This research plan will seek to further the understanding of human immunodeficiency virus type 1 (HIV-1) latency and cell-specific control of HIV-1 replication. A molecular model for HIV-1 latency, in which non- productive infections are characterized by an aberrant pattern of HIV-1 RNA expression, will be investigated in primary human cells from HIV-1-infected individuals. Lymphocytes and monocyte/macrophages from HIV-1-infected individuals, at various stages of disease, will be evaluated for evidence of HIV-1 proviral latency. These studies will be performed using a very sensitive quantitative RNA polymerase chain reaction (PCR). This aberrant pattern of HIV-1-RNA expression has recently been demonstrated, in our laboratory, to be present in the peripheral blood cells of certain HIV-1- infected individuals. Preliminary data suggests that this aberrant RNA pattern is significantly more common in asymptomatic HIV-1-infected individuals, as compared to those with AIDS. The association, or lack thereof, of this state of viral latency with T4 lymphocyte counts, parameters of viral replication, therapy, time since HIV-1-seroconversion, gender, clinical classification of HIV-1 infection and mode of initial HIV- 1 infection will be studied. Cell culture models, initially using the HIV-1-latently-infected monocytic and T-lymphocytic cell lines, U1 and ACH-2, will be evaluated to further understand the proximal cause(s) of HIV-1 proviral latency. The role of the extremely tight control of Nuclear Factor-kappaB (NF-kappaB) in HIV-1 latency will be investigated. As well, utilizing a sensitive UV crosslinking assay, the differences in NF-kappaB-moieties between uninfected and productively-infected cells, and HIV-1 latently-infected cells will be determined. Preliminary data suggests that a NF-kappaB-like complex, with a faster migration pattern on electrophoretic mobility shift assay (EMSA), is detected in latently HIV-1-infected cells, as compared to uninfected and productively-infected cells. Rev, the HIV-1-encoded protein which controls the rescue of HIV-1 genomic RNA from the nucleus, has been suggested to be a key control element in HIV-1 latency. This will be studied using the U1 and ACH-2 cells and recombinant retroviral-expression vectors, which will be engineered to produce functional and mutant Rev proteins upon infection. In this way, Rev's effect on HIV-1 latency, and its possible use in intracellular immunization protocols to maintain latency, will be evaluated. As well, the possible relationship of certain Rev-interacting cell-specific factors to the control and maintenance of HIV-1 proviral latency will be investigated, utilizing a RNA EMSA. HIV-1 infection of the human host has a long and variable course, characterized by a protracted period of relatively quiescent viral production. This proposal will focus on assessing HIV-1 proviral latency in vivo and will seek to explain some of the molecular mechanisms, cellular and viral determinants, involved in non-productive HIV-1 infection. It is planned that these interdependent studies will lead to preliminary insights into novel techniques to maintain a non-productive state of HIV-1 infection in vitro and, hopefully, in vivo.
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