Date of Graduation

Fall 2019

Degree

Master of Science in Cell and Molecular Biology

Department

Biomedical Sciences

Committee Chair

Amy Hulme

Keywords

HIV-1, reverse transcription, uncoating, nuclear import, cyclophilin a, microglia

Subject Categories

Cell Biology | Virology

Abstract

Parenchymal microglia represent a susceptible cell type to HIV infection and contribute to HIV Associated Neurocognitive Disorders (HAND). Currently, HIV host-protein interactions in microglia are understudied, but relevant to the design of antiviral drugs. HIV replication events rely on host and viral proteins to evade an immune response while improve replication success. Post-fusion the HIV capsid is released into the cytoplasm and begins trafficking towards the nucleus. During transit viral RNA is transcribed to DNA through reverse transcription (RT). In addition, the HIV capsid that protects the reverse transcription complex disassembles in a step termed uncoating. Once the pre-integration complex reaches the nuclear envelope host-interactions facilitate nuclear import. In the cytoplasm the capsid acts as an interface for protein interactions including cyclophilin A (CypA). CypA is a cytoplasmic peptidyl prolyl isomerase that binds the CypA binding loop of CA. CypA has been known to enhance reverse transcription and nuclear import in a cell type dependent manner. Recent work in the CHME3 human microglial cell line indicates CypA enhances end point infectivity. Therefore, the goal of this study was to identify which replication events were altered by CypA. To characterize which replication event is altered reverse transcription, uncoating, and nuclear import were assessed. qPCR, cell based, and confocal microscopy experiments were employed to detect deficient replication in absence of CypA binding. Blocking CypA interaction with CsA treatment resulted in a significant decrease in the completion of reverse transcription. qPCR analysis indicated an effect on early RT products was not detectable until after 2 hours post-infection. Further, by quantifying 2-LTR circles as an indirect measurement of nuclear import loss of CypA binding led to a decrease in nuclear DNA. Preliminary results for uncoating suggest that CypA may not drastically alter uncoating kinetics, though replicate data is needed. Based on these findings we conclude that late reverse transcription is enhanced by CypA interaction. This work establishes a foundation for understanding how CypA influences HIV in microglia while contributing to the larger field studying HIV-CypA interactions.

Copyright

© Zachary Michael Ingram

Open Access

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