PATHOGENIC MECHANISMS OF HIV

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A central project in the laboratory is the understanding of the mechanisms of HIV expression and pathogenicity and the application of this information to the development of new therapeutic and vaccine approaches. We have studied the complex interactions of cytokines, chemokines, and HIV receptors in primary cells; the role of the HIV accessory protein Vpr as a virus coactivator; and the regulation of the CCR5 gene, a receptor essential for HIV infection in humans. We study the function of several HIV-1 proteins, including Tat, Rev, and Vpr. We showed that the defect of Rev in rodent cells is caused by delayed import of Rev in the nucleus. The factors responsible for this effect are under investigation and may lead to new types of interventions targeted against the essential Rev factor. We have demonstrated that the HIV-1 Vpr protein is a transcriptional coactivator of nuclear receptors. This finding explains the promiscuous activating effects of Vpr on the expression of several viral and cellular genes. We found that Vpr binds directly to p300/CBP, an important coactivator of many cellular promoters. This binding leads to increased activation of such promoters. We identified multiple binding sites for CCAAT-enhancer-binding protein (C/EBPb) within the gene for chemokine receptor CCR5, suggesting that C/EBPb may be involved in CCR5 regulation. RNA analysis demonstrated a 5-fold increase in C/EBPb expression in circulating T-lymphocytes from HIV-1-infected individuals compared with healthy blood donors. Similarly, analysis of CCR5 expression in PBMCs demonstrated increased numbers of CCR5+ T lymphocytes in HIV-1-infected individuals as compared with healthy blood donors. The increase in CCR5+ T lymphocytes was found in all stages of the disease and correlated with the decline in CD4 counts. Our results suggest that C/EBPb is an important regulator of CCR5 expression and may play a relevant role in the pathogenesis of HIV disease. We have studied alternative mechanisms for the nuclear export of mRNAs. We identified a new RNA element from the mouse genome that is able to replace the Rev and RRE of HIV-1, using a mutated HIV-1 DNA proviral clone as a new type of molecular trap. The factors binding to this element are under investigation. This result showed the presence of additional RNA elements in the genome that are able to direct nuclear export and may define a distinct pathway for nucleocytoplasmic transport. We have also initiated the study of new HIV recombinants to understand the complex epidemiologic questions associated with HIV subtype recombination. Improved DNA vaccines against AIDS We have developed improved vaccine vectors for DNA vaccination against AIDS. We showed that improved expression leads to better immunogenicity of DNA vectors and also that gag DNA is very immunogenic after intramuscular DNA injection. We also showed that direction of the gag antigen towards the secretory pathway results in increased immune response. We continue to improve the methodology of DNA vaccination, aiming at efficient vaccination using combinations of vectors against multiple HIV antigens. Animal models We have developed a system to introduce any gene of interest by the Cre-loxP recombination system into predetermined genomic loci where gene expression is tightly regulated. Chromosomal loci that permit tight control of gene expression were identified in embryonic stem (ES) cells and analyzed extensively for expression in many tissues by creating mice from ES cells and breeding them with mice containing tetracycline-controlled transactivator (tTA). Only a small number of loci showed low basal activity and up to a 1000-fold inducibility. In general, inducibility correlated with tTA expression levels in different tissues. To further improve the stringency of gene regulation, the insulator sequence of chicken b-globin locus was introduced into the same genomic loci. The presence of insulator further reduced the basal activity of the luciferase indicator to less than one luciferase molecule per cell. This system allows the tight regulation of expression after gene transfer and is a valuable tool for gene function studies in transgenic animals.