The gp41 TM protein of HIV-1 includes several domains: an N-terminal ectodomain which is outside the lipid bilayer, a membrane-spanning hydro-phobic domain, and a C-terminal domain which is inside the lipid bilayer. A hydrophobic N-terminal sequence within the ectodomain has been implicated in the fusion of the virus with cell membranes after initial binding to CD4. Also within the ectodomain is a leucine zipper domain and a helical domain which have been reported to be involved in virus-cell fusion.5758 Within the ectodomain of gp41 is a region, amino acid residues 583-599, which was identified59 to be partially homologous to the immunosuppressive domain identified by Cianciolo et al.60-61 within the p15E TM of type C and D retroviruses. As will be discussed below, many of the studies of gp41-mediated immunosuppression have focussed on this putative immunosup-pressive region. Table III lists many of the activities described for gp41.
One of the first reports of immunosuppressive activity associated with gp41 was a 1988 study by Cianciolo et al.62 using a synthetic peptide (CS-3) corresponding to the putative immunosuppressive region of HIV-1 gp41. Using human PBMCs, they demonstrated up to 81% inhibition of anti-CD3-
TABLE III Potential Immunomodulating Activities Reported for HIV-1 gp41 Protein
In human PBMCs or lymphocytes
Inhibits mitogen and lymphokine-dependent proliferation 66, 68, 75
Inhibits antigendependent proliferation 62, 68, 75
Inhibits anti-CD3-stimulated proliferation 62, 65
Inhibits B lymphocyte proliferation 67
Inhibits lymphokine-activated killer activity 69
Inhibits production of IL2, IFN-a, Y 70, 74
Enhances production of IL-1P, TNF-a 70 In human monocytes or monocytic cell lines
Induces IL-6 or IL-10 76, 77
stimulated T cell proliferation, while they observed no effect on anti-Ig-stimulated B cell proliferation. These initial observations of T cell inhibition were later confirmed by a number of laboratories,63-68 Ruegg et al.,63 in addition to demonstrating inhibition of human lymphocyte proliferation with the synthetic peptide corresponding to the immunosuppressive region of gp41, also demonstrated inhibition of murine lymphocyte proliferation, which strengthened the significance of the similarities between the TM proteins of the murine and human viruses. This same group later reported that the gp41 immunosuppressive peptide could inhibit protein kinase C (PKC) and anti-CD3-stimulated Ca2+ influx and PKC-mediated phosphorylation of the CD3 gamma chain in Jurkat T cells.64 In another report, they demonstrated that human lymphoproliferation induced by the T cell activation molecules CD3, CD2, or CD28 were all inhibited by this gp41 peptide.65 In another study using peptides corresponding to this same immuno-suppressive sequence, Denner et al.66 confirmed both the inhibition of mitogen-induced and lymphokine-dependent T lymphocyte proliferation and interspecies reactivity for this sequence. They also reported that N-ter-minal octamers represented the minimal immunosuppressive domain and that, in addition to its immunosuppressive activity, the peptide could inhibit the cytopathic effect of HIV-1 on human MT4 cells, suggesting interference with viral replication. This latter result would seem to agree with the data of Wild et al.,51 who reported inhibition of viral infectivity with a gp41-encoded peptide (DP107) which partially overlapped the putative immunosuppres-sive region. Denner et al.61 later reported that the GP41 immunosuppressive peptide inhibited B lymphocyte stimulation by the B cell mitogen lipopoly-saccharide and by antibodies against cell surface immunoglobulins, a result which contrasts with the earlier report by Cianciolo et al.62 in which they observed no effects on B cells. Wang et al.68 also demonstrated inhibition of human lymphocyte proliferative responses to mitogens and recall antigens using a peptide which included the putative immunosuppressive domain, but which also included an additional C-terminal 10 amino acids encompassing a well-documented B cell epitope (amino acids 598-609). They reported inhibition of both CD4+ and CD8+ T cells, indicating that CD4 binding was not involved. Importantly, they reported that sera from HIV-infected patients, although reactive with the peptide, could not block the suppressive activity of the peptide, suggesting that the immunosuppressive domain is distinct from the immunogenic domain and is not itself immuno-genic.
Although many studies have focused on the putative immunosuppressive domain of gp41, which is partially homologous to type C retroviral TM p15E, other investigators have explored additional regions of the gp41 molecule using synthetic peptides. Cauda et al.69 found that lymphokine-activated killer (LAK) cell activity was reduced in HIV-1-infected patients and that HIV-1 gp41 synthetic peptides corresponding to amino acids 735752 and amino acids 846-860 were able to significantly inhibit LAK activity. They found that HIV-1-positive sera and supernatant fluids from cultured PBMCs from HIV-1-infected patients had similar activity, suggesting a possible role for a gp41-encoded protein. The following year this same group reported70 that the gp41 peptides studied earlier were also able to suppress the production of IFN-a, IFN-y, and IL-2 from normal human PBMCs while enhancing the production of IL-1 and TNF-a.
Although data demonstrating in vitro immunosuppression with gp41 peptides were intriguing, data generated with intact gp41 were slow to be reported. Qureshi et al.11 had identified potential cell-surface-binding proteins for HIV-1 gp41 using the CS-3 immunosuppressive peptide of gp41. Ebenbichler et al.12 confirmed and extended the results of Qureshi et al.11 using recombinant soluble gp41 (rsgp41) bound to Sepharose to affinity-purify cell-membrane-binding proteins. They characterized three different proteins of 44, 98, and 106 kDa which bound to rsgp41 and reported that their expression decreased from a T-lymphoid cell line to a monoblastoid cell line to a cell line representing mature monocytes. Chen et al.13 used rsgp41 and flow cytometry to identify which cells could bind gp41. They demonstrated rsgp41 binding to normal human PBMCs, preferentially to B lymphocytes and monocytes, and independent of gp120-binding sites on CD4 molecules. The binding was dose dependent and they found that rsgp41 bound more strongly to B cells (47%) and monocytes (44%) than to CD4+ T cells (10%) or CD8+ T cells (12%).
In 1993 Oh et al.14 reported that sera of AIDS patients contained factor(s), 30-50 kDa in size, that could exert significant inhibition on normal T lymphocyte and natural killer functions. These factor(s) inhibited the early stages of lymphocyte activation and, in addition to inhibiting production of IL-2, inhibited the transcription of IL2 mRNA. The authors determined by Western blotting that their immunosuppressive factor(s) shared epitopes with HIV-1 gp41. Although indirect, this was one of the first suggestions that native gp41 could exert immunosuppressive activity. Two years later Chen et al.75 reported that rsgp41 could inhibit ConA-, PHA-, and tetanus toxoid-stimulated proliferation of normal human peripheral blood lymphocytes, although the concentration required for 50% inhibition, 8 ^M, was quite high. They found no effect of rsgp41 on PWM-stimulated lymphocyte proliferation, although they had earlier73 reported that B cells bound rsgp41 to a greater extent than any other cell type.
A potential effector mechanism for immunosuppressive activity by gp41 was first suggested in 1995 by the studies of Takeshita et al.16 Using the human monocytic cell line THP-1, they found that recombinant gp41, but not gp120 or p24, induced significant IL6 expression in treated cells. Their studies also indicated that IL-10was produced, with delayed kinetics, following the induction of IL6, although the induction of IL-10 required that IFN-y be present. When the investigators added recombinant IL-10 to gp41-treated THP-1 cells, they inhibited the expression of IL6, and when an IL-10 neutralizing antibody was added, IL6 production was enhanced. These studies suggested that IL-10 induced by gp41 might play a role in cytokine dysregulation associated with HIV-1 infection.
More recently, Koutsonikolis et al.11 reported that recombinant HIV-1 gp41 induces the expression of IL-10mRNA in human PBMCs within 3 h of exposure. Importantly, the induction of IL-10 release in the supernatants <10 of recombinant gp41-treated PBMCs was observed at concentrations =S10 nM, suggesting that this might be a physiologically relevant phenomenon. The authors showed that recombinant gp41 induced IL-10 only in monocytes and not in T lymphocytes and confirmed earlier reports47-48-50 of gp120 induction of IL-10 in monocytes. They also found that gp120 induction of IL-10 was specific for monocytes and that there was no effect on T lymphocyte IL-10 production.
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