The understanding of human immunodeficiency virus type 1 (HIV-1) pathogenesis, in vivo, requires the ability to precisely quantitate HIV-1 proviral DNA and RNA in a variety of tissues and secretions. Although HIV- 1 has long been known to infect semen and cervical/vaginal secretions of HIV-1-seropositive individuals, the molecular mechanisms affecting the sexual transmission of HIV-1, in vivo, are poorly defined. Understanding the factors which affect the infectivity of HIV- 1-seropositive individuals is of critical importance. Utilizing two novel molecular techniques, developed in our laboratory, the precise HIV-1-proviral load in the cellular components of semen and cervical/vaginal secretions, and the HIV- 1 RNA levels in the genital secretions shall be determined. An in situ polymerase chain reaction (PCR) for HIV-1 DNA, which allows amplification of a segment of the HIV-1 genome in intact cells, and a quantitative HIV-1 reverse transcriptase PCR technique will be utilized. Total HIV-1 RNA, in both cell-associated and cell-free fractions, of these genital secretions shall be evaluated. As well, an aberrant pattern of HIV- l RNA, previously correlated with restricted viral replication, will be investigated in the cellular components of these genital secretions. HIV-1 proviral and HIV-1 RNA total levels and patterns, in genital secretions, will be correlated with other clinical markers of HIV- 1 infection and initial mode of HIV-1 infection. As well, the levels of HIV- l proviral-harboring cells and HIV-l RNA in genital secretions shall be followed, longitudinally, through disease progression and correlated with clinical parameters of genital inflammation and various treatment modalities using anti-retroviral agents. In addition, levels of HIV-1 proviral-containing cells in the peripheral blood will be assessed, utilizing in situ PCR, and correlated with HIV-l RNA and DNA levels in genital secretions. The cellular tropism of HIV- l strains and the presence of defective viral genomes, in genital secretions of select cohorts of patients, will be assessed via cloning and sequencing techniques. The hypotheses, that macrophage-tropic strains correlate with increased sexual transmission while partially defective viruses lead to aberrant viral RNA patterns and possibly decreased transmissibility, will be tested. To further assess correlation of molecular parameters, described above, with clinical infectivity of HIV-l-seropositive individuals, couples both discordant and concordant for HIV-1 infection will be evaluated. In these ways, it is suggested that a more precise understanding of the mechanisms involved in controlling the sexual transmissibility of HIV-1 will be gained.
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