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HIV infection is initiated by the selective interaction between the cellular receptor CD4 and gp120, the external envelope glycoprotein of the virus. We used analytical ultracentrifugation, titration calorimetry, and surface plasmon resonance biosensor analysis to characterize the assembly state, thermodynamics, and kinetics of the CD4-gp120 interaction. The binding thermodynamics were of unexpected magnitude; changes in enthalpy, entropy, and heat capacity greatly exceeded those described for typical protein-protein interactions. These unusual thermodynamic properties were observed with both intact gp120 and a deglycosylated and truncated form of gp120 protein that lacked hypervariable loops V1, V2, and V3 and segments of its N and C termini. Together with previous crystallographic studies, the large changes in heat capacity and entropy reveal that extensive structural rearrangements occur within the core of gp120 upon CD4 binding. CD spectral studies and slow kinetics of binding support this conclusion. These results indicate considerable conformational flexibility within gp120, which may relate to viral mechanisms for triggering infection and disguising conserved receptor-binding sites from the immune system.

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.

Human immunodeficiency virus (HIV) infects cells after binding of the viral envelope glycoprotein gp120 to the cell surface recognition marker CD4. gp120 is noncovalently associated with the HIV transmembrane envelope glycoprotein gp41, and this complex is believed responsible for the initial stages of HIV infection and cytopathic events in infected cells. Soluble constructs of CD4 that contain the gp120 binding site inhibit HIV infection in vitro. This is believed to occur by competitive inhibition of viral binding to cellular CD4. Here we suggest an alternative mechanism of viral inhibition by soluble CD4 proteins. We demonstrate biochemically and morphologically that following binding, the soluble CD4 proteins sT4, V1V2,DT, and V1[106] (amino acids 1-369, 1-183, and -2 to 106 of mature CD4) induced the release of gp120 from HIV-1 and HIV-1-infected cells. gp120 release was concentration-, time-, and temperature-dependent. The reaction was biphasic at 37 degrees C and did not take place at 4 degrees C, indicating that binding of soluble CD4 was not sufficient to release gp120. The appearance of free gp120 in the medium after incubation with sT4 correlated with a decrease in envelope glycoprotein spikes on virions and exposure of a previously cryptic epitope near the amino terminus of gp41 on virions and infected cells. The concentration of soluble CD4 proteins needed to induce the release of gp120 from virally infected cells also correlated with those required to inhibit HIV-mediated syncytium formation. These results suggest that soluble CD4 constructs may inactivate HIV by inducing the release of gp120. We propose that HIV envelope-mediated fusion is initiated following rearrangement and/or dissociation of gp120 from the gp120-gp41 complex upon binding to cellular CD4, thus exposing the fusion domain of gp41.

The envelope glycoprotein gp120 of primate immunodeficiency viruses initiates viral attachment to CD4+ cells by binding to the CD4 antigen on host cell surfaces. However, among different CD4+ cell types, different viruses display distinct host cell ranges and cytopathicities. Determinants for both of these biological properties have been mapped to the env gene. We have quantitatively compared the CD4 binding affinities of gp120 proteins from viruses exhibiting different host cell tropisms and cytopathicities. The viral proteins were produced by using a Drosophila cell expression system and were purified to greater than 90% homogeneity. Drosophila-produced gp120 from T-cell tropic human immunodeficiency virus type 1 (HIV-1) BH10 exhibits binding to soluble recombinant CD4 (sCD4) and syncytia inhibition potency identical to that of pure authentic viral gp120. Relative to the affinity of HIV-1 BH10 gp120 for sCD4, that of dual tropic HIV-1 Ba-L is 6-fold lower, that of restricted T-cell tropic simian immunodeficiency virus mac is 70-fold lower, and that of noncytopathic HIV-2 ST is greater than 280-fold lower. Thus, viruses that utilize CD4 for infection do so by using a remarkably wide range of envelope affinities. These differences in affinity may play a role in determining cell tropism and cytopathicity.

We have grown crystals of a soluble recombinant form of human CD4, a transmembrane glycoprotein found predominantly on the surface of helper T cells. Crystals composed of the entire extracellular portion of CD4 exhibit extensive polymorphism. Of the five crystal types that have been grown, the best diffracts to Bragg spacings of 4.9 A. Symmetry considerations and characterization of the asymmetric unit by volume-specific amino acid analysis lead to the suggestion that a tetramer is the fundamental unit of crystallization. The characterization also showed that several of the crystal types have unusually high solvent contents. Because high solvent content and weak diffraction are indicative of an extended flexible structure, we examined the molecular shape of the recombinant CD4 with ultracentrifugation and found that it has an axial ratio of roughly 6, when modeled as a prolate ellipsoid. These results, combined with crystal packing constraints, suggest dimensions of approximately 25 x 25 x 125 A for a monomer. The structural features deduced here may be relevant to the biological function of CD4 as a receptor mediating cell-cell and cell-virus interactions.

Sequence determination of the joining segment gene (J) cluster in the kappa chain (J kappa) in the embryonic context demonstrates that rat genome contains seven J kappa gene segments that expanded from an ancestral cluster of five J kappa genes. The rat J segments are separated by about 300 base pairs (bp) and are flanked 5' by the presumed variable region (V)/J recombination signal sequence and 3' by the RNA splicing signal. Two of the J gene segments designated J2A and J2B and their 5'-flanking spacer DNA bear striking homology to J2 and its 5'-flanking spacer. Thus, the unit of duplication was the entire J kappa coding region and 5' noncoding spacer (345 bp). The duplication probably occurred as two separate unequal crossing-over (UXO) events. The first UXO event can be confined to recombination within an identical stretch (14 bp long) located at the 3' ends of the coding regions of J1 and J2. The second event could involve a longer segment (372 bp) of tight homology generated by the first UXO event, thus increasing the probability of repeated expansion of the same DNA segment. The sequence homology among the rat duplicated segments (98-99%) is larger than the homology between the corresponding rat and mouse segments (89%), showing that the rat J kappa gene expansion must have occurred after rat and mouse divergence 10 X 10(6) yr ago. We estimate that the first and second UXO events occurred 2 X 10(6) and 1 X 10(6) yr ago, respectively. J3 of rat and mouse share the same mutation (G leads to C) in the RNA splicing signal that presumably inactivates J3. This mutation preceded divergence of the two species. A mutation in the first nucleotide of codon 96 has occurred in both duplicated segments, the only position along 345 bp where J2, J2A, and J2B differ from each other. This results in three different amino acids at position 96 not present in any other J kappa. These mutations are physiologically significant because they diversify the third complementarity-determining region (CDR3) and, thus, may reflect selective pressure to increase antibody diversity. The germ-line diversification of CDR3 was exercised within the last 1-2 X 10(6) yr.

Previous studies have identified human breast tumor particles possessing many of the features characteristic of RNA tumor viruses. In addition to the expected size (600 S) and density (1.16 g/ml) these include possession of an outer membrane and an inner one surrounding a "core" containing a DNA polymerase and a large-molecular-weight (70S) RNA possessing detectable homology to the RNAs of the mouse mammary tumor virus (MMTV) and of the Mason-Pfizer monkey virus (MPMV). We report here the purification and characterization of the DNA polymerase from the human breast cancer particles. Its key properties are very similar to those ofthe RNA-dependent DNA nucleotidyltransferase (reverse transcriptase) found in MMTV and MPMV. Thus like these viral enzymes, the purified human breast cancer DNA polymerase exhibits the following three features that together distinguish the known viral reverse transcriptases from normal cellular DNA polymerases: (i) a strong preference for oligo(dT)-poly(rA) over oligo(dT)-poly(dA) as a template for the synthesis of poly(dT); (ii) the acceptance of the highly specific oligo(dG)-poly(rCm) as a template for the formation of poly(dG); (iii) the ability to use a viral RNA (AMV) as a template to fashion a faithful DNA complementary copy; and (iv) its preference for Mg++ over Mn++. In summary, the data described here on the enzyme of the human breast cancer particles add further evidence of similarities to the viral agents associated with the corresponding malignancies in the mouse and monkey models. To date, an enzyme with these properties has not been detected in normal breast tissues or in benign tumors of the breast.

From previous studies, lymphocyte DNA from human leukemias and DNA from involved tissues of patients with Hodgkin's disease or Burkitt's lymphoma contain sequences that are absent from their normal counterparts. These sequences are related to those found in particulate elements associated with these neoplasias and possessing biochemical properties characteristic of RNA tumor viruses. Similar observations have been made of unique sequences related to those of the feline virus RD-114 and found in spontaneous mastocytomas in cats. Here we extend these results to the classical murine model of virus-induced leukemias. Splenic DNA from BALB/c mice with leukemia induced by Rauscher leukemia virus (RLV) possess some RLV-related sequences that do not exist in normal BALB/c DNA. Furthermore, these leukemia-specific sequences were absent in all other mouse strains examined, including AKR, a strain with a high incidence of spontaneous leukemia. The DNA of all noninfected mouse strains possesses considerable homology with the RLV genome. Temperature denaturation studies indicate, however, that although the RLV-related sequences found in all normal mice are similar to each other, they are not exactly homologous with RLV sequences. We conclude that RLV-induced leukemia in BALB/c results in the insertion of RLV sequences into cellular DNA that itself possesses only partial homology with the RLV genome.