HIV-1 INFECTION IN FETAL BRAIN CELL CULTURES AND PEDIATRIC AIDS BRAIN TISSUE

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We have established an in vitro model of latent HIV-1 infection in human fetal astrocytes. Several weeks following infection or transfection, cocultivation with uninfected lymphocytes or stimulation with the cytokines TNF-alpha and IL1-beta will increase viral production from this cell type. We have demonstrated that phorbol 12-myristate 13-acetate (PMA) also increases HIV-1 p24 production from the primary human astrocyte. Using electrophoretic mobility shift assay (EMSA) in combination with supershift studies using specific antibodies, we demonstrated that PMA, like TNF-alpha increases the p50/p65 form of NF- kB. Furthermore, we also showed that the protein kinase inhibitor H7 inhibits PMA and TNF-alpha associated increases in HIV-1 expression at a time when it has little to no inhibitory effect on the associated increases in p50/p65 NF-kB. Thus, unless p50/p65 NF-kB or its binding is affected by H7 in a manner that cannot be resolved by EMSA, an increase in this form of NF-kB is not always sufficient to increase HIV-1 expression from the astrocyte. The laboratory is also investigating the ability of specific RNases to inhibit the multiplication of HIV-1 in lymphocyte cell lines. Onconase and bovine seminal RNase were shown to block infection of HIV-1 in productivity infected cell lines. This block appears from an intracellular mechanism of RNase activity. In addition to the regulation of NF-kB in infected astrocyte cultures, the viral rev protein also seems to be a target for cellular control. Using EMSA assays, astrocytes produce a factor which binds the RRE-rev complex. This factor also responds to cytokine and PMA stimulation. Both the NF-kB and the rev binding factor appear to be limited in concentration compared with cells highly susceptible to HIV-1 infection. These data point to a mechanism of HIV-1 latent infection in brain involving reduced levels of cellular factors necessary for productive HIV-1 multiplication. The role of the astrocyte as a reservoir of HIV-1 in the brain is being investigated using the in vitro model of infection.