Purification of Human Immunodeficiency Virus Type 1 Preintegration Complexes by Velocity Gradient Centrifugation.
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Authors
Donnellan, Meghan
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2009-12-11 , 2009-12-11
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Thesis
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en_US
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Abstract
The early events of human immunodeficiency virus type 1 (HIV-1) infection are mediated by two large nucleoprotein complexes (NPCs), the reverse transcription complex (RTC) and the preintegration complex (PIC). These complexes are responsible for the conversion of viral RNA into viral DNA, transport through the cytoplasm, import into the nucleus, and the subsequent integration of the viral genomic DNA into the host chromosomal DNA. Several viral proteins are associated with the NPCs, including reverse transcriptase (RT), integrase (IN), viral protein R (Vpr), capsid (CA), matrix (MA), and nucleocapsid (NC). Previous studies of these complexes have yielded conflicting data, partially due to inconsistent production and purification methods. Here I examined several infection protocols and determined overnight (O/N) infection of C8166-45 cells via spinoculation to be the optimal production method to yield active PICs. I also evaluated the efficiency of several velocity gradient centrifugation (VGC) parameters for purifying NPCs. The sedimentation patterns of NPCs were determined by analyzing the distribution of known interacting viral proteins in gradient fractions. My results indicate that short velocity gradient centrifugation is preferred to extended centrifugation. Shorter centrifugation lengths separate the two NPCs instead of concentrating them as increased centrifugation does. Increased centrifugation also results in the dissociation of the complexes. These methods for the production and purification of HIV-1 nucleoprotein complexes will facilitate future proteomic analyses to identify novel HIV-1 cellular factors and hopefully lead to new targets for antiretroviral treatments.
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Creighton University
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Donnellan-thesis.pdf