Long-term nonprogressive (LTNP) HIV-1 infection presents an opportunity to study attenuation of HIV in vivo. Molecular determinants of LTNP infection are relevant to pathogenesis and vaccines. Because HIV-1 in plasma represents replicating virus, and multiple regions of the genome can affect pathogenesis we developed an RT-PCR-based technique to obtain full-length genomic clones from plasma HIV-1 RNA. We then analyzed the HIV-1 sequence changes that occurred when an individual underwent a transition from LTNP to rapidly progressive infection and found a major difference in the serial sequences that explains the change in clinical states. The long-term objectives are: 1) to identify molecular determinants of HIV-1 pathogenesis and attenuation in vivo using novel approaches. The work will focus on complete HIV-1 RNA sequences from individuals in a large cohort (NIH Women's Interagency HIV Study and others) before and after their transition from LTNP to progressive infection; and 2) to find HIV-1 sequences which may serve as new target sites in the design of vaccines and anti-viral agents.
We aim to perform the following studies on a cohort of at least 33 untreated LTNP patients and controls: 1) Clone, sequence, and analyze by computational biology full-length plasma HIV-1 RNA genomes from the entire cohort. We will first focus on the 6 individuals in the cohort who have undergone a transition from LTNP to progressive infection; by comparing genomes obtained before and after the transition, we aim to identify sequences associated with attenuation and pathogenesis. We shall then analyze genomes from the remainder of the cohort, seeking previously identified and new mutations associated with attenuation of pathogenesis. 2) Functionally evaluate the sequences associated with attenuation or pathogenesis by constructing recombinants with infectious molecular HIV-1 clones; 3) Measure the infectivity of the clones by transfection and phenotypically characterize the virus produced; 4) Determine the HIV-1 co-receptor utilization pattern of the clones and viral isolates by performing infectivity assays and viral envelope fusion assays. The results will be correlated with the clinical, virologic, and immunogenetic characteristics of each patient in an effort to identify determinants of pathogenesis and attenuation in vivo.
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