Efforts to discover new anti-HIV-1 therapies and to elicit effective anti-HIV-1 immunity have increasingly focused on human factors that regulate HIV-1 replication and pathogenesis. Cyclophilin A, a peptidyl-prolyl isomerase that regulates protein-folding and is itself a receptor for the immunosuppressive drug cyclosporine, binds HIV-1 Gag via the CA domain. The interaction stimulates HIV-1 reverse transcription in human cells but inhibits it in owl monkey (OMK) or macaque cells. These phenotypes appear to result from effects of cyclophilin A on HIV-1 CA accessibility to species-specific Lvl/Refl host restriction factors. Also, via a novel conformational switch, cyclophilin A inhibits the Itk tyrosine kinase, suggesting that Gag binding to cyclophilin A might contribute to HIV-1 pathogenesis by disrupting CD4 v T cell effector function. These and other observations made since the discovery of the Gag/cyclophilin A interaction in 1993 serve as the basis for current specific aims.
Aim 1. Several complementary approaches will be used to clone OMK Lvl and human Refl, including a functional screen of a cDNA library from owl monkey cells, a modified two-hybrid screen using a CA-fusion bait that saturates OMK restriction, and subtraction libraries from cell line subclones selected for loss of restriction.
Aim 2. As203, m-Cl-CCP, and PKl1195 all disrupt mitochondrial membrane potential and decrease Refl restriction. Select primate species will be challenged to determine if the drugs also inhibit Lvl. Preliminary data suggest that cyclophilin A protects HIV-1 from Refl; to test this hypothesis the effect of the drugs on HIV-1 infectivity will be examined in contexts where cyclophilin A or Refl are disrupted. Detailed biochemical and cell biological effects of the drugs will be characterized and contrasted with those of cyclosporine.
Aim 3. Under pressure from cyclosporine, HIV-1 acquires mutations in sequences encoding a type II turn in CA that are sufficient to confer cyclosporine-resistance. In certain human cell lines, these mutants replicate poorly and are cyclosporine-dependent. Evidence that this is due to a new restriction activity present in certain individuals will be sought. Group O viruses have alterations in the same gag sequences, are cyclosporine-resistant, and will be examined for sensitivity to the same activity.
Aim 4. It will be determined if HIV-1 Gag disrupts the cyclophilin A-Itk interaction and causes elevated Th2 cytokine and IL-2 expression from effector/memory CD4* T cells as is seen in cyclophilin A knockout mice. These immunologic abnormalities would be expected to contribute to HIV-1 replication and pathogenesis.
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