Linker-Scanning Mutational Analysis of Tanscriptional Activity of the Human Immunodeficiency Virus Type 1 Long Terminal Repeat in Human Placental Trophoblast
Kickland, Erich C.
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The vertical transmission of the Human Immunodeficiency Virus Type 1 (HIV -1) is defined as the transmission of the virus from infected mother to fetus. In order to infect the fetal blood supply in utero, the virus must pass through the placenta. The virus collects in the placental trophoblast, and only certain strains are able to pass from that point into the fetal blood supply, because the placental trophoblast is not a strong host for HIV replication. This report analyzes transcription factors present in placental trophoblast which may be critically important to HIV replication in trophoblast. This is done through the use of a set of 27 linker-scanning mutants created by Dr. Steven L. Zeichner in Philadelphia. These mutants replace 18 bp at a time in subsequent order along the U3 and R regions of the viral long terminal repeat (LTR). The U3 and R regions of the LTR are primarily regions of viral transcriptional control. By transfecting the mutated LTR's into human placental trophoblasts, it is possible to analyze the transcriptional activity of each mutant in the cell type specific environment. Monitoring of the transcriptional activity is achieved by the use of a luciferase reporter gene system. This reporter gene produces luciferase, which cleaves the substrate D(-)-luciferin to produce visible light that is measured with a luminometer. By analyzing the data from the luminometer, it is possible to see which mutants have the lowest light emission, and therefore which mutants have the lowest transcriptional activity. Since it is known in which particular mutant each specific known or potential transcription control region lies, it is possible to discern which control regions are critical to HIV-1 replication in placental trophoblast. This can be accomplished by determining which mutated regions have significantly lower activity when compared to the wild type LTR. For each of the 27 mutants, a set of JAR cells were transfected in duplicate. JAR cells are analogous to first trimester trophoblast, and are used for comparison purposes. Time constraints considered, it was not possible to run another set of transfections in term placental trophoblast. The readings taken from the luminometer (in units of photons emitted) for the JAR cells luciferase assay were very sporadic even between duplicates of the same sample. Attempts to normalize the data by factoring in cellular debris concentration using spectrophotometer analysis did not help to even out the numbers. Due to the unreliable nature of the readings, the data were inconclusive concerning the determination of important transcriptional control elements. Eventual determination of which control regions are important to viral replication will assist in deciphering the mechanisms by which HIV can replicate in placental trophoblast and hopefully enrich current understandings of vertical transmission of HIV.
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