We have induced defensive cellular responses by attenuated infection with HIV-1 mutated in the vif gene. Resistance was induced in transformed human T cells by co-cultivation with HIV-1 resistant PBL, which were latently infected with vif-negative HIV-1. The resulting latently infected transformed T cell clones were found to secrete a soluble HIV-1 Resistance Factor (HRF), a new factor that confers resistance to HIV-1 upon uninfected cells by blocking HIV-1 transcription. HRF activity in HRF(+) clones was shown initially through their resistance to productive HIV-1 infection and their block to TAT activated transcription of a marker gene. More detailed studies indicate that HRF prevents formation of NF-kB/DNA complex in nuclear extracts of cells that have been exposed to supernatants of HRF(+) cells. This observation prompted the development of Rapid Suppression Assay (RSA) that measures the activity of HRF by inhibition of phorbol myristate induction of HIV-1 in ACH-2 cells. In this application we propose to (1) isolate and purify HRF active in RSA from cell supernatants through sequential gel exclusion chromatographic steps. Purified HRF will be subjected to N'-terminal sequencing, yielding sequences for primer design. (2) We shall construct a cDNA library from HRF(+) cells, identify a full-length cDNA using HRF sequence based primers, and clone HRF into a Baculovirus expression vector or a mammalian expression vector for high level expression. Anti-HRF antibodies will be raised against recombinant HRF. (3) In parallel we will employ state of the art technologies to identify up-regulated genes in HRF(+) clones using cDNA expression arrays. In this approach we will search known genes involved in hematology, immunology and transcription regulation to complement biochemical isolation and identification. (4) The activity of recombinant HRF will be tested in the optimal dose for inhibition of a panel of primary isolates in different cell types. (5) HRF expression in Iymphocytes from HIV-1 infected persons will be tested by RT-PCR and the levels of expression will be correlated to the disease state of the donor. Having T cell clones producing HRF creates a unique opportunity to isolate, characterize and apply this protein for treatment of HIV-1 infection in culture, and in long term goals to design new therapeutics against AIDS and to understand cellular protective responses to infection by attenuated HIV-1.
