Mutant CD4 Molecules Research Articles

Coreceptor Function of Mutant Human CD4 Molecules without Affinity to gp120 of Human Immunodeficiency Virus

Despite extensive mutational studies on the human CD4 molecule and its affinity to human immunodeficiency virus (HIV) envelope glycoprotein gp120, coreceptor functions of such mutant molecules have only been examined by indirect measurement of their affinity to class II major histocompatibility complex (MHC) molecules. In this report, coreceptor functions of mutant human CD4 molecules, which have no or reduced affinity to an HIV envelope protein, gp120, were assessed in a murine T cell receptor/class II MHC recognition system. The substitution of human C" beta strand with the murine homologous segment resulted in the loss of the coreceptor function as well as in the complete loss of gp120 binding capacity, corroborating the consensus that Phe-43 in C" beta strand plays crucial roles in both situations. However, simultaneous replacement of the C'-C" loop along with the C" beta strand by homologous murine segments rescued the coreceptor function, whereas gp120 binding capacity remained negative. Further analysis indicated that insertion of lysine between Gly-41 and Ser-42 can partially compensate for the coreceptor function lost by the Phe-43 --> Val mutation. Although the coreceptor function of these mutant CD4 molecules in a human T cell recognition system is yet to be determined, these observations necessitate a re-evaluation of the role played by Phe-43 in coreceptor function. Examination of the sensitivities of the mutant CD4 molecules expressed on HeLa cells to infection by a T cell-tropic HIV-1 strain indicated that only those mutants that had completely lost gp120 binding capacity were resistant to the infection. All mutants having whole C" substitution, irrespective of additional substitutions or their coreceptor functions, were resistant to the infection.

Mutant CD4 Molecules with Improved Binding to HIV Envelope Protein gp120 Selected by Phage Display

Phage-display methodology has been used to select variant CD4 proteins exhibiting increased binding to the surface envelope glycoprotein, gp120, of Human Immunodeficiency Virus Type-1. To facilitate the selection, a library of mutant CD4 proteins was constructed by cloning a PCR-generated error prone population of the first two domains of CD4 into the phagemid expression vector pHEN1. Phage displaying CD4 in functional form were confirmed by Western blot with CD4-specific antibody and by phage ELISA on immobilized gp120. Biopanning of CD4 phage on immobilized gp120 followed by individual characterization identified five clones with increased binding to gp120. All of the selected variants had one or two amino acid substitutions within the V1 domain of CD4, notably at positions 15, 27, 30, 50, and 66 located in the strands surrounding the main binding loop. Variants which exhibited increased binding to recombinant gp120in vitrowere also shown to have an increased capacity for virus neutralization broadly in line with theirin vitrobinding activity.

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.

Kinetic and structural analysis of mutant CD4 receptors that are defective in HIV gp120 binding.

The T-cell antigen coreceptor CD4 also serves as the receptor for the envelope glycoprotein gp120 of HIV. Extensive mutational analysis of CD4 has implicated residues from a portion of the extracellular amino-terminal domain (D1) in gp120 binding. However, none of these proteins has been fully characterized biophysically, and thus the precise effects on molecular structure and binding interactions are unknown. In the present study, we produced soluble versions of three mutant CD4 molecules (F43V, G47S, and A55F) and characterized their structural properties, thermostability, and ability to bind gp120. Crystallographic and thermodynamic analysis showed minimal structural alterations in the F43V and G47S mutant proteins, which have solvent-exposed mutant side chains. In contrast, some degree of disorder appears to exist in the folded state of A55F, as a result of mutating a buried side chain. Real time kinetic measurements of the interaction of the mutant proteins with gp120 showed affinity decreases of 5-fold for G47S, 50-fold for A55F, and 200-fold for F43V. Although both rate constants for the binding reaction were affected by these mutations, the loss in affinity was mainly due to a decrease in on rates, with less drastic changes occurring in the off rates. These observations suggest the involvement of conformational adaptation in the CD4-gp120 interaction. Together, the structural and kinetic data confirm that F43V is a critical residue in gp120 recognition site, which may also include main chain interactions at residue Gly-47.

Suggested binding mechanism of the HIV-gp120 to its CD4 receptor

The molecular recognition and attachment of the CD4 molecule and the HIV envelope glycoprotein (gp120) might be described as a consecutive three-step molecular recognition process. 1. (a) Long range interaction: electrostatic pre-orientation, 2. (b) short range interaction: electronic attachment followed by a ‘Locking-in’ (via aromatic ring orientation) and 3. (c) internal interaction (induced fit): conformational readjustment of the protein molecules. On the basis of the preliminary investigations (X-ray structures of CD4 and biological studies of CD4 and gp120 point mutants) we described a computational model. This approach consists of empirical calculations as well as ab initio level of quantum chemistry. The conformational analysis of the wild type and mutant CD4 molecules was supported by molecular mechanics and dynamics (Amber force field). The latter analysis involves the application of a novel method, the Amino Acid Conformation Assignment of Proteins (ACAP) software, developed for the notation of secondary protein structures. According to the cardinal role of the electrostatic factors during this interaction, several ab initio investigations were performed for better understanding of the recognition process on submolecular level. Using the above mentioned computational model, we could interpret the basic behaviours and predict some additional features of CD4-gp120 interaction, in spite of the missing gp120 X-ray structure.

Analysis of endoproteolytic cleavage and intracellular transport of human immunodeficiency virus type 1 envelope glycoproteins using mutant CD4 molecules bearing the transmembrane endoplasmic reticulum retention signal

We investigated endoproteolytic processing of the human immunodeficiency virus (HIV) envelope glycoprotein precursor, gp160, as well as envelope-mediated membrane fusion in the presence of CD4 molecules that were either partially or fully retained in the endoplasmic reticulum (ER). Pulse-chase analyses revealed that gp160 formed complexes with CD4 molecules, and gp160 in the complex was endoproteolytically cleaved to gp120 and gp41 in the secretory pathway. The gp120/gp41 complex thus generated was properly targeted to the plasma membrane in cells expressing gp160 and wild-type CD4 or mutant CD4 molecules that were partially retained in the ER. Additionally, membrane fusion (syncytium) assays were performed to monitor the presence or absence of gp120/gp41 complexes at the cell surface of cotransfected cells and demonstrated that the HIV-1 envelope glycoprotein-mediated membrane fusion was appreciably reduced in the presence of wild-type CD4 or either one of the mutant CD4 molecules. Reduction in the formation of syncytia appears to be due predominantly to saturation of the CD4 binding site on the gp120/gp41 complex at the cell surface of cotransfected cells, but partial retention of the complex in the ER could also partly account for the reduction. However, the intracellular gp120/gp41 complex generated in cells expressing gp160 and CD4 mutant having the transmembrane ER retention signal (KKTC) was completely retained in the ER and hence could not participate in membrane fusion events at the plasma membrane. Taken together, these data suggest that the endoproteolytic cleavage of gp160 occurs in the ER or cis-Golgi network, and ER retention strategies can potentially be used in preventing the spread of HIV-1 infection in permissive cells.

The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits.

The lymphocyte glycoprotein CD4 is constitutively internalized and recycled in nonlymphoid cells, but is excluded from the endocytic pathway in lymphocytic cells (Pelchen-Matthews, A., J. E. Armes, G. Griffiths, and M. Marsh. 1991. J. Exp. Med. 173: 575-587). Inhibition of CD4 endocytosis is dependent on CD4 expressing an intact cytoplasmic domain and is only observed in cells where CD4 can interact with the protein tyrosine kinase p56lck, a member of the src gene family. We have expressed p56lck, p60c-src, or chimeras of the two proteins in CD4-transfected NIH-3T3 or HeLa cells. Immunoprecipitation of CD4 and in vitro kinase assays showed that p56lck and the lck/src chimera, which contains the NH2 terminus of p56lck, can associate with CD4. In contrast, p60c-src and the src/lck chimera, which has the NH2 terminus of p60c-src, do not associate with CD4. Endocytosis assays using radioiodinated anti-CD4 monoclonal antibodies demonstrated that coexpression of CD4 with p56lck, but not with p60c-src, inhibited CD4 endocytosis, and that the extent of the inhibition depended directly on the relative levels of CD4 and p56lck expressed. The uptake of mutant CD4 molecules which cannot interact with p56lck was not affected. Measurement of the fluid-phase endocytosis of HRP or the internalization of transferrin indicated that the effect of p56lck was specific for CD4, and did not extend to other receptor-mediated or fluid-phase endocytic processes. Immunogold labeling of CD4 at the cell surface and observation by electron microscopy demonstrated directly that p56lck inhibits CD4 endocytosis by preventing its entry into coated pits.

Requirement for association of p56 lck with CD4 in antigen-specific signal transduction in T cells

The T cell-specific transmembrane glycoprotein CD4 interacts with class 11 MHC molecules via its external domain and is associated with the tyrosine kinase p56 lck via a cysteine motif in its cytoplasmic domain. We have assessed the ability of CD4 to synergize with the antigen-specific T cell receptor (TCR) for induction of transmembrane signals that result in lymphokine production. Mutant CD4 molecules were introduced into T cells that lacked endogenous CD4 but expressed TCRs specific for lysozyme peptides or the superantigen SEA bound to Ab or A bm12 class II MHC molecules. With either ligand, T cell activation occurred only when CD4 was associated with p56 lck . These results demonstrate that residues within the cytoplasmic domain of CD4 are required for its coreceptor function in TCR-mediated signal transduction and strongly support the notion that the association of CD4 with p56 lck is critical in this process.

HIV infection does not require endocytosis of its receptor, CD4

The T cell surface molecule CD4 interacts with class II MHC molecules on the surface of target cells as well as with the envelope glycoprotein of human immunodeficiency virus (HIV). Internalization of CD4 molecules is observed after exposure of CD4+ T cells to either phorbol esters or appropriate antigen-bearing target cells. To determine whether HIV entry proceeds via receptor-mediated endocytosis or direct viral fusion with the cell membrane, we have constructed two mutants in the cytoplasmic domain of the CD4 protein that severely impair the ability of CD4 molecules to undergo endocytosis. Quantitative infectivity studies reveal that HeLa cell lines expressing wild-type or mutant CD4 molecules are equally susceptible to HIV infection. In addition, HIV binding does not lead to CD4 endocytosis. These studies indicate that although the CD4 molecule can be internalized, HIV entry proceeds via direct fusion of the viral envelope with the cell membrane.