Human megakaryocytes have a CD4 molecule capable of binding human immunodeficiency virus-1

Most human megakaryocytes (MGKs) express the CD4 antigen on their surface. Approximately 25% have a CD4 receptor density comparable to that of CD4+ T cells (Basch et al, Proc Natl Acad Sci USA 87:8085, 1990). In these studies, we show: (1) the presence of mRNA for CD4 in human MGKs; (2) the binding of human immunodeficiency virus-1 (HIV-1) to human MGKs; (3) the inhibition of binding by anti-CD4 (Leu3a) antibody or rCD4; (4) the infection of a human MGK line, CHRF-288 with HIV-1; and (5) inhibition of infection with anti-CD4. Human MGKs have mRNA for CD4 as shown by in situ hybridization with an RNA probe synthesized from a 3-kb cDNA sequence of plasmid pSP65.T4.8 containing the full-length CD4 sequence. MGKs (23% +/- 17%) bound HIV-1, as determined by anti-gp120 and anti-CD41 staining. Binding to human MGKs could be inhibited 55% to 75% with anti-CD4 or rCD4, respectively. Infection of a CD4+ MGK line (CHRF-288) could be accomplished with HIV- 1, as determined by proviral DNA polymerase chain reaction and p24 production. Preincubation with anti-CD4 inhibited apparent proviral DNA infection by 100% and p24 production by 65% to 70%. Thus, human MGKs have a CD4 receptor capable of binding HIV-1. Using this receptor, HIV- 1 can infect cells representative of the MGK lineage.     

The CD4 receptor plays essential but distinct roles in HIV-1 infection and induction of apoptosis in primary bone marrow GPIIb/IIIa+ megakaryocytes and the HEL cell line

Summary. We investigated whether cells belonging to the megakaryocyte lineage could be infected in vitro with human immunodeficiency virus type-1 (HIV-1). Primary GPIIb/IIIa⁺ bone marrow (BM) cells and HEL continuous cell line were first phenotypically characterized for the presence of megakaryocytic markers and CD4 antigen, then challenged in vitro with the laboratory strain IIIB of HIV-1. Both GPIIb/IIIa⁺ BM and HEL cells expressed significant levels of CD4 receptor (<50%) and were efficiently infected with HIV-1, as judged by the presence of proviral DNA after polymerase chain reaction analysis and by quantitative evaluation of gag p24 antigen in the culture supernatants. Of note, infection with HIV-1 in both primary BM megakaryocytes and HEL cells was specifically blocked by soluble recombinant CD4. To ascertain whether the CD4 receptor was essential for infection of megakaryocytic cells, HEL were subcloned into CD4⁺ and CD4 cells. Although unfractionated and CD4⁺ HEL cells were productively infected with HIV-1, CD4 HEL cells could not be infected. Infection of HEL cells did not induce gross cytotoxic effects or a significant increase of apoptosis. On the other hand, treatment of unfractionated or CD4⁺ HEL cells with cross-linked recombinant env gp120 or Leu3a anti-CD4 monoclonal antibody markedly (P< 0.01) increased the degree of apoptosis with respect to HEL cells infected with HIV-1 or treated with cross-linked gag p24 or anti-GPIIb/IIIa antibody. Taken together, these data indicate that the CD4 receptor represents the main route of infection in cells belonging to the megakaryocytic lineage. Moreover, an inappropriate engagement of CD4 by either free env gpl20 or anti-CD4 monoclonal antibody could be more relevant than a direct infection with HIV-1 in the induction of the frequent BM megakaryocyte abnormalities found in HIV-1 seropositive thrombocytopenic patients.     

Unliganded HIV-1 gp120 core structures assume the CD4-bound conformation with regulation by quaternary interactions and variable loops

The HIV-1 envelope (Env) spike (gp120(3)/gp41(3)) undergoes considerable structural rearrangements to mediate virus entry into cells and to evade the host immune response. Engagement of CD4, the primary human receptor, fixes a particular conformation and primes Env for entry. The CD4-bound state, however, is prone to spontaneous inactivation and susceptible to antibody neutralization. How does unliganded HIV-1 maintain CD4-binding capacity and regulate transitions to the CD4-bound state? To define this mechanistically, we determined crystal structures of unliganded core gp120 from HIV-1 clades B, C, and E. Notably, all of these unliganded HIV-1 structures resembled the CD4-bound state. Conformational fixation with ligand selection and thermodynamic analysis of full-length and core gp120 interactions revealed that the tendency of HIV-1 gp120 to adopt the CD4-bound conformation was restrained by the V1/V2- and V3-variable loops. In parallel, we determined the structure of core gp120 in complex with the small molecule, NBD-556, which specifically recognizes the CD4-bound conformation of gp120. Neutralization by NBD-556 indicated that Env spikes on primary isolates rarely assume the CD4-bound conformation spontaneously, although they could do so when quaternary restraints were loosened. Together, the results suggest that the CD4-bound conformation represents a "ground state" for the gp120 core, with variable loop and quaternary interactions restraining unliganded gp120 from "snapping" into this conformation. A mechanism of control involving deformations in unliganded structure from a functionally critical state (e.g., the CD4-bound state) provides advantages in terms of HIV-1 Env structural diversity and resistance to antibodies and inhibitors, while maintaining elements essential for entry.     

The susceptibility to X4 and R5 human immunodeficiency virus-1 strains of dendritic cells derived in vitro from CD34(+) hematopoietic progenitor cells is primarily determined by their maturation stage.

Dendritic cells (DC) were sorted on day 8 from cultures of CD34(+) cells with stem cell factor/Flt-3 ligand/ granulocyte-macrophage colony-stimulating factor (GM-CSF)/tumor necrosis factor-alpha (TNF-alpha)/interleukin-4 (IL-4). Exposing immature CCR5(+)CXCR4(lo/-) DC to CCR5-dependent human immunodeficiency virus (HIV)-1Ba-L led to productive and cytopathic infection, whereas only low virus production occurred in CXCR4-dependent HIV-1LAI-exposed DC. PCR analysis of the DC 48 hours postinfection showed efficient entry of HIV-1Ba-L but not of HIV-1LAI. CD40 ligand- or monocyte-conditioned medium-induced maturation of HIV-1Ba-L-infected DC reduced virus production by about 1 Log, while cells became CCR5(-). However, HIV-1Ba-L-exposed mature DC harbored 15-fold more viral DNA than their immature counterparts, ruling out inhibition of virus entry. Simultaneously, CXCR4 upregulation by mature DC coincided with highly efficient entry of HIV-1LAI which, nonetheless, replicated at the same low level in mature as in immature DC. In line with these findings, coculture of HIV-1Ba-L-infected immature DC with CD3 monoclonal antibody-activated autologous CD4(+) T lymphocytes in the presence of AZT decreased virus production by the DC. Finally, whether they originated from CD1a+CD14(-) or CD1a-CD14(+) precursors, DC did not differ as regards permissivity to HIV, although CD1a+CD14(-) precursor-derived immature DC could produce higher HIV-1Ba-L amounts than their CD1a-CD14(+) counterparts. Thus, both DC permissivity to, and capacity to support replication of, HIV is primarily determined by their maturation stage.     

CD4 Expression by erythroid precursor cells in human bone marrow

Flow cytometry was used to assess CD4 expression in 62 consecutive bone marrow specimens from patients with a variety of clinical conditions. Using a lysed-whole-blood technique for labeling with monoclonal antibodies, two populations of CD4+ cells were identified within the lymphocyte/blast-cell fraction in 58 (94%) of these specimens. These consisted of (1) a population of T helper cells with high density expression of CD4 and (2) a second population of cells with low-density expression of CD4, which ranged from 1% to 36% of the gated cells. This latter population was present regardless of age, sex, or clinical condition including 21 of 21 specimens (100%) categorized as unremarkable bone marrows both morphologically and by flow cytometry and in four of four patients (100%) with human immunodeficiency virus- type 1 (HIV-1) infection. Coexpression of the erythroid lineage marker, glycophorin A, with the majority of cells in this second population was demonstrated in all 11 randomly selected samples using two-color flow cytometric analysis. These cells also expressed low levels of the myeloid markers, CD13 and CD33, but CD34 expression could not be demonstrated. These results provide evidence for expression of CD4 on cells of erythroid lineage in human marrow, and offer a potential mechanism for direct infection of erythroid precursor cells and deranged erythropoiesis in patients with HIV-1 infection.     

Expression of human CD4 in transgenic mice does not confer sensitivity to human immunodeficiency virus infection.

Transfection of the human CD4 molecule into mouse cells does not confer susceptibility to human immunodeficiency virus type 1 (HIV-1) infection. Expression of the human CD4 molecule in transgenic mice was seen to offer some new possibilities. However, transgenic mouse T cells expressing either the human CD4 receptor, or a hybrid human/mouse CD4 receptor alone or in conjunction with human major histocompatibility complex class I molecules, were refractory to in vitro HIV-1 infection. In addition, no infection was observed after in vivo HIV inoculation to mice of these various transgenic lines. Injection of recombinant gp160 viral protein to the transgenic mice did not alter their T and B cell populations. The existence of a dominant block in mouse cells that prevents HIV entry is discussed.     

Cervical and prostate primary epithelial cells are not productively infected but sequester human immunodeficiency virus type 1

Primary prostate and cervical epithelial cells and epithelial cell lines were examined for human immunodeficiency virus type 1 (HIV-1) infection or transmission to peripheral blood mononuclear cells (PBMC). Neither cell-free nor cell-associated HIV-1 infected primary epithelial cells or cell lines. Pretreatment of HIV-1 to enhance CD4-independent entry did not augment infection. Cell surface expression was detected for galactosyl ceramide but not for CC-chemokine receptor 5, CXC-chemokine receptor 4, or CD4. The ability to transfer HIV-1 to resting or activated PBMC was tested by culturing with rinsed or trypsinized and replated HIV-1-exposed epithelial cells. Virus was not recovered from the rinsed or replated cocultures with resting PBMC; however, activated PBMC recovered HIV-1 from rinsed epithelial cells and rarely from replated epithelial cells. Although urogenital epithelial cells are not infected, these data suggest that they can transfer virus to activated immune cells and have implications for sexual transmission of HIV-1.     

SPC3, a synthetic peptide derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120, inhibits HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms.

The third variable region (V3 loop) of gp120, the HIV-1 surface envelope glycoprotein, plays a key role in HIV-1 infection and pathogenesis. Recently, we reported that a synthetic multibranched peptide (SPC3) containing eight V3-loop consensus motifs (GPGRAF) inhibited HIV-1 infection in both CD4+ and CD4- susceptible cells. In the present study, we investigated the mechanisms of action of SPC3 in these cell types--i.e., CD4+ lymphocytes and CD4- epithelial cells expressing galactosylceramide (GalCer), an alternative receptor for HIV-1 gp120. We found that SPC3 was a potent inhibitor of HIV-1 infection in CD4+ lymphocytes when added 1 h after initial exposure of the cells to HIV-1, whereas it had no inhibitory effect when present only before and/or during the incubation with HIV-1. These data suggested that SPC3 did not inhibit the binding of HIV-1 to CD4+ lymphocytes but interfered with a post-binding step necessary for virus entry. In agreement with this hypothesis, SPC3 treatment after HIV-1 exposure dramatically reduced the number of infected cells without altering gp120-CD4 interaction or viral gene expression. In contrast, SPC3 blocked HIV-1 entry into CD4-/GalCer+ human colon epithelial cells when present in competition with HIV-1 but had no effect when added after infection. Accordingly, SPC3 was found to inhibit the binding of gp120 to the GalCer receptor. Thus, the data suggest that SPC3 affects HIV-1 infection by two distinct mechanisms: (i) prevention of GalCer-mediated HIV-1 attachment to the surface of CD4-/GalCer+ cells and (ii) post-binding inhibition of HIV-1 entry into CD4+ lymphocytes.     

Cell-dependent mechanisms restrict the HIV type 1 coreceptor activity of US28, a chemokine receptor homolog encoded by human cytomegalovirus

Several members of the chemokine receptor family are used together with CD4 for HIV-1 entry into target cells. The human cytomegalovirus US28 gene encodes a chemokine receptor homolog that has been reported to function as an HIV-1 coreceptor. However, studies of US28 have given conflicting results regarding its ability to mediate HIV-1 entry. We examined the ability of US28 to function as an HIV-1 coreceptor in various cell lines and found that its coreceptor activity is highly cell dependent. US28 could function as a coreceptor for HIV-1 entry in HeLa and U87 cells but not in COS-1 and Cf2Th cells. In COS-1 cells, US28 was expressed on the cell surface and could mediate cell-cell fusion with HIV-1 Env-expressing cells, suggesting that the block to infection may result from a defect in virus internalization or postentry steps. In Cf2Th cells, US28 was expressed at high levels intracellularly but was not transported to the cell surface. The block in US28 coreceptor function in COS-1 and Cf2Th cells was coreceptor dependent, since CCR5, CXCR4, and other coreceptors can mediate HIV-1 entry in these cell lines. HIV-1 viruses pseudotyped with the MuLV or VSV Env entered and replicated at similar efficiency in COS-1 and U87 cells in single-cycle infections, suggesting that postentry and other early events in the HIV-1 life cycle are not intrinsically inefficient in COS-1 cells. These results identify two distinct mechanisms that can restrict the HIV-1 coreceptor activity of US28 in a cell- and coreceptor-dependent manner, and help to explain the existing controversy regarding the ability of US28 to mediate HIV-1 entry.     

A nonneutralizing anti-HIV Type 1 antibody turns into a broad neutralizing antibody when expressed on the surface of HIV type 1-susceptible cells. II. Inhibition of HIV type 1 captured and transferred by DC-SIGN

Previously, we demonstrated that the expression of a nonneutralizing human anti-HIV-1 gp41 scFv on the surface of HIV-1-susceptible cells markedly inhibits HIV-1 replication and HIV-1 envelope-mediated cell-cell fusion. The inhibition is at the level of viral entry, specific for the HIV-1 envelope, and independent of virus tropism. In the previous studies, cell-free viruses of laboratory-adapted HIV-1 strains from subtype B were used to infect human CD4 T cell lines. To further test the effectiveness of this membrane-bound scFv (m-scFv) on HIV-1 infection, in this study, we carried out experiments to determine whether the m-scFv can neutralize infection of primary isolates from various HIV-1 subtypes and whether the m-scFv can neutralize HIV-1 captured and transferred by DC-SIGN on the surface of monocytic cell lines or DCs. We demonstrated that the m-scFv markedly inhibits primary isolates derived from various subtypes and significantly blocks HIV-1 captured and transferred by DC-SIGN on monocytic cell lines and on human DCs. Therefore, a nonneutralizing antibody acts as a broad neutralizing antibody when expressed on the cell surface, which significantly inhibits infection of both cell-free and DC-SIGN-captured and transferred virus. Our studies further point out the potential use of m-scFv as a inhibitor against HIV-1 transmission as well as a tool to dissect the mechanism of HIV-1 entry via DC-SIGN capture and transfer to CD4 T cells.     

Characterization of CD4-induced epitopes on the HIV type 1 gp120 envelope glycoprotein recognized by neutralizing human monoclonal antibodies

The entry of human immunodeficiency virus (HIV-1) into target cells typically requires the sequential binding of the viral exterior envelope glycoprotein, gp120, to CD4 and a chemokine receptor. CD4 binding exposes gp120 epitopes recognized by CD4-induced (CD4i) antibodies, which can block virus binding to the chemokine receptor. We identified three new CD4i antibodies from an HIV-1-infected individual and localized their epitopes. These epitopes include a highly conserved gp120 beta-strand encompassing residues 419-424, which is also important for binding to the CCR5 chemokine receptor. All of the CD4i antibodies inhibited the binding of gp120-CD4 complexes to CCR5. CD4i antibodies and CD4 reciprocally induced each other's binding, suggesting that these ligands recognize a similar gp120 conformation. The CD4i antibodies neutralized laboratory-adapted HIV-1 isolates; primary isolates were more resistant to neutralization by these antibodies. Thus, all known CD4i antibodies recognize a common, conserved gp120 element overlapping the binding site for the CCR5 chemokine receptor.     

Combination of HIV-1-specific CD4 Th1 cell responses and IgG2 antibodies is the best predictor for persistence of long-term nonprogression

BACKGROUND: Strong T cell and antibody responses to human immunodeficiency virus (HIV), low virus production, and some genetic traits have been individually associated with nonprogression of HIV infection, but the best correlate with protection against disease progression remains unknown. METHODS: We prospectively followed 66 untreated long-term nonprogressors and analyzed relationships between HIV-1-specific CD4 T helper (Th) 1 and CD8 T cell responses and HIV-1-specific antibodies, HIV-1 RNA and proviral DNA loads, host genes, and CD4 Th1 cell counts at entry into the study and 4 years later. RESULTS: HIV-1 p24-specific CD4 Th1 cell proliferation, interferon (IFN)- gamma production, and IFN- gamma -producing cell frequencies at entry significantly and negatively correlated with HIV-1 RNA and proviral DNA loads and were independent of CD4 Th1 cell counts and host genes. HIV-1 Gag-specific IFN- gamma -producing CD8 T cell frequencies correlated with HIV-1 proviral DNA loads but not with RNA loads. Only high frequencies of HIV-1 p24-specific CD4 Th1 cells combined with HIV-1 gp41-specific IgG2 antibodies significantly predicted persistence of high CD4 Th1 cell counts. CONCLUSION: HIV-1-specific CD4 Th1 responses combined with IgG2 antibodies and IFN- gamma -producing CD4 Th1 cells are better predictors of long-term nonprogression than are virus parameters, host genes, or HIV-1-specific CD4 Th1 or CD8 T cell proliferation.     

The leukotriene B4 receptor functions as a novel type of coreceptor mediating entry of primary HIV-1 isolates into CD4-positive cells

The recently cloned human chemoattractant receptor-like (CMKRL)1, which is expressed in vivo in CD4-positive immune cells, has structural homology with the two chemokine receptors C-C chemokine receptor (CCR)5 and C-X-C chemokine receptor (CXCR)4, which serve as the major coreceptors necessary for fusion of the HIV-1 envelope with target cells. In view of the structural similarity, CMKRL1 was tested for its possible function as another HIV-1 coreceptor after stable expression in murine fibroblasts bearing the human CD4 receptor. The cells were infected with 10 primary clinical isolates of HIV-1, and entry was monitored by semiquantitative PCR of viral DNA. The efficiency of the entry was compared with the entry taking place in CD4-positive cells expressing either CCR5 or CXCR4. Seven of the isolates used CMKRL1 for viral entry; they were mainly of the syncytium-inducing phenotype and also used CXCR4. Entry efficiency was higher with CMKRL1 than with CXCR4 for more than half of these isolates. Three of the ten isolates did not use CMKRL1; instead, entry was mediated by both CCR5 and CXCR4. The experiments thus indicate that CMKRL1 functions as a coreceptor for the entry of HIV-1 into CD4-positive cells. In the course of this study, leukotriene B₄ was shown to be the natural ligand for this receptor (now designated BLTR), which therefore represents a novel type of HIV-1 coreceptor along with the previously identified chemokine receptors. BLTR belongs to the same general chemoattractant receptor family as the chemokine receptors but is structurally more distant from them than are any of the previously described HIV-1 coreceptors.     

Internalization of human immunodeficiency virus type I and other retroviruses by megakaryocytes and platelets

Direct infection of megakaryocytes and platelets by human immunodeficiency virus type I (HIV-I) or other retroviruses has not been demonstrated. To determine whether this could occur, murine bone marrow was co-cultivated with the amphotropic retrovirus-producing cell line PA317-N2, and freshly isolated normal human bone marrow and platelets were co-cultivated with HIV-infected H9 cells. In each case, ultrastructural analyses showed viruses within megakaryocytes and platelets. In murine specimens, the uptake of retrovirus was avid at all stages of differentiation. In human specimens, viral uptake was less frequent. These results suggest that direct infection of megakaryocytes could play a role in the pathophysiology of HIV-associated disease. In addition, these observations suggest that cells of the megakaryocyte lineage could serve as target cells in gene transfer experiments using retroviral-based vectors.     

Human immunodeficiency virus (HIV) type 1 can superinfect HIV-2-infected cells: pseudotype virions produced with expanded cellular host range.

In studies on viral interference, cloned T-cell lines chronically infected with human immunodeficiency virus (HIV) type 1 or HIV-2 were inoculated with several strains of these two AIDS retrovirus subtypes. HIV-2UC1-infected cells, which still express the CD4 receptor, could be superinfected with a variety of HIV-1 and HIV-2 strains. This event was accompanied by cytopathic effects in the cells and production of pseudotype virions with an expanded cellular host range. HIV-1- or HIV-2-infected clonal cell lines, which did not express CD4, could not be superinfected by any HIV strains but were coinfected after transfection of molecular clones into the persistently infected cells. These observations indicate that viral interference with HIV occurs at the cell surface and involves a down-modulation of the CD4 molecule. If the CD4 protein is expressed, superinfection can take place, and phenotypically mixed virus particles are produced. Since HIV-1 and HIV-2 dually infected individuals have been detected, these in vitro observations may have relevance to the in vivo state.