The objective of this proposal is to characterize the role of the LTR in the establishment of virus load in cats infected with the relatively virulent FIV-pPPR. Infection of naive cats with molecularly cloned FIV-pPPR results in a productive virus infection with altered CD4+: CD8+ T-cell ratios within 17 months of infection. FIV-pPPR has been found to replicate in primary feline PBMC, primary feline blood-derived macrophages, and feline T-cells. The promoter function of the LTR of FIV-pPPR have been characterized and found to be activated through specific regulatory domains (i.e., AP-1 and ATF sites) by multiple cell-signaling pathways including the protein kinase C and the protein kinase A pathways. The role of regulatory regions within the FIV LTR in FIV replication will be assessed with infectious FIV-pPPR proviruses containing site-directed mutations and deletions in the U3 domain of the LTR. Replication of these LTR mutant viruses will be tested in feline T-cell lines, primary feline PBMC, and primary feline macrophages. Replication of FIV LTR mutant viruses in primary feline PBMC and macrophages in varying activation states as well as resting states will also be assessed. Replication of FIV-pPPR LTR mutant viruses will be tested in vivo by infection of specific pathogen free cats and assessment of virus load in inoculated cats. Virus load will be assessed in PBMC and peripheral lymph nodes sampled from infected cats by semi-quantitative analysis of viral DNA and viral RNA by polymerase chain reaction. Infected cats will be assessed for virus load and for clinical pathological alterations (i.e., CD4+ and CD8+ T-cell counts) during early infection and in later stages of infection. Infectious LTR mutant viruses with which infection is characterized by a significantly reduced virus load, will be assessed in pilot studies as potential vaccines. The opportunity for direct assessment of viral determinants of pathogenesis of disease in vivo is provided by animal models such as FIV. FIV infection in cats presents with immune deficits and CD4+ T-cell depletion very similar to those observed in humans infected with HIV. Because of the accessibility of cats, the FIV model provides an economically feasible working animal model for dissection of the molecular pathogenesis of lentivirus-induced immunodeficiency, testing novel vaccine strategies, and testing of therapeutic agents targeting specific viral determinants.
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