Cloning and characterization of MRI, a modulator of retroviral infection.

Abstract

Although significant progress has been made in elucidating the life cycle of HIV and identifying host cell proteins which interact with HIV, a much greater understanding of these processes is necessary in order to eradicate this disease. The studies presented in this dissertation further examine these processes and identifies a new host cell protein, MRI, which appears to be involved in the poorly understood step of viral uncoating.The first part of my project involved the creation, characterization, and utilization of a novel toxic retroviral vector encoding barnase, a non-specific protease derived from Bacillus amyloliquefaciens. In conjunction with an inhibitor of barnase (barstar), I showed that barnase-based retroviral vectors were capable of inducing cell death in a wide variety of cell types and that the presence of barstar was essential for the creation of high titer retroviral vectors. The second part of my project was to was to create a heavily mutagenized hamster cell line, V79-4, and repeatedly challenge this cell line with the toxic retroviral vector in order to isolate clonal populations of cells resistant to retrovirally mediated transduction. Ultimately, I isolated two cell lines, 31-2 and 67-1, which we characterized further. 31-2 was determined to be least five fold resistant to both MLV- and lenti-viral at a stage in viral replication post reverse transcription. 67-1 was minimally ten fold resistant to both MLV- and lenti-viral infection at a stage between viral entry and uncoating. We demonstrated that the mutation in 67-1 implicated the proteasome and identified a gene we designated MRI that reversed the inhibition of infection in the mutated 67-1 line. The implication of MRI in the retroviral lifecycle identifies a new protein that could facilitate the creation of novel host cell based therapy for HIV.