The human immunodeficiency virus (HIV) uses the human CD4 glycoprotein as a receptor for infection of susceptible cells. Cells expressing a series of mutated forms of the CD4 gene have shown a variability in their ability to support replication of three HIV type 1 (HIV-1) and three HIV-2 strains. Moreover, when different stages of virus production were examined by a variety of assays, a consistent delay was observed in all cell lines containing CD4 mutants compared with those with intact full-length CD4. Ceils expressing the CD4.415 mutant (modffied at the serine 415 corresponding to a phosphorylation site of the cytoplasmic domain) showed only a minimal effect on virus replication. Cells expressing CD4.403 and CD4.401 mutants (lacking the whole cytoplasmic domain) manifested a moderate delay in production of virus progeny. The most substantial effect on HIV replication was observed in cells expressing a chimeric hybrid containing sequences corresponding to the first 177 residues of the N-terminal CD4 fused to CD8 sequences encoding the hinge, transmembrane, and cytoplasmic domains of the human CD8. Furthermore, in a cell-to-cell contact assay, fusion was absent when the CD4 proximal membrane domain was replaced by the CD8 counterpart. In addition, a strong correlation between the down-modulation of the surface CD4 and HIV expression was observed. These observations suggest that in addition to the known binding region, other domains of CD4 could play an important role in regulating HIV entry of cells.

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In neurodegenerative diseases associated with AIDS, reactive astrocytosis plays a central role in the neurotoxicity of the brain parenchyma. Whereas the HIV-1 nef gene is overexpressed during restricted HIV-1 infection of human astrocytes, our previous results have demonstrated that nef expressed in human U251MG glial cells activates the sphingomyelin pathway triggered by TNF-alpha, increasing ceramide production. Since ceramide is an important regulatory molecule of programmed cell death induced by TNF-alpha, we examined whether nef could alter TNF-alpha-induced apoptosis in the U251MG human astrocytoma cell line. Transfection studies indicated that nef could both prevent apoptosis and promote cell proliferation in response to TNF-alpha stimulation. MAPK and JNK activities were further analyzed in order to elucidate signaling cascades subsequent to the upregulation of ceramide production. After TNF-alpha treatment, both kinases were shown to be preferentially activated in the presence of nef. These experiments strongly suggest that the HIV-1 Nef protein might modulate the sensitivity of astrocytes to inflammatory molecules, thus contributing to the development of neurodegenerative diseases associated with AIDS.