Aymeric de Parseval

Binding of Recombinant Feline Immunodeficiency Virus Surface Glycoprotein to Feline Cells: Role of CXCR4, Cell-Surface Heparans, and an Unidentified Non-CXCR4 Receptor

ABSTRACT To address the role of CXCR4 in the cell-surface attachment of the feline immunodeficency virus (FIV), a soluble fusion protein, gp95-Fc, consisting of the surface glycoprotein (SU, gp95) of either a primary (PPR) or cell line-adapted (34TF10) FIV strain was fused in frame with the Fc domain of human immunoglobulin G1. The recombinant SU-immunoadhesins were used as probes to investigate the cellular binding of FIV SU. In agreement with the host cell range properties of both viruses, binding of 34TF10 gp95-Fc was observed for all cell lines tested, whereas PPR gp95-Fc bound only to primary feline T cells. 34TF10 gp95-Fc also bound to Jurkat and HeLa cells, consistent with the ability of FIV to use human CXCR4 as a fusion receptor. As expected, 34TF10 gp95-Fc binding to Jurkat cells was blocked by addition of stromal cell-derived factor 1α (SDF-1α), as was binding to the 3201 feline lymphoma cell line. However, SDF-1α, RANTES, macrophage inflammatory protein 1β, and heparin all failed to inhibit the binding of either gp95-Fc to primary T cells, suggesting that a non-CXCR4 receptor is involved in the binding of FIV SU. In this regard, an unidentified 40-kDa protein species from the surface of primary T cells but not Jurkat and 3201 cells specifically coprecipitated with both gp95-Fc. Yet another type of binding of 34TF10 gp95-Fc to adherent kidney cells was noted. SDF-1α failed to block the binding of 34TF10 gp95-Fc to either HeLa, Crandel feline leukemia, or G355-5 cells. However, binding was severely impaired in the presence of soluble heparin, as well as after enzymatic removal of surface heparans or on cells deficient in heparan expression. These overall findings suggest that in addition to CXCR4, a non-CXCR4 receptor and cell-surface heparans also play an important role in FIV gp95 cell surface interactions on specific target cells.

A Highly Conserved Arginine in gp120 Governs HIV-1 Binding to Both Syndecans and CCR5 via Sulfated Motifs

HIV-1 has maximized its utilization of syndecans. It uses them as in cis receptors to infect macrophages and as in trans receptors to infect T-lymphocytes. In this study, we investigated at a molecular level the mechanisms that control HIV-1-syndecan interactions. We found that a single conserved arginine (Arg-298) in the V3 region of gp120 governs HIV-1 binding to syndecans. We found that an amine group on the side chain of this residue is necessary for syndecan utilization by HIV-1. Furthermore, we showed that HIV-1 binds syndecans via a 6-O sulfation, demonstrating that this binding is not the result of random interactions between basic residues and negative charges, but the result of specific contacts between gp120 and a well defined sulfation in syndecans. Surprisingly, we found that Arg-298, which mediates HIV-1 binding to syndecans, also mediates HIV-1 binding to CCR5. We postulated that HIV-1 recognizes similar motifs on syndecans and CCR5. Supporting this hypothesis, we obtained several lines of evidence that suggest that the 6-O sulfation recognized by HIV-1 on syndecans mimics the sulfated tyrosines recognized by HIV-1 in the N terminus of CCR5. Our finding that CCR5 and syndecans are exploited by HIV-1 via a single determinant echoes the mechanisms by which chemokines utilize these two disparate receptors and suggests that the gp120/chemokine mimicry may represent a common strategy in microbial pathogenesis.

Human Immunodeficiency Virus Envelope (gp120) Binding to DC-SIGN and Primary Dendritic Cells Is Carbohydrate Dependent but Does Not Involve 2G12 or Cyanovirin Binding Sites: Implications for Structural Analyses of gp120-DC-SIGN Binding

ABSTRACT The calcium-dependent lectin, DC-SIGN, binds to human immunodeficiency virus (HIV) (and simian immunodeficiency virus) gp120 and mediates the binding and transfer of HIV from monocyte-derived dendritic cells (MDDCs) to permissive T cells. However, it has been recently reported that DC-SIGN binding to HIV gp120 may be carbohydrate independent. Here, we formally demonstrate that gp120 binding to DC-SIGN and MDDCs is largely if not wholly carbohydrate dependent. Endo-β-N-glucosaminidase H (EndoH) treatment of gp120-Fc under conditions that maintained wild-type CD4 binding—and the full complement of complex glycans—significantly decreased (>90%) binding to DC-SIGN expressing cell lines, as well as to MDDCs. Any residual binding of EndoH-treated gp120-Fc to DC-SIGN was completely competed off with mannan. Mutational analysis indicated that no single glycosylation site affected the ability of gp120-Fc to bind DC-SIGN. To further guide our efforts in mapping the DC-SIGN binding sites on gp120, we used two well-characterized HIV inhibitory agents (2G12 monoclonal antibody and cyanovirin) that bind to high-mannose sugars on gp120. We showed that 2G12 and DC-SIGN bound to nonoverlapping sites in gp120 because (i) 2G12 did not block soluble gp120 or virion binding to DC-SIGN, (ii) 2G12 bound to gp120-Fc that was prebound to cell surface DC-SIGN, and (iii) gp120-Fc mutants that lack glycosylation sites involved in 2G12s epitope were also fully capable of binding DC-SIGN. These data were substantiated by the inability of cyanovirin to block gp120-Fc binding to DC-SIGN. Cyanovirin has been shown to effectively compete for 2G12 binding to gp120. Indeed, high concentrations of cyanovirin dramatically enhanced gp120-Fc binding to cell surfaces in the presence or absence of DC-SIGN. We provide evidence that this enhancement may be due to cyanovirins ability to bridge gp120 to mannosylated cell surface proteins. These results have implications for antiviral therapeutics and for ongoing efforts to finely map the glycan structures on gp120 responsible for DC-SIGN binding.

Structural mapping of CD134 residues critical for interaction with feline immunodeficiency virus.

CD134 is a primary binding receptor for feline immunodeficiency virus (FIV), and with CXCR4 facilitates infection of CD4+ T cells. Human CD134 fails to support FIV infection. To delineate the regions important for defining virus specificity of CD134, we exchanged domains between human and feline CD134. The binding site for FIV surface glycoprotein (SU) is located in domain 1, in a region distinct from the natural ligand (CD134L)-binding site. Mutagenesis showed that Asp60 and Asp62 are required for interaction with FIV, and modeling studies localized these two residues to the outer edge of domain 1. Substitutions S60D and N62D, in conjunction with H45S, R59G and V64K, imparted both FIV SU binding and receptor function to human CD134. Finally, we demonstrated that soluble CD134 facilitates infection of CD134− CXCR4+ target cells in a manner analogous to CD4 augmentation of HIV infection.

Molecular mechanisms of FIV infection.

Feline immunodeficiency virus (FIV) is an important viral pathogen worldwide in the domestic cat, which is the smallest animal model for the study of natural lentivirus infection. Thus, understanding the molecular mechanisms by which FIV carries out its life cycle and causes an acquired immune deficiency syndrome (AIDS) in the cat is of high priority. FIV has an overall genome size similar to HIV, the causative agent of AIDS in man, and shares with the human virus genomic features that may serve as common targets for development of broad-based intervention strategies. Specific targets include enzymes encoded by the two lentiviruses, such as protease (PR), reverse transcriptase (RT), RNAse H, and integrase (IN). In addition, both FIV and HIV encode Vif and Rev elements essential for virus replication and also share the use of the chemokine receptor CXCR4 for entry into the host cell. The following review is a brief overview of the current state of characterization of the feline/FIV model and development of its use for generation and testing of anti-viral agents.

Sequential CD134-CXCR4 interactions in feline immunodeficiency virus (FIV): soluble CD134 activates FIV Env for CXCR4-dependent entry and reveals a cryptic neutralization epitope.

ABSTRACT Recombinant soluble CD134 (sCD134) facilitated feline immunodeficiency virus (FIV) entry into CXCR4-positive, cell surface CD134-negative target cells. sCD134-activated entry was dose dependent and CXCR4 dependent. We used the sCD134 activation system to explore the neutralization by four anti-V3 monoclonal antibodies (MAbs). V3 MAbs weakly neutralized FIV infection using target cells expressing both CD134 and CXCR4 but potently inhibited sCD134-activated entry into target cells expressing CXCR4 alone. These findings provide direct evidence for a sequential interaction of FIV Env with CD134 and CXCR4 and reveal the presence of a cryptic epitope in V3 that is masked in the mature envelope oligomers.

Human Immunodeficiency Virus Type 1 and Influenza Virus Exit via Different Membrane Microdomains

ABSTRACT Directed release of human immunodeficiency virus type 1 (HIV-1) into the cleft of the virological synapse that can form between infected and uninfected T cells, for example, in lymph nodes, is thought to contribute to the systemic spread of this virus. In contrast, influenza virus, which causes local infections, is shed into the airways of the respiratory tract from free surfaces of epithelial cells. We now demonstrate that such differential release of HIV-1 and influenza virus is paralleled, at the subcellular level, by viral assembly at different microsegments of the plasma membrane of HeLa cells. HIV-1, but not influenza virus, buds through microdomains containing the tetraspanins CD9 and CD63. Consequently, the anti-CD9 antibody K41, which redistributes its antigen and also other tetraspanins to cell-cell adhesion sites, interferes with HIV-1 but not with influenza virus release. Altogether, these data strongly suggest that the bimodal egress of these two pathogenic viruses, like their entry into target cells, is guided by specific sets of host cell proteins.

Factors That Increase the Effective Concentration of CXCR4 Dictate Feline Immunodeficiency Virus Tropism and Kinetics of Replication

ABSTRACT The surface glycoprotein (gp95) of the feline immunodeficiency virus (FIV) binds in a strain-specific manner to several cell surface molecules, including CXCR4, heparan sulfate proteoglycans (HSPGs), DC-SIGN, and a 43-kDa cell surface receptor on T cells recently identified as CD134 by M. Shimojima et al. (Science 303:1192-1195, 2004). CXCR4 is the entry receptor in all known cases, and the other molecules act as binding receptors to help facilitate infection. In this report, we confirm and extend the findings regarding CD134 as a primary receptor for FIV. In addition, we show that temperature critically influences the binding properties of FIV gp95 to CXCR4 and HSPGs. The data show that gp95 of the field strain FIV-PPR bound to CXCR4 at 22°C, whereas binding was not detected at 4°C. In contrast, binding of the laboratory adapted FIV-34TF10 gp95 was observed at either 4°C or 22°C, albeit at increased levels at the higher temperature. The level of CXCR4 increased after the temperature was switched from 4 to 22°C, whereas the level of HSPGs decreased, resulting in higher binding of gp95 from both strains to CXCR4 and lower binding of gp95 of FIV-34TF10 to HSPGs (FIV-PPR gp95 does not bind to these molecules). The findings also show that HSPGs facilitate the CXCR4-mediated infectivity of CrFK and G355-5 cells by FIV-34TF10. These two nonlymphoid cell lines express very low levels of CXCR4 and are permissive to FIV-34TF10 but not to productive infection by FIV-PPR. However, overexpression of human CXCR4 in CrFK or G-355-5 cells resulted in extensive cell fusion and infection by FIV-PPR. Taken together, these findings indicate that factors that increase the effective concentration of CXCR4 enhance FIV infectivity and may involve (i) temperature or ligand-induced conformational changes in CXCR4 that enhance SU binding, (ii) coreceptor interactions with gp95 that either alter gp95 conformation to enhance CXCR4 binding and/or raise the localized concentration of receptor or ligand, or (iii) direct increase in CXCR4 concentration via overexpression.

Specific Interaction of Feline Immunodeficiency Virus Surface Glycoprotein with Human DC-SIGN

ABSTRACT DC-SIGN, a specific C-type lectin expressed on dendritic cells, binds and transmits multiple strains of primate immunodeficiency viruses to susceptible cells. Here, we report that human DC-SIGN also captures feline immunodeficiency virus via high-affinity (1 nM), Ca2+-dependent, d-mannose-inhibited binding to the major envelope glycoprotein, gp95.

Feline Immunodeficiency Virus OrfA Is Distinct from Other Lentivirus Transactivators

ABSTRACT The feline immunodeficiency virus (FIV) accessory factor, OrfA, facilitates transactivation of transcription directed by elements of the viral long terminal repeat (LTR). In order to map OrfA domains required for this transactivation, we used N- and C-terminal deletion constructs of the protein, expressed in a Gal4-based transactivation system. The results demonstrated that FIV OrfA, unlike other lentiviral transactivators such as visna virus Tat, is unable to transactivate from minimal promoter-based reporters and requires additional elements of the viral LTR. Stable CrFK-based cell lines were prepared that expressed OrfA to readily detectable levels and in which we were able to demonstrate 32-fold transactivation of an LTR-chloramphenicol acetyltransferase construct. Transactivation was heavily dependent on the presence of an ATF site within the viral LTR. Changing the translation initiation codon context substantially increased the level of production of OrfA from a bicistronic message that also encodes Rev. In the presence of a more favorable context sequence, the upstream expression of OrfA increased 21-fold, with only a 0.5-fold drop in downstream Rev expression. This suggests that Rev translation may occur via an internal ribosomal entry site rather than by leaky scanning.