The overall goal of this application is to determine if particular HIV-1 genotypes, harbored in CMS reservoirs and compartments, are linked to the pathogenesis of HIV-associated dementia (HAD). The current study will evaluate cloned full-length HIV-1 genomes from the brains of patients who died with early, intermediate, and late stage HAD in the pre and post HAART era as well as controls. Newly developed molecular analytical tools will allow us to determine if a specific HIV evolutionary pathway occurs in the CMS of patients that develop HAD. Additionally, we will systematically address the on-going dispute of whether or not a specific "signature sequence" evolves during HAD pathogenesis. Molecular substitution of both ancestral HAD and non-HAD sequences and sequences containing HAD-specific signature patterns (if found) into HIV molecular clones will allow us to test the effects that the genomes have on macrophage function in vitro and allow histochemical identification of modified macrophages within diseased areas of the brain. In preliminary studies we found that an original/earliest form of HIV gp120-V3 sequence in a patient with HAD was in the meninges. This sequence was clearly the origin of all of the other sequences evaluated, including those in four other areas of the brain, and in the periphery, notably the seminal vesicles and lymph nodes. Within the brain, the most evolved form of HIV was in the sub-cortical white matter, an area of most intense involvement with classical histological changes associated with HAD. The preliminary results section has validated the novel phylogenetic approach that we will employ in the current application. Specific aims include: 1) To quantitate HIV DMA levels and sequence full-length HIV genomes from anatomically distinct regions of the brains of patients who died with HIV disease as compared to controls. Up to 100,000 HIV sequences will be evaluated from more than 20 autopsy derived specimens (through the ACSR) using a novel software, HIVbase, a sequence specific relational database that allows large scale storage and rapid processing of DNA sequence data. 2) To compare the phylodynamics of viral populations in each tissue sequenced in Specific AIM1 for all patients. This specific aim will test whether viral sequences evolve in parallel with disease pathogenesis and potentially identify disease specific "signature sequences". 3) Test whether HIV viral sequence migration within the CMS occurs in a programmatic and disease specific manner. 4) Perform molecular substitution studies utilizing HAD specific "signature sequences" to test effects on in vitro macrophage function and attempt to localize those cells in vivo in brains from patients with HAD. Evaluate the pathogenic potential of ancestral sequences involved in early stage infection. 5) If HAD specific "signature sequences" are identified in the above specific aims, quantitative analytic approaches will be employed to test whether those sequences can be identified in the blood of patients with HAD as compared to controls. The ultimate goal will be to determine whether specific HIV sequences contribute to HAD pathogenesis and whether those sequences can be identified in the blood of patients at risk for HAD.