Abstract
The human immunodeficiency virus type 1 (HIV-1) lifecycle is complex and the virus interacts with many host cellular proteins for productive replication. Mass spectrometry analysis is a powerful tool to identify cellular proteins important for HIV replication and pathogenesis. Prior proteomic studies demonstrate that HIV-1 infection perturbs the host cell proteome. |The nuclear compartment of cells is critical for HIV replication: integration of viral DNA into the host genome is essential to virus replication and pathogenesis; numerous host transcription factors are required for HIV-1 gene expression; and subtle changes to the nuclear proteome either through translocation of cytosolic proteins or induction of regulatory factors can contribute to evasion of the host immune response. However, to date no study has looked specifically at the changes in the host nuclear proteome during HIV infection. My initial studies examined the changes in the nuclear proteome of HIV-1 infected T-cells.|On a global level, the cellular changes in response to virus infection are profound. Cells must attempt to inhibit infection as well as signal the surrounding environment to the presence of the pathogen. The results of both efforts cause changes in the expression and/or localization of host cell factors including signaling molecules, RNAs, and proteins. In the second study we examined the changes in protein expression and localization in HIV-1 infected T-cells by analyzing the cytosolic, membrane/organelle, and nuclear fractions using Sequential Window Acquisition of all Theoretical fragment-ion spectra Mass Spectrometry (SWATH-MS) proteomic approach. In addition, factors identified as potential HIV-1 interacting proteins were evaluated for biological relevance in a cell culture model.|Cyclophilin B (CypB) is a member of the peptidyl prolyl isomerase family that is an intracellular chaperon for prolactin and IRF3. It predominantly localizes to the ER, but also contains a nuclear localization signal and can be secreted from cells. It has previously been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1) and several proteomic and genetic studies identified it as a potential factor involved in HIV replication. In the nuclear proteome study described in Chapter II, we found that nuclear levels of CypB increased during HIV replication. In Chapter IV we show that ectopic over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. The hydrophobic N-terminus of the protein is required for the effect. The N-terminus of CypB contains an ER leader sequence necessary for secretion as well as a nuclear localization signal. Passive transfer experiments showed that secreted CypB did not impact HIV infection. However the deletion of the N-terminus resulted in a pronounced mislocalization of CypB from the ER. Combined, these experiments indicate that intracellular CypB regulates a pathway of HIV nuclear import. |Viral proteins pose a tempting therapeutic target; however the fast viral turnover and inherent genetic variability drive the rapid evolution of resistance mechanisms to therapies directed at only viral proteins. Host cellular factors represent a unique target for drug development. These studies have identified potential factors essential to HIV-1 replication and provide new insights into protein changes at the cellular level following infection.