Atomic structure of a fragment of human CD4 containing two immunoglobulin-like domains.

The structure of an N-terminal fragment of CD4 has been determined to 2.4 A resolution. It has two tightly abutting domains connected by a continuous beta strand. Both have the immunoglobulin fold, but domain 2 has a truncated beta barrel and a non-standard disulphide bond. The binding sites for monoclonal antibodies, class II major histocompatibility complex molecules, and human immunodeficiency virus gp120 can be mapped on the molecular surface.     

Two monoclonal antibodies against different antigens using the same VH germ-line gene

Immunoglobulin diversity seems to arise largely by three mechanisms: (1) the existence of several germ-line genes, which must be rearranged before expression--that is, V and J for the light (L) chains, V, D and J for the heavy (H) chains; (2) somatic events, including mutations and gene conversion; and (3) combinatorial association of heavy and light chains, leading to the proposal that random pairing of p X H and q X L chains might generate p X q antibody molecules expressing discrete specificities. As heavy and light chains derived from the same immunoglobulin molecule would frequently reassociate preferentially, it is likely that only a fraction of potential heavy--light pairs actually provides "valid' antibodies. As a consequence of combinatorial heavy--light chain pairing, antibodies of discrete specificities sharing the same VH region, associated with distinct light chains (or vice versa) should be encountered. We report here that two heavy chains, derived from the same VH germ-line gene, may be present in anti-NP or anti--GAT antibodies, depending on their association with a specific lambda or kappa light chain, respectively.     

Substitution of murine for human CD4 residues identifies amino acids critical for HIV-gp120 binding

Human CD4 is the receptor for the gp120 envelope glycoprotein of human immunodeficiency virus and is essential for virus entry into the host cell. Sequence analysis of CD4 has suggested an evolutionary origin from a structure with four immunoglobulin-related domains. Only the two NH2-terminal domains are required to mediate gp120 binding. The extracellular segment of murine CD4 has an overall 50% identity with its human counterpart at the amino-acid level, but fails to bind gp120. To define those residues of human CD4 critical for gp120 binding, we have taken advantage of this species difference and substituted all non-conserved murine for human CD4 residues between amino-acid positions 27-167. We used oligonucleotide-directed mutagenesis to create each of 16 individual mutant human CD4 molecules containing from 1-4 amino-acid substitutions. Introduction of as few as three amino acids into corresponding positions of human CD4 abrogates gp120 binding. Furthermore, these critical residues are located in domain I with a contribution from domain II. Modelling studies using the three-dimensional coordinates of the V kappa Bence-Jones REI homodimer localize the site in domain I to the C" beta strand within CDR2 but projecting away from the homologues of principle antigen-binding regions CDR 1 and 3.     

Insights into IgA-mediated immune responses from the crystal structures of human FcalphaRI and its complex with IgA1-Fc

Immunoglobulin-alpha (IgA)-bound antigens induce immune effector responses by activating the IgA-specific receptor FcalphaRI (CD89) on immune cells. Here we present crystal structures of human FcalphaRI alone and in a complex with the Fc region of IgA1 (Fcalpha). FcalphaRI has two immunoglobulin-like domains that are oriented at approximately right angles to each other. Fcalpha resembles the Fcs of immunoglobulins IgG and IgE, but has differently located interchain disulphide bonds and external rather than interdomain N-linked carbohydrates. Unlike 1:1 FcgammaRIII:IgG and Fc epsilon RI:IgE complexes, two FcalphaRI molecules bind each Fcalpha dimer, one at each Calpha2-Calpha3 junction. The FcalphaRI-binding site on IgA1 overlaps the reported polymeric immunoglobulin receptor (pIgR)-binding site, which might explain why secretory IgA cannot initiate phagocytosis or bind to FcalphaRI-expressing cells in the absence of an integrin co-receptor.     

Lymphocyte activation by HIV-1 envelope glycoprotein

Cell activation by phytohaemagglutinin, phorbol ester and by the supernatant of phytohaemagglutinin-stimulated peripheral blood mononuclear cells induces the expression and cytopathic effects of latent human immunodeficiency virus type-1 (HIV-1) in vitro. The lymphocyte surface protein CD4 has been identified as a receptor for HIV-1 and binds the viral envelope glycoprotein (gp120). In the light of evidence indicating that one natural function of CD4 is as a growth factor receptor, we examined the ability of native gp120 to activate resting CD4-bearing lymphocytes. Our results indicate that gp120 has innate biological activity as a result of a specific interaction with CD4, inducing increases in intracellular levels of inositol trisphosphate and of calcium, and in interleukin-2 receptor expression and cell motility.     

Four ATP-binding sites in the midregion of the beta heavy chain of dynein.

The 'motor' proteins of eukaryotic cells contain specialized domains that hydrolyse ATP to produce force and movement along a cytoskeletal polymer (actin in the case of the myosin family; microtubules in the case of the kinesin family and dyneins). There are motor-protein superfamilies in which each member has a conserved force-generating domain joined to a different 'tail' which conveys specific attachment properties. The minus-end-directed microtubule motors, the dyneins, may also constitute a superfamily of force-generating proteins with distinct attachment domains. Axonemal outer-arm dynein from sea urchin spermatozoa is a multimeric protein consisting of two heavy chains (alpha and beta) with ATPase activity, three intermediate chains and several light chains. Here I report the sequence of cloned complementary DNA encoding the beta heavy chain of a dynein motor molecule. The predicted amino-acid sequence reveals four ATP-binding consensus sequences in the central domain. The dynein beta heavy chain is thought to associate transiently with a microtubule during ATP hydrolysis, but the ATP-dependent microtubule-binding sequence common to the kinesin superfamily is not found in the dynein beta heavy chain. These unique features distinguish the dynein beta heavy chain from other motor protein superfamilies and may be characteristic of the dynein superfamily.     

The gene product Murr1 restricts HIV-1 replication in resting CD4+ lymphocytes

Although human immunodeficiency virus-1 (HIV-1) infects quiescent and proliferating CD4+ lymphocytes, the virus replicates poorly in resting T cells. Factors that block viral replication in these cells might help to prolong the asymptomatic phase of HIV infection; however, the molecular mechanisms that control this process are not fully understood. Here we show that Murr1, a gene product known previously for its involvement in copper regulation, inhibits HIV-1 growth in unstimulated CD4+ T cells. This inhibition was mediated in part through its ability to inhibit basal and cytokine-stimulated nuclear factor (NF)-kappaB activity. Knockdown of Murr1 increased NF-kappaB activity and decreased IkappaB-alpha concentrations by facilitating phospho-IkappaB-alpha degradation by the proteasome. Murr1 was detected in CD4+ T cells, and RNA-mediated interference of Murr1 in primary resting CD4+ lymphocytes increased HIV-1 replication. Through its effects on the proteasome, Murr1 acts as a genetic restriction factor that inhibits HIV-1 replication in lymphocytes, which could contribute to the regulation of asymptomatic HIV infection and the progression of AIDS.     

HIV requires multiple gp120 molecules for CD4-mediated infection

Binding of glycoprotein gp120 to the T cell-surface receptor CD4 is a crucial step in CD4-dependent infection of a target cell by the human immunodeficiency virus (HIV). Blocking some or all gp120 molecules on the viral surface should therefore inhibit infection. Consequently, competitive receptor inhibitors, such as soluble synthetic CD4 (sCD4), synthetic CD4 peptides and immunoglobulins, have been investigated in vitro and in vivo, but little is known about the molecular mechanisms of these inhibitors. We have now quantitatively examined blocking by soluble CD4 in the hope of gaining insight into the complex process of viral binding, adsorption and penetration. At low sCD4 concentrations, the inhibition in three HIV strains is proportional to the binding of gp120. The biological association constant (gp120-sCD4 Kassoc) for HIV-2NIHZ is (8.5 +/- 0.5) x 10(7) M-1, whereas Kassoc for HIV-1HXB3 (1.4 +/- 0.2) and HIV-1MN (1.7 +/- 0.1) x 10(9) M-1 are 15-20-fold larger. For all three viral strains, the biological Kassoc from infectivity assays is comparable to the chemical Kassoc. The inhibitory action of sCD4 at high concentrations, however, is not fully explained by simple proportionality with the binding to gp120. Positive synergy in blocking of infection occurs after about half the viral gp120s molecules are occupied, and is identical for all three viral strains, despite the large differences in Kassoc. Our method of measuring the viral-cell receptor Kassoc directly from infectivity assays is applicable to immunoglobulins, to other viruses and to assays using primary or transformed cell lines.     

Inhibition of HIV replication by pokeweed antiviral protein targeted to CD4+ cells by monoclonal antibodies

Functional impairment and selective depletion of CD4+ T cells, the hallmark of AIDS, are at least partly caused by human immunodeficiency virus (HIV-1) type 1 binding to the CD4 molecule and infecting CD4+ cells. It may, therefore, be of therapeutic value to target an antiviral agent to CD4+ cells to prevent infection and to inhibit HIV-1 production in patients' CD4+ cells which contain proviral DNA. We report here that HIV-1 replication in normal primary CD4+ T cells can be inhibited by pokeweed antiviral protein, a plant protein of relative molecular mass 30,000, which inhibits replication of certain plant RNA viruses, and of herpes simplex virus, poliovirus and influenza virus. Targeting pokeweed antiviral protein to CD4+ T cells by conjugating it to monoclonal antibodies reactive with CD5, CD7 or CD4 expressed on CD4+ cells, increased its anti-HIV potency up to 1,000-fold. HIV-1 replication is inhibited at picomolar concentrations of conjugates of pokeweed antiviral protein and monoclonal antibodies, which do not inhibit proliferation of normal CD4+ T cells or CD4-dependent responses. These conjugates inhibit HIV-1 protein synthesis and also strongly inhibit HIV-1 production in activated CD4+ T cells from infected patients.     

HIV infection of primate lymphocytes and conservation of the CD4 receptor

The CD4 T-lymphocyte differentiation antigen is an essential component of the cell surface receptor for human immunodeficiency viruses (HIVs) causing AIDS (acquired immune deficiency syndrome) (refs 1-3). Peripheral blood lymphocytes of apes, New World and Old World monkeys express cell surface antigens homologous to CD4 of human T-helper lymphocytes. The cells of several of these species can be infected in short term culture with diverse strains of the type-1 or type-2 human immunodeficiency viruses (HIV-1 and HIV-2). HIV-1 is the prototype AIDS virus, and HIV-2 is the second type of AIDS virus, prevalent in West Africa. Infection of the primate cells correlates with evolutionary conservation on CD4 of one particular epitope cluster, and is inhibited by treatment of the cells with monoclonal antibodies to this epitope. The capacity of HIV to replicate in simian cells may provide a means for evaluating antiviral drugs and vaccines.     

Correlation between fragmented immunoglobulin genes and heavy chain deletion mutants.

It is generally accepted that the variable (V) and constant (C) regions of immunoglobulin (Ig) chains are under separate genetic control. The notion that the different domains and interdomain regions are also under the control of independent genetic units was initially based on the clearcut results obtained by studying the primary structure of deletion mutants and received definitive support from direct analysis of cloned heavy (H) and light (L) chain genes. Here we present additional studies carried out on two selected gamma 3 deletion mutants which indicate the genetic control of human H chains may be even more complex than previously believed.     

Macrophage and T cell-line tropisms of HIV-1 are determined by specific regions of the envelope gp120 gene

Strains of human immunodeficiency virus type 1 (HIV-1) display a high degree of biological heterogeneity which may be linked to certain clinical manifestation of AIDS. They vary in their ability to infect different cell types, to replicate rapidly and to high titre in culture, to down-modulate the CD4 receptor, and to cause cytopathic changes in infected cells. Some of these in vitro properties correlate with pathogenicity of the virus in vivo. To map the viral determinants of the cellular host range of HIV-1, recombinant viruses were generated between biologically active molecular clones of HIV-1 isolates showing differences in infection of primary peripheral blood macrophages and established T-cell lines. We report here that a specific region of the envelope gp120 gene representing 159 amino-acid residues of glycoprotein gp120 seems to determine macrophage tropism, whereas an overlapping region representing 321 amino-acid residues determines T cell-line tropism. These studies provide a basis for relating functional domains of the HIV-1 env gene to pathogenic potential.     

Effect of recombinant soluble CD4 in rhesus monkeys infected with simian immunodeficiency virus of macaques

The CD4 molecule is a high-affinity cell-surface receptor for the human immunodeficiency virus (HIV-1) and a soluble truncated form of CD4 produced by recombinant DNA technology is a potent inhibitor of HIV-1 replication and HIV-1-induced cell fusion in vitro. Rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIVMAC), a virus closely related to HIV-1, develop an AIDS-like syndrome, and so provide an important model for the evaluation of potential AIDS therapies. We have assessed the therapeutic effect of recombinant soluble CD4 in SIVMAC-infected rhesus monkeys. Virus was readily isolated from peripheral blood lymphocytes and bone marrow cells of these animals before starting treatment with soluble CD4, but became difficult to isolate soon after treatment had begun. Moreover the diminished growth of both granulocyte-macrophage and erythrocyte progenitor colonies from the bone marrow of these monkeys rose to normal levels during treatment. These findings indicate that soluble CD4 could prove valuable in the treatment of AIDS.     

Unusual intron in the immunoglobulin domain of the newly isolated murine CD4 (L3T4) gene

The T-cell surface glycoprotein, CD4, is expressed predominantly on helper T cells and is thought to play a major role in cell-cell interactions. Monoclonal antibodies against CD4 have been shown to block numerous T-cell functions; moreover, recent results suggest that the CD4 molecule may be involved in transmembrane signal transduction. The human CD4 glycoprotein has also been shown to form at least part of the receptor for the AIDS virus, HIV-1. Elucidation of the functions of CD4 will be facilitated by the ability to manipulate the protein by genetic means. Because the mouse system is well suited for a variety of functional studies, we have isolated, sequenced and expressed cDNA clones encoding the murine CD4 (L3T4) glycoprotein. Comparison of the mouse and human CD4 sequences reveals striking evolutionary conservation of the cytoplasmic domain, suggesting that this region is essential for CD4 function. In addition, both the human and mouse CD4 gene contain a large intron in the coding region of the V-like domain. As no other members of the immunoglobulin gene superfamily have been shown to contain similarly placed introns, this finding may have important implications regarding the evolution of this gene family in particular and of introns in general.     

Expression and function of CD4 in a murine T-cell hybridoma

The CD4 (T4) antigen was originally described as a phenotypic marker specific for helper T cells, and has recently been shown to be the receptor for the human immunodeficiency virus (HIV). Functional studies using monoclonal antibodies directed at CD4 and major histocompatibility complex (MHC) class II molecules led to the suggestion that CD4 binds to the MHC class II molecules expressed on stimulator cells, enhancing T-cell responsiveness by increasing the avidity of T cell-stimulator cell interaction and/or by transmitting a positive intracellular signal. But recent evidence that antibodies to CD4 inhibit T-cell responsiveness in the absence of any putative ligand for CD4 has been interpreted as suggesting that antibody-mediated inhibition may involve the transmission of a negative signal via the CD4 molecule instead. We have infected a murine T-cell hybridoma that produces interleukin 2 (IL-2) in response to human class II HLA-DR antigens with a retroviral vector containing CD4 cDNA. The resulting CD4-expressing hybridoma cell lines produce 6- to 20-fold more IL-2 in response to HLA-DR antigens than control cell lines. Furthermore, when antigen levels are suboptimal, the response of the cell lines is entirely CD4-dependent. The data presented here clearly demonstrate that CD4 can enhance T-cell responsiveness and may be crucial in the response to suboptimal levels of antigen.     

Soluble CD4 blocks the infectivity of diverse strains of HIV and SIV for T cells and monocytes but not for brain and muscle cells

The CD4 antigen has been subverted as a receptor by the human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV). Several groups have reported that recombinant, soluble forms of the CD4 molecule (sCD4) block the infection of T lymphocytes by HIV-1, as CD4 binds the HIV envelope glycoprotein, gp120, with high affinity. We now report that sCD4 blocks diverse strains of HIV-1, HIV-2 and SIV, but is less effective for HIV-2. The blocking effect is apparent even after adsorption of virions to CD4 cells. Soluble CD4 prevents HIV infection of T-lymphocytic and myelomonocytic cell lines, but neither sCD4 nor anti-CD4 antibodies inhibit infection of glioma and rhabdomyosarcoma cell lines.     

Phosphorylation and dephosphorylation of the high-affinity receptor for immunoglobulin E immediately after receptor engagement and disengagement.

Triggering of mast cells and basophils by immunoglobulin E (IgE) and antigen induces various biochemical signals, including tyrosine kinase activation, which lead to cell degranulation and the release of mediators of the allergic reaction. The high-affinity receptor for IgE (Fc epsilon RI) responsible for initiating these events is a complex structure composed of an IgE-binding alpha-chain, a beta-chain and a homodimer of gamma-chains. It has been assumed that beta and gamma, which have extensive cytoplasmic domains, play an important but undefined role in coupling Fc epsilon RI to signal transduction mechanisms. Here we show that Fc epsilon RI engagement induces immediate in vivo phosphorylation on beta (tyrosine and serine) and gamma (tyrosine and threonine) by at least two different non-receptor kinases. We take advantage of unique features of this receptor system to demonstrate that the phosphorylation signal is restricted to activated receptors and is immediately reversible upon receptor disengagement by undefined phosphatases. Rapid phosphorylation and dephosphorylation may be a general mechanism to couple and uncouple activated receptors to other effector molecules. This could be particularly relevant to other multimeric receptors containing Fc epsilon RI gamma-chains or the related zeta and eta chains such as the T-cell antigen receptor (TCR) and the low-affinity receptor for immunoglobulin G (Fc gamma RIII, CD16).     

The synthesis and in vivo assembly of functional antibodies in yeast

The yeast Saccharomyces cerevisiae can synthesize, process and secrete higher eukaryotic proteins. We have investigated the expression of immunoglobulin chains in yeast and demonstrate here the synthesis, processing and secretion of light and heavy chains, the glycosylation of heavy chain, the intracellular localization of these foreign proteins by immunofluorescence, and the detection of functional antibodies in cells co-expressing both chains. This may provide the basis of a microbial fermentation process for the production of monoclonal antibodies. The co-expression of light and heavy chains in Escherichia coli has been reported but functional antibodies were not assembled in vivo. Furthermore, only low-level assembly of these chains was found in vitro.     

Identification of human CD4 residues affecting class II MHC versus HIV-1 gp120 binding

Interactions of CD4 with the class II major histocompatibility complex (MHC) are crucial during thymic ontogeny and subsequently for helper and cytotoxic functions of CD4+CD8- T lymphocytes. CD4 is the receptor for the T-lymphotropic human immunodeficiency virus and binds its envelope glycoprotein, gp120. The residues involved in gp120 binding have been localized to a region within the immunoglobulin-like domain I of CD4, which corresponds to CDR2 of an immunoglobulin variable region, but the CD4 residues important in MHC class II interaction have not been characterized. Here, using a cell-binding assay dependent specifically on the CD4-MHC class II association, we analyse the effects of mutations in CD4 on class II versus gp120 binding. Mutations in CDR2 that destroy gp120 binding affect CD4-MHC class II binding similarly. In addition, binding of soluble gp120 to CD4-transfected cells abrogates their ability to interact with class II-bearing B lymphocytes. In contrast, other mutations within domains I or II that have no effect on gp120 binding eliminate or substantially decrease class II interaction. Thus, the CD4 binding site for class II MHC is more complex than the gp120 binding site, possibly reflecting a broader area of contact with the former ligand and a requirement for appropriate juxtaposition of the two N-terminal domains. The ability of gp120 to inhibit the binding of class II MHC to CD4 could be important in disrupting normal T-cell physiology, acting both to inhibit immune responses and to prevent differentiation of CD4+CD8+ thymocytes into CD4+CD8- T lymphocytes.