Biological characterization of a simian immunodeficiency virus-like retrovirus (HTLV-IV): evidence for CD4-associated molecules required for infection.

We have analyzed a number of biological features of HTLV-IV, a retrovirus indistinguishable from a macaque isolate of simian immunodeficiency virus (SIV), and compared this virus with several strains of human immunodeficiency virus type 1 (HIV-1). Although HTLV-IV was found to be similar to HIV-1 in its tropism for CD4+ lymphocytes, its effects on CD4 expression and the ability of its externalized envelope molecule to form a complex directly with the CD4 molecule, a number of striking differences were noted. Unlike with HIV-1, the range of cells susceptible to HTLV-IV infection and syncytia formation was restricted to a subset of CD4+ cell lines, particularly those that coexpressed CD4 with human leukocyte antigen (HLA) class II antigens. An analysis of the patterns of HTLV-IV infection with B x T somatic cell hybrids indicated that for this virus, molecules in addition to CD4 were probably required to facilitate infection and cell fusion. Additional studies of HTLV-IV infection of Sup-T1 cells, which are exquisitely sensitive to cytopathic effects induced by HIV-1, demonstrated that HTLV-IV infection could occur in the absence of cytopathic effects and, remarkably, with minimal or no downmodulation of the CD4 molecule from the cell surface. The failure of HTLV-IV infection to reduce the expression of several CD4 epitopes suggested that the HTLV-IV envelope produced by Sup-T1 cells was altered in its ability to interact with or bind to CD4. Additional differences were also noted in the size of the transmembrane envelope molecule of HTLV-IV produced by Sup-T1 cells, indicating that cell-specific alterations in processing of the HTLV-IV envelope occurred during the production of virus in this cell line. Understanding the basis for these biological differences between HTLV-IV and the HIV-1 viruses may help to elucidate more general mechanisms for pathogenesis of other members of the SIV and HIV families of retroviruses.

T4 endocytosis and phosphorylation induced by phorbol esters but not by mitogen or HIV infection.

The T4 (CD4) molecule has been shown to facilitate the interactions of T cells with HLA class II determinants, to function as a signal transducing molecule, and to serve as a receptor for HIV. Recent studies demonstrated that both phorbol esters and antigen stimulation induced the rapid and transient modulation and phosphorylation of T4 on an IL-2-dependent line of cloned peripheral blood T4+ cells. In the current study, we define the kinetics of T4 phosphorylation and internalization induced by phorbol esters and determine the extent to which this metabolic pathway is required for T cell proliferation, activation, and HIV infection. On both peripheral blood T4+ cells and the T cell line Sup-T1, the modulation and internalization of surface T4 induced by phorbol 12, 13-dibutyrate (PDB) was preceded by rapid and transient phosphorylation. On both cell types, by 48 h, T4 was reexpressed on the cell surface in a nonphosphorylated form and was shown to be resistant to phosphorylation and internalization when these cells were reexposed to PDB. In contrast, T4 on the surface of PBL was neither phosphorylated nor down-modulated when PBL were stimulated by PHA, indicating that these effects were not simply the result of T cell activation or proliferation. In additional studies, we demonstrate that this pathway for T4 phosphorylation and internalization is not required for HIV infection by showing that 1) the binding of the HIV gp 120 envelope to T4 does not induce phosphorylation of T4, 2) Sup-T1 cells that are rendered resistant to phorbol ester-induced T4 internalization and phosphorylation by prolonged culture in PDB remain highly susceptible to HIV infection, and 3) clones of HIV-producing cells expressing high levels of surface T4 that is complexed with viral envelope remain susceptible to PDB-induced modulation of T4. This observation suggests that, at least on lymphoid cells, HIV penetration does not occur exclusively by R-mediated endocytosis.

Nonrandom association of cellular antigens with HTLV-III virions.

Retroviruses are known to incorporate cellular antigens as they bud from infected cells. To identify the cellular antigens that associate with the AIDS-retrovirus, we evaluated a preparation of HTLV-III antigens with a panel of monoclonal antibodies reactive with a variety of antigens expressed on the H9 T-cell line used to produce the virus. Only monoclonal antibodies that identified HLA class-II antigens, beta-2 microglobulin, and a single anti-HLA class-I antibody were reactive in an ELISA of solubilized HTLV-III virus. No reactivity was seen with 11 monoclonal antibodies to T-cell antigens or with five antibodies to determinants on HLA class-I A or B molecules. These data suggest that on H9 cells the association of budding HTLV-III virions with cellular antigens may be a nonrandom process in which some HLA antigens, particularly class-II antigens, are selectively incorporated into the viral envelope. It is possible that a selective association of HLA class II antigens with budding HTLV-III virions may also occur for T cells infected in vivo, and could have relevance for the pathogenesis of this virus.

Alterations in T4 (CD4) protein and mRNA synthesis in cells infected with HIV.

Cells infected with the human immunodeficiency virus (HIV) show decreased expression of the 58-kilodalton T4 (CD4) antigen on their surface. In this study, the effect of HIV infection on the synthesis of T4 messenger RNA (mRNA) and protein products was evaluated in T-cell lines. Metabolically labeled lysates from the T4+ cell line Sup-T1 were immunoprecipitated with monoclonal antibodies to T4. Compared with uninfected cells, HIV-infected Sup-T1 cells showed decreased amounts of T4 that coprecipitated with both the 120-kilodalton viral envelope and the 150-kilodalton envelope precursor molecules. In four of five HIV-producing T-cell lines studied, the steady-state levels of T4 mRNA were also reduced. Thus, the decreased T4 antigen on HIV-infected cells is due to at least three factors: reduced steady-state levels of T4-specific mRNA, reduced amounts of immunoprecipitable T4 antigen, and the complexing of available T4 antigen with viral envelope gene products. The data suggested that the T4 protein produced after infection may be complexed with viral envelope gene products within infected cells. Retroviral envelope-receptor complexes may thus participate in a general mechanism by which receptors for retroviruses are down-modulated and alterations in cellular function develop after infection.

Relation of human T lymphotropic virus type III antibodies to T lymphocyte subset abnormalities in hemophiliac patients.

The relationship of T lymphocyte subset abnormalities and the presence of antibodies to the human T lymphotropic virus type III (HTLV-III) was evaluated in 66 adult patients with hemophilia. Positive test results for antibodies to HTLV-III were observed in 62 percent of patients with hemophilia A and 9 percent of patients with hemophilia B. Patients with HTLV-III antibodies had lower percentages and numbers of T helper (T4) cells and increased percentages of T suppressor (T8) cells compared with percents in patients without antibodies to HTLV-III. The mean T4/T8 ratio for antibody-negative patients was 1.45 compared with 0.65 for antibody-positive patients (p less than 0.0005). These abnormalities were more apparent among hemophiliac patients who received factor VIII concentrates. The strong association of lymphocyte subset abnormalities and seroreactivity to this virus suggests that infection by HTLV-III has occurred in this population rather than a passive exposure to viral antigens contained in factor concentrates.

Persistent noncytopathic infection of normal human T lymphocytes with AIDS-associated retrovirus.

Infection of normal peripheral blood T cells by the acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV) was evaluated in long-term cultures of helper-inducer T cells (T4 cells). Cells that were inoculated with ARV and maintained in medium supplemented with interleukin-2 remained productively infected with this virus for more than 4 months in culture, although they showed no cytopathic effects characteristic of acute ARV infection. The presence of replicating virus was demonstrated by reverse transcriptase activity of culture fluids and by viral antigens and budding particles detected on cells by immunofluorescence and electron microscopy. Virus produced in these cultures remained infectious and could induce cytopathic effects and viral antigens in uninfected lymphoid cells. The finding that normal lymphocytes may be productively infected by an AIDS retrovirus in the absence of cell death suggests that a range of biologic effects may occur after infection in vivo.