The retrovirus HIV-1 establishes lifelong infections, facilitated by integration, the covalent insertion of the viral cDNA into the host cell genome. After integration, the viral genes are expressed by host cell machinery. This dissertation focuses on patterns of HIV-1 integration site selection and of viral mRNA splicing, both of which are optimized to drive appropriate viral gene expression, and ultimately, virion production. In an effort to uncover determinants of integration site selection we investigated the role of the nuclear pore, interaction with which promotes HIV-1 integration efficiency. We found that the nuclear transport protein, Transportin-3, and the pore component, RanBP2, were important for viral targeting to chromatin rich in genes. We next asked which viral factors determine this bias. The HIV-1 CA protein has been shown to bind RanBP2. We used CA mutants to show that the preference for gene dense chromatin partially mapped to this interaction. We also sought to identify chromatin features that most strongly attract HIV-1 particles, and to this end we characterized normal distributions of HIV-1 integration sites in primary human T cells at unprecedented depth. In another group of studies, we investigated viral mRNA populations and asked whether their calibration was sensitive to differences between cells. A series of RNA elements interact with host factors to regulate splicing of the HIV-1 pre-RNA transcript. We found that production or degradation of certain mRNAs differed between T cells and a human osteosarcoma cell line and among human donors. Such differences might have ties to cell cycle control. We also detected viral strain-specific splicing patterns. The patterns of integration and mRNA populations described in this dissertation reflect the importance of cellular factors in the HIV-1 replication cycle. These studies aid in the identification of therapeutic targets, while revealing much unexplored complexity in the viral steps of nuclear import, integration and mRNA processing.
|Advisor:||Bushman, Frederic D.|
|Commitee:||Bates, Paul, Collman, Ronald G., Hoxie, James A., You, Jianxin|
|School:||University of Pennsylvania|
|Department:||Cell and Molecular Biology|
|School Location:||United States -- Pennsylvania|
|Source:||DAI-B 74/03(E), Dissertation Abstracts International|
|Keywords:||Capsid, High throughput sequencing, Hiv, Integration, Nuclear pore, Splicing|
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