BIOLOGICAL EFFECTS OF HIV-1 GENETIC VARIABILITY

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Human immunodeficiency virus type 1 (HIV-1), the etiological agent of acquired immunodeficiency syndrome (AIDS) and related complexes, displays a high degree of genetic and biological variability. The diversity of HIV-1 has implications for tracking the epidemiology of AIDS, for understanding the pathology of the virus, and for the development of therapeutic, preventive, and diagnostic reagents. There are important questions to be answered relating genetic differences to the biology of the virus, including its latency and its ability to infect and replicate in different cell types. Sequences involved in regulating most aspects of the HIV-1 life cycle, in particular viral gene expression and integration, are located within the long terminal repeats (LTRs) at the 5' and 3' ends of linear viral molecules. The direct contribution of genetic variability in cic-acting transcriptional regulatory elements within HIV-1 LTRs to phenotypic variability in vitro and in vivo has not been extensively evaluated. Integration into the host-cell genome involves covalent linkage between HIV-1 LTRs and cellular DNA. In general, unintergrated linear or circular DNA forms of retroviruses occur only as short-lived replicative intermediates. However, unintegrated viral forms accumulate in cultured cells infected with HIV-1 and appear to correlate with in vitro cytopathicity. The prevalence of unintegrated HIV-1 DNA within cells of infected individuals and its relationship, if any, to HIV-1 pathogenesis has not been determined. The proposed studies are specially aimed at: (1) evaluating directly in cells from infected individuals the extent of sequence heterogeneity within HIV-1 LTRs; (2) assessing the prevalence of unintegrated forms of HIV-1 in infected individuals; and (3) determining the biological effects of natural genetic variability in functional assays. An important aspect of the experimental design is evaluation of genetic diversity directly in infected individuals without selection for in vitro growth of HIV-1 isolates. The studies will focus on clinically ill pediatric AIDS patients and their asymptomatic, seropositive mothers to assess the generation of HIV-1 diversity over time between individuals with different clinical status infected with related viruses. Sequencing LTRs after enzymatic amplification using the polymerase chain reaction (PCR) will assess the extent of LTR variability. Transient expression of an indicator gene under control of variant LTRs will evaluate the biological effects of sequence diversity of LTR promoter function and cell-type specific gene expression. Proposed experiments are designed to test the hypotheses that natural genetic variability occurs in HIV-1 LTRS; that LTR diversity develops in vivo within and between individuals infected with related viruses; and that genetic diversity not only contributes to functional variability of LTRS, but is also associated within the clinical status of individuals infected with HIV-1.