Endocytosis and recycling of the HIV coreceptor CCR5.

The chemokine receptor CCR5 is a cofactor for the entry of R5 tropic strains of human immunodeficiency viruses (HIV)-1 and -2 and simian immunodeficiency virus. Cells susceptible to infection by these viruses can be protected by treatment with the CCR5 ligands regulated on activation, normal T cell expressed and secreted (RANTES), MIP-1alpha, and MIP-1beta. A major component of the mechanism through which chemokines protect cells from HIV infection is by inducing endocytosis of the chemokine receptor. Aminooxypentane (AOP)-RANTES, an NH(2)-terminal modified form of RANTES, is a potent inhibitor of infection by R5 HIV strains. AOP-RANTES efficiently downmodulates the cell surface expression of CCR5 and, in contrast with RANTES, appears to prevent recycling of CCR5 to the cell surface. Here, we investigate the cellular basis of this effect. Using CHO cells expressing human CCR5, we show that both RANTES and AOP-RANTES induce rapid internalization of CCR5. In the absence of ligand, CCR5 shows constitutive turnover with a half-time of 6-9 h. Addition of RANTES or AOP-RANTES has little effect on the rate of CCR5 turnover. Immunofluorescence and immunoelectron microscopy show that most of the CCR5 internalized after RANTES or AOP-RANTES treatment accumulates in small membrane-bound vesicles and tubules clustered in the perinuclear region of the cell. Colocalization with transferrin receptors in the same clusters of vesicles indicates that CCR5 accumulates in recycling endosomes. After the removal of RANTES, internalized CCR5 recycles to the cell surface and is sensitive to further rounds of RANTES-induced endocytosis. In contrast, after the removal of AOP-RANTES, most CCR5 remains intracellular. We show that these CCR5 molecules do recycle to the cell surface, with kinetics equivalent to those of receptors in RANTES-treated cells. However, these recycled CCR5 molecules are rapidly reinternalized. Our results indicate that AOP-RANTES-induced changes in CCR5 alter the steady-state distribution of the receptor and provide the first evidence for G protein-coupled receptor trafficking through the recycling endosome compartment.

CD4-Chemokine receptor hybrids in human immunodeficiency virus type 1 infection.

Most human immunodeficiency virus (HIV) strains require both CD4 and a chemokine receptor for entry into a host cell. In order to analyze how the HIV-1 envelope glycoprotein interacts with these cellular molecules, we constructed single-molecule hybrids of CD4 and chemokine receptors and expressed these constructs in the mink cell line Mv-1-lu. The two N-terminal (2D) or all four (4D) extracellular domains of CD4 were linked to the N terminus of the chemokine receptor CXCR4. The CD4(2D)CXCR4 hybrid mediated infection by HIV-1(LAI) to nearly the same extent as the wild-type molecules, whereas CD4(4D)CXCR4 was less efficient. Recombinant SU(LAI) protein competed more efficiently with the CXCR4-specific monoclonal antibody 12G5 for binding to CD4(2D)CXCR4 than for binding to CD4(4D)CXCR4. Stromal cell-derived factor 1 (SDF-1) blocked HIV-1(LAI) infection of cells expressing CD4(2D)CXCR4 less efficiently than for cells expressing wild-type CXCR4 and CD4, whereas down-modulation of CXCR4 by SDF-1 was similar for hybrids and wild-type CXCR4. In contrast, the bicyclam AMD3100, a nonpeptide CXCR4 ligand that did not down-modulate the hybrids, blocked hybrid-mediated infection at least as potently as for wild-type CXCR4. Thus SDF-1, but not the smaller molecule AMD3100, may interfere at multiple points with the binding of the surface unit (SU)-CD4 complex to CXCR4, a mechanism that the covalent linkage of CD4 to CXCR4 impedes. Although the CD4-CXCR4 hybrids yielded enhanced SU interactions with the chemokine receptor moiety, this did not overcome the specific coreceptor requirement of different HIV-1 strains: the X4 virus HIV-1(LAI) and the X4R5 virus HIV-1(89. 6), unlike the R5 strain HIV-1(SF162), infected Mv-1-lu cells expressing the CD4(2D)CXCR4 hybrid, but none could use hybrids of CD4 and the chemokine receptor CCR2b, CCR5, or CXCR2. Thus single-molecule hybrid constructs that mimic receptor-coreceptor complexes can be used to dissect coreceptor function and its inhibition.

Chemokine receptor trafficking and viral replication.

Chemokines and chemokine receptors have emerged as crucial factors controlling the development and function of leukocytes. Recent studies have indicated that, in addition to these essential roles, both chemokines and chemokine receptors play critical roles in viral infection and replication. Not only are chemokine receptors key components of the receptor/fusion complexes of primate immunodeficiency viruses, but chemokines can also influence virus entry and infection. Many viruses, in particular herpesviruses, encode chemokines and chemokine receptors that influence the replication of both the parent virus and other unrelated viruses. The cell surface expression of the chemokine receptors is regulated through their interaction with membrane trafficking pathways. Ligands induce receptor internalization and downmodulation through endocytosis, and recycling is regulated within endosomes. Part of the mechanism through which chemokines protect cells from HIV infection is through ligand-induced internalization of the specific chemokine receptor co-receptors. In addition, mechanisms may exist to regulate the trafficking of newly synthesized receptors to the cell surface. Here we discuss aspects of the mechanisms through which chemokine receptors interact with membrane-trafficking pathways and the influence of these interactions on viral replication.

B cell antigen receptor engagement inhibits stromal cell-derived factor (SDF)-1alpha chemotaxis and promotes protein kinase C (PKC)-induced internalization of CXCR4.

The entry of B lymphocytes into secondary lymphoid organs is a critical step in the development of an immune response, providing a site for repertoire shaping, antigen-induced activation and selection. These events are controlled by signals generated through the B cell antigen receptor (BCR) and are associated with changes in the migration properties of B cells in response to chemokine gradients. The chemokine stromal cell-derived factor (SDF)-1alpha is thought to be one of the driving forces during those processes, as it is produced inside secondary lymphoid organs and induces B lymphocyte migration that arrests upon BCR engagement. The signaling pathway that mediates this arrest was genetically dissected using B cells deficient in specific BCR-coupled signaling components. BCR-induced inhibition of SDF-1alpha chemotaxis was dependent on Syk, BLNK, Btk, and phospholipase C (Plc)gamma2 but independent of Ca2+ mobilization, suggesting that the target of BCR stimulation was a protein kinase C (PKC)-dependent substrate. This target was identified as the SDF-1alpha receptor, CXCR4, which undergoes PKC- dependent internalization upon BCR stimulation. Mutation of the internalization motif SSXXIL in the COOH terminus of CXCR4 resulted in B cells that constitutively expressed this receptor upon BCR engagement. These studies suggest that one pathway by which BCR stimulation results in inhibition of SDF-1alpha migration is through PKC-dependent downregulation of CXCR4.

Lack of p56lck expression correlates with CD4 endocytosis in primary lymphoid and myeloid cells.

In cell lines the endocytic properties of CD4 are regulated through its association with the src-family tyrosine kinase p56lck. In lymphoid cell lines expressing p56lck, CD4 is restricted to the cell surface and undergoes only limited internalization. Phosphorylation of the cytoplasmic domain of CD4 causes p56lck to dissociate and activates an endocytosis signal leading to the internalization of CD4 through clathrin-coated pits. In p56lck-negative transfected cell lines CD4 is constitutively internalized, but internalization is inhibited when p56lck is expressed in these cells. We now demonstrate that these endocytic properties of CD4 determined in transfected cell lines hold true for CD4 naturally expressed on myeloid cell lines (HL-60 and U937), as well as on primary lymphocytes, monocytes and macrophages isolated from human blood. CD4 showed limited internalization on p56lck-positive lymphocytes, but was rapidly internalized in p56lck-negative monocytes and macrophages. Surprisingly, rapid internalization of CD4 was seen with the lymphocytes from one unidentified donor. In these cells we failed to detect p56lck expression by Western blotting.

Differential regulation of CXCR4 and CCR5 endocytosis.

The chemokine receptors CCR5 and CXCR4 are major co-receptors/receptors for the CD4-dependent and CD4-independent entry of human and simian immunodeficiency viruses. The chemokines that bind and activate these receptors can inhibit the entry of viruses that use the respective co-receptor molecules. Chemokine-induced co-receptor internalisation is a significant component of the mechanism through which chemokines inhibit virus entry. CXCR4 internalisation is induced by the CXCR4 ligand stromal cell derived factor-1 (SDF-1), phorbol esters and, in T cells, cellular activation. Here we show that CXCR4 endocytosis can be mediated through either one of two distinct internalisation signals. A COOH-terminal serine rich domain is required for ligand- but not phorbol ester- induced CXCR4 internalisation. However, a Ser/IleLeu motif, similar to that required for the endocytosis of CD4 and the T cell receptor/CD3 complex, is required for phorbol ester-induced, but not ligand-induced, CXCR4 endocytosis. By contrast, CCR5 internalisation is induced by the beta-chemokine RANTES but not by phorbol esters. CCR5 lacks the Ser/IleLeu sequence required for phorbol ester-induced uptake of CXCR4. Together these results indicate that distinct mechanisms can regulate CXCR4 and CCR5 endocytosis and trafficking.

Aminooxypentane-RANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity.

CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH2-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.

Phorbol esters and SDF-1 induce rapid endocytosis and down modulation of the chemokine receptor CXCR4.

The chemokine receptor CXCR4 is required, together with CD4, for entry by some isolates of HIV-1, particularly those that emerge late in infection. The use of CXCR4 by these viruses likely has profound effects on viral host range and correlates with the evolution of immunodeficiency. Stromal cell-derived factor-1 (SDF-1), the ligand for CXCR4, can inhibit infection by CXCR4-dependent viruses. To understand the mechanism of this inhibition, we used a monoclonal antibody that is specific for CXCR4 to analyze the effects of phorbol esters and SDF-1 on surface expression of CXCR4. On human T cell lines SupT1 and BC7, CXCR4 undergoes slow constitutive internalization (1.0% of the cell surface pool/min). Addition of phorbol esters increased this endocytosis rate >6-fold and reduced cell surface CXCR4 expression by 60 to 90% over 120 min. CXCR4 was internalized through coated pits and coated vesicles and subsequently localized in endosomal compartments from where it could recycle to the cell surface after removal of the phorbol ester. SDF-1 also induced the rapid down modulation (half time approximately 5 min) of CXCR4. Using mink lung epithelial cells expressing CXCR4 and a COOH-terminal deletion mutant of CXCR4, we found that an intact cytoplasmic COOH-terminal domain was required for both PMA and ligand-induced CXCR4 endocytosis. However, experiments using inhibitors of protein kinase C indicated that SDF-1 and phorbol esters trigger down modulation through different cellular mechanisms. SDF-1 inhibited HIV-1 infection of mink cells expressing CD4 and CXCR4. The inhibition of infection was less efficient for CXCR4 lacking the COOH-terminal domain, suggesting at least in part that SDF-1 inhibition of virus infection was mediated through ligand-induced internalization of CXCR4. Significantly, ligand induced internalization of CXCR4 but not CD4, suggesting that CXCR4 and CD4 do not normally physically interact on the cell surface. Together these studies indicate that endocytosis can regulate the cell-surface expression of CXCR4 and that SDF-1-mediated down regulation of cell-surface coreceptor expression contributes to chemokine-mediated inhibition of HIV infection.

Transduction of activation signal that follows HIV-1 binding to CD4 and CD4 dimerization involves the immunoglobulin CDR3-like region in domain 1 of CD4.

The role of CD4 during the human immunodeficiency virus type 1 (HIV-1) life cycle in T cells is not restricted to binding functions. HIV-1 binding to CD4 also triggers signals that lead to nuclear translocation of NF-kappaB and are important to the productive infection process. In addition to its cytoplasmic tail, in the ectodomain, the immunoglobulin (Ig) CDR3-like region of CD4 domain 1 seemed to play a role in this cascade of signals. We demonstrate in this work that the structural integrity of the CDR3-like loop is required for signal transduction. Substitutions of negatively charged residues by positively charged residues within the CDR3-like loop either inhibited NF-kappaB translocation after HIV-1 and gp120-anti-gp120 immune complexes binding to E91K,E92K mutants or induced its constitutive activation for E87K,D88K mutants. Moreover, A2.01-3B cells expressing the E91K,E92K mutant exhibited a lower HIV-1Lai replication. These cells, however, expressed p56(lck), demonstrated NF-kappaB translocation upon PMA stimulation, bound HIV-1Lai envelope glycoprotein with high affinity, and contained HIV-1 DNA 24 h after exposure to virus. E91K, E92K, and E87K,D88K mutant CD4 molecules were unable to bind a CD4 synthetic aromatically modified exocyclic, CDR3.AME-(82-89), that mimics the CDR3-like loop structure and binds to native cell surface CD4. This result together with molecular modeling studies indicates that the CDR3.AME-(82-89) analog binds to the CDR3-like loop of CD4 and strongly suggests that this region represents a site for CD4 dimerization. The negative charges on the CDR3-like loop thus appear critical for CD4-mediated signal transduction most likely related to CD4 dimer formation.

Role of CD4 epitopes outside the gp120-binding site during entry of human immunodeficiency virus type 1.

CD4 is the primary receptor for human immunodeficiency virus (HIV). The binding site for the surface glycoprotein of HIV type 1 (HIV-1), gp120, has been mapped to the C'-C" region of domain 1 of CD4. Previously, we have shown that a mutant of rat CD4, in which this region was exchanged for that of human CD4, is able to mediate infection of human cells by HIV-1, suggesting that essential interactions between HIV and CD4 are confined to this region. Our observations appeared to conflict with mutagenesis and antibody studies which implicate regions of CD4 outside the gp120-binding site in postbinding events during viral entry. In order to resolve this issue, we have utilized a panel of anti-rat CD4 monoclonal antibodies in conjunction with the rat-human chimeric CD4 to distinguish sequence-specific from steric effects. We find that several antibodies to rat CD4 inhibit HIV infection in cells expressing the chimeric CD4 and that this is probably due to steric hinderance. In addition, we demonstrate that replacement of the rat CDR3-like region with its human homolog does not increase the affinity of the rat-human chimeric CD4 for gp120 or affect the exposure of gp41 following binding to CD4, providing further evidence that this region does not play a crucial role during entry of virus.

Selective effects of electrostatic changes in the CD4 CDR-3-like loop on infection by different human immunodeficiency virus type 1 isolates.

The role of the CDR-3-like loop of the first domain of the CD4 molecule in infection by the human immunodeficiency virus type 1 (HIV-1) is controversial. In an attempt to determine whether the strong negative charge in the CDR-3-like loop influences HIV-1 infection we have substituted by mutagenesis negative for positively charged residues at position 87/88 and 91/92. These mutations were shown to have no obvious effect on CD4 conformation outside of the CDR-3-like loop. Infection of cells expressing the E87K/D88K substitution mutant resulted in a selective reduction in infectivity for certain HIV-1 viruses compared to cells expressing wile-type CD4. Viruses Hx10, HxB2, and MN were 4- to 13-fold less efficient at infecting the E87K/D88K mutant, whereas SF2, RF, and NDK yielded an efficiency of infection similar to, or slightly greater than, that of the wild type. To investigate the step at which infectivity was selectively reduced, we compared early events in the life cycles of Hx10 and SF2 viruses using PCR entry and gp120-binding assays. Both gp120 binding and virus entry were reduced for Hx10 on the mutant CD4-expressing cells as compared to wild-type CD4-expressing cells, whereas no difference was seen in either assay with SF2. Although relatively small in magnitude, the contribution of the CDR-3-like loop to the overall CD4-gp120 interaction may serve to modify the binding and entry of certain virus isolates.

Class I major histocompatibility complex-restricted cytotoxic T lymphocytes specific for Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines against which they were raised.

We have raised CD8+ cytotoxic T lymphocytes (CTL) from three Epstein-Barr virus-seropositive donors by incubating peripheral blood lymphocytes with irradiated autologous B95.8-strain EBV-transformed B lymphoblastoid cells (LCL). However, to detect lysis in a standard 51Cr release assay of the LCL against which these CTL were raised, superinfection with recombinant vaccinia expressing the appropriate EBV protein or incubation with the peptide epitope was necessary. The untreated LCL were not lysed, even though Western blotting demonstrated that they expressed the EBV antigens containing the CTL epitopes. We have found CTL of this phenotype that are restricted by human leukocyte antigen-A2, -A3, -B7, or -B39, and which recognize the EBV latent proteins, EBV nuclear antigen (EBNA)-3A, EBNA-3C, or terminal protein. During these experiments, we identified a new human leukocyte antigen-A3-restricted EBNA-3A epitope, residues 603-611, RLRAEAGVK. We raised a spontaneous LCL, transformed by endogenous EBV, from one donor, but this was also not lysed. For at least one of the epitopes, CTL from another donor lysed the LCL without superinfection or addition of peptides. We conclude that the CTL were unable to achieve a high enough avidity of interaction with untreated LCL to trigger effector function, although the LCL were able to stimulate them to grow in vitro for up to 4 mo. To assess whether a small percentage of the LCL might possess a higher antigen density, we used an assay of tumor necrosis factor release from a CTL clone, which was able to detect antigen-bearing cells representing only 1% of a stimulating LCL population. Nevertheless, the untreated autologous LCL line failed to stimulate tumor necrosis factor release.

Antisense oligonucleotides in solution or encapsulated in immunoliposomes inhibit replication of HIV-1 by several different mechanisms.

Phosphodiester and phosphorothioate oligonucleotides in alpha and beta configurations directed against the initiation codon region of the HIV-1 rev gene were evaluated for their ability to inhibit HIV-1 replication in acutely and chronically infected human CEM cells. Encapsulation in antibody-targeted liposomes (immunoliposomes) permitted intracellular delivery and distinction between oligonucleotide-mediated inhibition of viral entry and intracellular effects on viral RNA. Our results are consistent with four mechanisms of antiviral activity for these antisense oligonucleotides: (i) interference with virus-mediated cell fusion by free but not liposome-encapsulated phosphorothioate oligonucleotides of any sequence; (ii) interference with reverse transcription in a sequence non-specific manner by phosphorothioate oligonucleotides in alpha and beta configurations; (iii) interference with viral reverse transcription in a sequence-specific and RNase-H-independent manner by alpha and beta phosphodiester oligonucleotides; (iv) interference with viral mRNA in a sequence-specific and RNase-H-dependent manner by beta-phosphorothioate oligonucleotides.

Factors involved in entry of the human immunodeficiency virus type 1 into permissive cells: lack of evidence of a role for CD26.

It has been proposed recently that the cell surface peptidase CD26 acts in concert with CD4, the human immunodeficiency virus (HIV) primary receptor molecule, to mediate HIV entry into permissive cells. We have failed to detect significant levels of CD26 cell surface expression and enzymatic activity in a number of commonly propagated human CD4+ cell lines, although CD26 mRNA was present at very low levels, as detected by reverse transcription PCR. No relationship existed between the expression of CD26 and the ability of these cells to be infected with HIV or to fuse to form syncytia. We have tested two inhibitors of CD26 enzymatic activity and several anti-CD26 monoclonal antibodies and found that they inhibit neither HIV infection nor HIV-induced syncytium formation. NIH 3T3 cells stably transfected with the cDNAs for human CD4 and CD26 expressed these molecules at the cell surface and had CD26 enzymatic activity. Inoculation of the double transfectants with HIV did not result in virus entry above the background level, as verified by PCR amplification of viral DNA. We were unable to recover infectious virus from the HIV-inoculated NIH 3T3 double transfectants either by transfer of supernatants or by cocultivation with human CD4+ indicator cells. Moreover, the transfectants did not fuse with HIV-infected cells to form syncytia, nor were syncytia observed in HIV-inoculated cultures. These results are inconsistent with the CD26 molecule being a cofactor for entry of HIV in CD4+ cells.

Human and simian immunodeficiency viruses: virus-receptor interactions.

The major cellular receptor for the primate immunodeficiency viruses is the CD4 molecule. As well as mediating virion attachment to the cell surface, CD4 is thought to activate the viral fusion pathway. CD4 is not, however, sufficient for viral entry; other molecules are probably involved, and in certain circumstances these may substitute for CD4. Viral tropism and cytopathogenicity are also influenced by receptor interactions.