Alveolar Macrophage Host Defenses

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DESCRIPTION (provided by applicant): Alveolar macrophages (AM) are a key reservoir of human immunodeficiency virus-1 (HIV-1) infection in the lung. In the course of studies attempting to alter the genetic repertoire of human AM in order to understand HIV-1 interaction with these cells, we made the novel observation that treatment of human AM with modified adenovirus gene transfer vectors (Ad) prior to HIV- 1 infection blocks subsequent HIV- 1 replication, even in the absence of an Ad transgene, The purpose of this proposal is to understand the mechanism of Ad inhibition of HIV-1 replication in human AM at the molecular level. The specific aims of the proposal are to understand: 1. The step(s) in the HIV-1 lifecycle that are being inhibited by Ad; 2. The Ad component(s) that are responsible for causing the inhibition; and 3. The intracellular pathway(s) in AM that are being modulated to mediate this effect of Ad on HIV-1 replication. To accomplish these goals, experiments will be performed to assess the effect of timing of Ad treatment of AM relative to HIV-1 infection on the inhibitory effect in comparison with specific inhibitors of various steps in the HIV-1 life cycle. Transcription from the HIV-1 LTR will be compared with the results of p24 ELISA under conditions of the various inhibitors. A TAQMAN PCR assay will be used to quantify the synthesis of HIV-1 specific DNA. To ascertain the Ad components responsible, Ad variants such as empty capsids and vectors deficient in specific Ad genes such as E4 will be used. The intracellular pathways responsible for mediating the inhibitory effect of Ad on HIV-1 replication will be modeled in cell lines deficient in relevant signal transduction molecules such as STAT-1. Correlation will be made with changes in AM gene expression detected by gene chip technology and confirmed with Northern and Western analysis. Taken together, these studies will provide insight into the mechanism of Ad inhibition of HIV-1 replication in human AM.