These experiments will identify the initial cellular targets after vaginal mucosal infection by simian immunodeficiency virus (SIV). We will first develop replication-defective helper-free SIV vectors expressing a readily detectable reporter gene, bacterial beta galactosidase. The vectors will be important for precise elucidation of the pattern of initial infection after vaginal exposure, since they will not spread beyond the first cells infected. Coupling this information with time course studies on vaginal infection by replication-competent SIV will allow us to trace the cellular pathway of infection beginning from the initially infected cells through spread within the vaginal mucosa and adjoining tissues. Additional experiments will determine the effect of in vitro SIV phenotype on target cells and efficiency of vaginal infection. The role of SIV accessory proteins in infection of primary macaque cells in vitro and in vivo will also be investigated. The proposed experiments combine the expertise of the P.I. in molecular virology with the expertise of the co-P.I. in vaginal mucosal infection by SIV in macaques. Specific experiments include: 1) Development of helper-free replication-defected SIV-beta-gal vectors. Experiments will include molecular cloning of vectors, including those with mutant accessory proteins (vpr, vpx, and nef). SIV-based retroviral packaging plasmids will be generated, and helper-free vector stocks will be prepared by transient transfection of 293T cells. 2) Infection of primary rhesus macaque TH, macrophages and Langerhans cells with SIV-based vectors and replication-competent SIV will be carried out. The role of accessory proteins in infection will be explored. 3) To identify the first cells infected, the helper-free beta-gal vectors will be used to vaginally infect female rhesus macaques. Staining of histology sections or purified cells with X-gal and cell-specific MAbs will be carried out. The pathway of infection spread will be determined by carrying out time course infections with wild-type SIV. Comparative experiments with different vectors will also be used to explore the question of which initially infected cells progress to productive infection. 4) The effect of in vitro SIV phenotypes of the pattern of initial virus infection in vivo will be studied. Variant viruses and beta-gal vectors that show different phenotypes (macrophage vs. T-lymphotrophic; NSI vs. SI) will be generated and used in vaginal infections as in the previous aim. The role of SIV accessory proteins in infection in the animal will also be investigated.
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