The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain

The isolation of clones encoding the human surface protein T4, and the expression of the T4 gene in new cellular environments, have enabled us to examine the role of this protein in the pathogenesis of AIDS. Our studies support a mechanism of AIDS virus infection that initially involves the specific interaction of the AIDS virus with T4 molecules on the cell surface. This association can be demonstrated on T4+ transformed T and B lymphocytes as well as epithelial cells. Furthermore, the presence of T4 on the surface of all human cells examined is sufficient to render these cells susceptible to AIDS virus infection. Our data suggest that the T4-AIDS virus complex is then internalized by receptor-mediated endocytosis. Finally, we find that the T4 gene is expressed in the brain as well as in lymphoid cells, providing an explanation for the dual neurotropic and lymphotropic character of the AIDS virus. In this manner, a T lymphocyte surface protein important in mediating effector cell-target cell interactions has been exploited by a human retrovirus to specifically target the AIDS virus to populations of T4+ cells.  

Monoclonal antibody analysis of keratin expression in epidermal diseases: a 48- and 56-kdalton keratin as molecular markers for hyperproliferative keratinocytes

The polypeptide composition of epidermal keratin varies in disease. To better understand the biological meaning of these variations, we have analyzed keratins from a number of human epidermal diseases by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The results reveal a continuous spectrum of keratin expression ranging from one closely resembling the normal in vivo pattern to one almost identical to cultured epidermal keratinocytes. Specifically, a 50-kilodalton (kd) (AE1-positive) and a 58-kd (AE3-positive) keratin are present in all diseases, supporting the concept that they represent "permanent" markers for keratinocytes. A 56.5-kd (AE1) and a 65-67-kd (AE3) keratin, previously shown to be markers for keratinization, are expressed only by lesions retaining a keratinized morphology. A 48-kd (AE1) and a 56-kd (AE3) keratin are present in all hyperproliferative (para- or nonkeratinized) disorders, but not in normal abdominal epidermis or in ichthyosis vulgaris which is a nonhyperproliferative disease. These two keratins have previously been found in various nonepidermal keratinocytes undergoing hyperproliferation, suggesting that these keratins are not epidermis-specific and may represent markers for hyperproliferative keratinocytes in general. In various epidermal diseases, there is a reciprocal expression of the (keratin) markers for hyperproliferation and keratinization, supporting the mutual exclusiveness of the two cellular events. Moreover, our results indicate that, as far as keratin expression is concerned, cultured human epidermal cells resemble and thus may be regarded as a model for epidermal hyperplasia. Finally, the apparent lack of any major, disease-specific keratin changes in the epidermal disorders studied so far implies that keratin abnormalities probably represent the consequence, rather than the cause, of these diseases.  

Identification of the residues in human CD4 critical for the binding of HIV

The CD4 molecule is a T cell surface glycoprotein that interacts with high affinity with the envelope glycoprotein of the human immunodeficiency virus, HIV, thus serving as a cellular receptor for this virus. To define the sites on CD4 essential for binding to gp120, we produced several truncated, soluble derivatives of CD4 and a series of 26 substitution mutants. Quantitative binding analyses with the truncated proteins demonstrate that the determinants for high affinity binding lie solely with the first 106 amino acids of CD4 (the V1 domain), a region having significant sequence homology to immunoglobulin variable regions. Analysis of the substitution mutants further defines a discrete binding site within this domain that overlaps a region structurally homologous to the second complementarity-determining region of antibody variable domains. Finally, we demonstrate that the inhibition of virus infection and virus-mediated cell fusion by soluble CD4 proteins depends on their association with gp120 at this binding site.  

Human immunodeficiency virus infection of CD4-bearing cells occurs by a pH-independent mechanism.

The effect of weak bases (NH4Cl and amantadine) and carboxylic ionophores (monensin) on the infection of CD4 (T4) positive human cell lines by HIV-1 is examined. These reagents, which raise the pH of acidic intracellular organelles, fail to inhibit HIV-1 entry and the events leading to viral protein synthesis at concentrations inhibitory for low pH-dependent fusogenic enveloped viruses. The infectivity of VSV (HIV-1) pseudotypes is unaffected by weak bases at concentrations causing 95% plaque reduction of VSV in its own envelope. HIV-1 dependent cell--cell fusion (syncytium formation) occurs in medium maintained at pH 7.4-7.6, and virions are not irreversibly inactivated by incubation in acid medium. Our results show that HIV-1 entry and membrane fusion do not require exposure to low pH. The production of infectious HIV-1 particles, however, is inhibited in cells treated with NH4Cl.  

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.  

High-titer packaging cells producing recombinant retroviruses resistant to human serum.

Novel retroviral protein expression constructs were designed to retain minimal retroviral sequences and to express dominant selectable markers by reinitiation of translation after expression of the viral genes. HT1080 cells were selected as producer cells for their ability to release high-titer viruses that are resistant to inactivation by human serum. Two HT1080-based packaging cell lines which produce Moloney murine leukemia virus cores with envelope glycoproteins of either amphotropic murine leukemia virus (FLYA13 line) or cat endogenous virus RD114 (FLYRD18 line) are described. Direct comparison with previous retroviral packaging systems indicated that 100-fold-higher titers of helper-free recombinant viruses were released by the FLYA13 and FLYRD18 lines.  

Primary, syncytium-inducing human immunodeficiency virus type 1 isolates are dual-tropic and most can use either Lestr or CCR5 as coreceptors for virus entry.

A panel of primary syncytium-inducing (SI) human immunodeficiency virus type 1 isolates that infected several CD4+ T-cell lines, including MT-2 and C8166, were tested for infection of blood-derived macrophages. Infectivity titers for C8166 cells and macrophages demonstrated that primary SI strains infected macrophages much more efficiently than T-cell line-adapted HIV-1 strains such as LAI and RF. These primary SI strains were therefore dual-tropic. Nine biological clones of two SI strains, prepared by limiting dilution, had macrophage/C8166 infectivity ratios similar to those of their parental viruses, indicating that the dual-tropic phenotype was not due to a mixture of non-SI/macrophage-tropic and SI/T-cell tropic viruses. We tested whether the primary SI strains used either Lestr (fusin) or CCR5 as coreceptors. Infection of cat CCC/CD4 cells transiently expressing Lestr supported infection by T-cell line-adapted strains including LAI, whereas CCC/CD4 cells expressing CCR5 were sensitive to primary non-SI strains as well as to the molecularly cloned strains SF-162 and JR-CSF. Several primary SI strains, as well as the molecularly cloned dual-tropic viruses 89.6 and GUN-1, infected both Lestr+ and CCR5+ CCC/CD4 cells. Thus, these viruses can choose between Lestr and CCR5 for entry into cells. Interestingly, some dual-tropic primary SI strains that infected Lestr+ cells failed to infect CCR5+ cells, suggesting that these viruses may use an alternative coreceptor for infection of macrophages. Alternatively, CCR5 may be processed or presented differently on cat cells so that entry of some primary SI strains but not others is affected.  

Human immunodeficiency virus type 2 infection and fusion of CD4-negative human cell lines: induction and enhancement by soluble CD4.

We describe human immunodeficiency type 2 (HIV-2) strains which induce cell-to-cell fusion and infect certain CD4- human cell lines. Soluble CD4 (sCD4) induces or enhances fusion by most HIV-2 strains tested. Soluble CD4-immunoglobulin G chimeras and conjugates of sCD4 and antibody to the third domain of CD4 block HIV-2 fusion of CD4- cells. We conclude that HIV-2 can enter CD4- cells via an alternative cell surface receptor to CD4. While some strains entered efficiently, others retained a dependency on an interaction with sCD4 to initiate changes in the virion envelope required for membrane fusion.