception, viruses with sufficient SU and TM to allow quantification were found to have approximately equivalent molar amounts of SU and TM. The quantity of SU associated with virions and the SU:TM ratios were not significantly changed during multiple freeze-thaw cycles or purification through sucrose gradients. Exposure of purified HIV-1 and SIV to temperatures of 55°C or greater for 1 h resulted in loss of most of the SU from the virus but retention of TM. Incubation of purified virus with soluble CD4 at 37°C resulted in no appreciable loss of SU from either SIV or HIV-1. These results indicate that the association of SU and TM on the purified virions studied is quite stable. These findings suggest that incorporation of SU-TM complexes into the viral membrane may be the primary factor determining the quantity of SU associated with SIV and HIV-1 virions, rather than shedding of SU from mature virions.

Antigenic conservation and immunogenicity of the HIV coreceptor binding site

Immunogenic, broadly reactive epitopes of the HIV-1 envelope glycoprotein could serve as important targets of the adaptive humoral immune response in natural infection and, potentially, as components of an acquired immune deficiency syndrome vaccine. However, variability in exposed epitopes and a combination of highly effective envelope-cloaking strategies have made the identification of such epitopes problematic. Here, we show that the chemokine coreceptor binding site of HIV-1 from clade A, B, C, D, F, G, and H and circulating recombinant form (CRF)01, CRF02, and CRF11, elicits high titers of CD4-induced (CD4i) antibody during natural human infection and that these antibodies bind and neutralize viruses as divergent as HIV-2 in the presence of soluble CD4 (sCD4). 178 out of 189 (94%) HIV-1–infected patients had CD4i antibodies that neutralized sCD4-pretreated HIV-2 in titers (50% inhibitory concentration) as high as 1:143,000. CD4i monoclonal antibodies elicited by HIV-1 infection also neutralized HIV-2 pretreated with sCD4, and polyclonal antibodies from HIV-1–infected humans competed specifically with such monoclonal antibodies for binding. In vivo, variants of HIV-1 with spontaneously exposed coreceptor binding surfaces were detected in human plasma; these viruses were neutralized directly by CD4i antibodies. Despite remarkable evolutionary diversity among primate lentiviruses, functional constraints on receptor binding create opportunities for broad humoral immune recognition, which in turn serves to constrain the viral quasispecies.

Analysis of neutralization specificities in polyclonal sera derived from human immunodeficiency virus type 1-infected individuals.

ABSTRACT During human immunodeficiency virus type 1 (HIV-1) infection, patients develop various levels of neutralizing antibody (NAb) responses. In some cases, patient sera can potently neutralize diverse strains of HIV-1, but the antibody specificities that mediate this broad neutralization are not known, and their elucidation remains a formidable challenge. Due to variable and nonneutralizing determinants on the exterior envelope glycoprotein (Env), nonnative Env protein released from cells, and the glycan shielding that assembles in the context of the quaternary structure of the functional spike, HIV-1 Env elicits a myriad of binding antibodies. However, few of these antibodies can neutralize circulating viruses. We present a systematic analysis of the NAb specificities of a panel of HIV-1-positive sera, using methodologies that identify both conformational and continuous neutralization determinants on the HIV-1 Env protein. Characterization of sera included selective adsorption with native gp120 and specific point mutant variants, chimeric virus analysis, and peptide inhibition of viral neutralization. The gp120 protein was the major neutralizing determinant for most sera, although not all neutralization activity against all viruses could be identified. In some broadly neutralizing sera, the gp120-directed neutralization mapped to the CD4 binding region of gp120. In addition, we found evidence that regions of the gp120 coreceptor binding site may also be a target of neutralizing activity. Sera displaying limited neutralization breadth were mapped to the immunogenic V3 region of gp120. In a subset of sera, we also identified NAbs directed against the conserved, membrane-proximal external region of gp41. These data allow a more detailed understanding of the humoral responses to the HIV-1 Env protein and provide insights regarding the most relevant targets for HIV-1 vaccine design.

HIV vaccine research: the way forward.

The need to broaden research directed at answering fundamental questions in HIV vaccine discovery through laboratory, nonhuman primate (NHP), and clinical research has recently been emphasized. In addition, the importance of attracting and retaining young researchers, developing better NHP models, and more closely linking NHP and clinical research is being stressed. In an era of a level budget for biomedical research at the U.S. National Institutes of Health (NIH), HIV/AIDS vaccine research efforts will need to be carefully prioritized such that resources to energize HIV vaccine discovery can be identified. This article summarizes progress and challenges in HIV vaccine research, the priorities arising from a recent summit at NIAID, and the actions needed, some already under way, to address those priorities.

Differential N-Linked Glycosylation of Human Immunodeficiency Virus and Ebola Virus Envelope Glycoproteins Modulates Interactions with DC-SIGN and DC-SIGNR

ABSTRACT The C-type lectins DC-SIGN and DC-SIGNR [collectively referred to as DC-SIGN(R)] bind and transmit human immunodeficiency virus (HIV) and simian immunodeficiency virus to T cells via the viral envelope glycoprotein (Env). Other viruses containing heavily glycosylated glycoproteins (GPs) fail to interact with DC-SIGN(R), suggesting some degree of specificity in this interaction. We show here that DC-SIGN(R) selectively interact with HIV Env and Ebola virus GPs containing more high-mannose than complex carbohydrate structures. Modulation of N-glycans on Env or GP through production of viruses in different primary cells or in the presence of the mannosidase I inhibitor deoxymannojirimycin dramatically affected DC-SIGN(R) infectivity enhancement. Further, murine leukemia virus, which typically does not interact efficiently with DC-SIGN(R), could do so when produced in the presence of deoxymannojirimycin. We predict that other viruses containing GPs with a large proportion of high-mannose N-glycans will efficiently interact with DC-SIGN(R), whereas those with solely complex N-glycans will not. Thus, the virus-producing cell type is an important factor in dictating both N-glycan status and virus interactions with DC-SIGN(R), which may impact virus tropism and transmissibility in vivo.

EXPRESSION OF CXCR4, THE RECEPTOR FOR STROMAL CELL-DERIVED FACTOR-1 ON FETAL AND ADULT HUMAN LYMPHOHEMATOPOIETIC PROGENITORS

Stromal cell‐derived factor‐1 (SDF‐1) is a CXC chemokine produced by stromal cells that acts as a chemoattractant for human CD34+ progenitor cells. We investigated the expression of CXCR4, the receptor for SDF‐1, on CD34+ cells from different hematopoietic sites and developmental stages. CXCR4 was detected by flow cytometry on 37 % of fetal bone marrow (BM) [gestation weeks (gw) 14 – 23] and 40 % of adult BM CD34+ cells. Interestingly, in fetal liver CD34+ cells, CXCR4 was expressed at lower levels at later stages (9 %, gw 20 – 23) compared to early stages of development (39 %, gw 7.5 – 18), suggesting a development‐related change in the migratory capacity of progenitors. CXCR4 was detected at similar levels on both phenotypically primitive and committed progenitors from fetal and adult sites. However, B cell lineage progenitor and precursor cells expressed CXCR4 at the highest density (80 % of BM CD34+/CD10+ pro‐B cells are CXCR4+). CXCR4 was also expressed in the fetal thymus in early T cell precursors and found to be down‐regulated during T cell maturation. Finally, we found that stem cell factor, alone or in combination with other cytokines, can up‐modulate CXCR4 expression on CD34+ cells by three‐ to fourfold. In conclusion, our results suggest that CXCR4 may play an important role in the local and systemic trafficking of human CD34+ cells as well as in human B lymphopoiesis and that its expression can be modulated by cytokines.