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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National Institute of Allergy and Infectious Diseases (NIAID)
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AIDS and Related Research Study Section 3 (ARRC)
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University of California Irvine
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Wang, Yichuan; Kim, Steven S; Lu, Ding et al. (2004) Use of a replication-defective vector to track cells initially infected by SIV in vivo: infected mononuclear cells rapidly appear in the draining lymph node after intradermal inoculation of rhesus monkeys. AIDS Res Hum Retroviruses 20:1298-305
Abel, Kristina; Alegria-Hartman, Michelle J; Rothaeusler, Kristina et al. (2002) The relationship between simian immunodeficiency virus RNA levels and the mRNA levels of alpha/beta interferons (IFN-alpha/beta) and IFN-alpha/beta-inducible Mx in lymphoid tissues of rhesus macaques during acute and chronic infection. J Virol 76:8433-45
Marthas, M L; Lu, D; Penedo, M C et al. (2001) Titration of an SIVmac251 stock by vaginal inoculation of Indian and Chinese origin rhesus macaques: transmission efficiency, viral loads, and antibody responses. AIDS Res Hum Retroviruses 17:1455-66
Greenier, J L; Miller, C J; Lu, D et al. (2001) Route of simian immunodeficiency virus inoculation determines the complexity but not the identity of viral variant populations that infect rhesus macaques. J Virol 75:3753-65
Abel, K; Alegria-Hartman, M J; Zanotto, K et al. (2001) Anatomic site and immune function correlate with relative cytokine mRNA expression levels in lymphoid tissues of normal rhesus macaques. Cytokine 16:191-204
Kim, S S; Kothari, N; You, X J et al. (2001) Generation of replication-defective helper-free vectors based on simian immunodeficiency virus. Virology 282:154-67
Kim, S S; You, X J; Harmon, M E et al. (2001) Use of helper-free replication-defective simian immunodeficiency virus-based vectors to study macrophage and T tropism: evidence for distinct levels of restriction in primary macrophages and a T-cell line. J Virol 75:2288-300
Hu, J; Gardner, M B; Miller, C J (2000) Simian immunodeficiency virus rapidly penetrates the cervicovaginal mucosa after intravaginal inoculation and infects intraepithelial dendritic cells. J Virol 74:6087-95
Hu, J; Miller, C J; O'Doherty, U et al. (1999) The dendritic cell-T cell milieu of the lymphoid tissue of the tonsil provides a locale in which SIV can reside and propagate at chronic stages of infection. AIDS Res Hum Retroviruses 15:1305-14
Miller, C J; Hu, J (1999) T cell-tropic simian immunodeficiency virus (SIV) and simian-human immunodeficiency viruses are readily transmitted by vaginal inoculation of rhesus macaques, and Langerhans' cells of the female genital tract are infected with SIV. J Infect Dis 179 Suppl 3:S413-7

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