The specific aims of this proposal are: 1) to compare the pathogenesis of HIV in lymphocytes and monocytes/macrophages (mo/m0) with emphasis on virus attachment, penetration, maturation and release; and 2) to study the effects of antiviral agents, either synthetic or natural products, on HIV replication in lymphocytes and/or mo/mo/, especially in persistently -infected macrophages. The experimental design will rely mainly on ultrastructural approaches. 1) Pathogenesis of HIV in lymphocytes and mo/mo/: HIV-infected primary cultures of lymphocytes and mo/mo/will be monitored for HIV attachment during the first 3-4 hours after infection and for viral replication on days 7, 10, 14 (acute) and 40 (chronic) post-infection by: a) assaying supernatant for reverse transcriptase activity and HIV antigen levels (ELISA); and b) processing cell pellets of lymphocytes and mo/mo/monolayers for EM and IEM. Dually-infected mo/mo/or H9 cells with HIV and HCMV will also be monitored as in b). A detailed search for pseudotype virions will be made using immunogold-labelling EM and/or immunoferritin EM techniques. 2) Antiviral drug assays: Primary cultures of lymphocytes and mo/mo obtained form the same donors will be treated with polybrene and then infected with HIV-1 or HIV-2, after which various concentrations of antiviral drugs will be added to the culture medium. Drug inhibition of HIV replication in infected cultures will be determined as describe in 1). In EM studies, special attention will be given to the effects of drugs on HIV attachment and penetration during the early states of infection, i.e. the first 4 hours after infection; and on inhibition of HIV release on days 7, 10 and 14 post-infection. The ultrastructural information obtained from these basic studies should expand our knowledge of the pathogenesis of HIV infection and the mechanisms of antiviral drug action. This information may lead to improved strategies for the treatment of AIDS and related disorders.
