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CRISPR-Cas9 Analysis of the HIV-host Interaction Network

出版	McGill University Libraries, 2019
URL	http://books.google.com.hk/books?id=EEAyzgEACAAJ&hl=&source=gbs_api

註釋"As an obligate intracellular pathogen, the human immunodeficiency virus type 1 (HIV-1) relies on host cellular components for replication. The virus has developed an arsenal of methods to productively reproduce within cells while evading host defenses, such as hijacking the cellular membrane trafficking pathway. This pathway facilitates the transport and delivery of molecular cargo within intracellular membrane-bound vesicles and is exploited by HIV-1 for its own intracellular trafficking purposes; however, the cellular players involved in this process remain to be elucidated. To investigate the interactions between HIV-1 and members of the intracellular trafficking circuits, we employed a sophisticated CRISPR-Cas9 arrayed genetic screen causing loss-of-function in 140 genes of the membrane trafficking family. The screen was performed in the HIV-1 reporter cell line, TZM-bl, which was modified to stably express the Cas9 endonuclease. Our screen focused on host factors that significantly alter HIV-1 infectivity, measured by Tat-dependent LTR-driven luciferase activity, and virion production, quantified by p24 capsid protein present in the cell-free supernatant. Of the 140 queried genes, we have uncovered 10 candidate genes that significantly inhibited HIV-1 replication compared to the non-targeting control (selected threshold e"2-fold decrease in viral infectivity or virus production) and proved to be viable knockouts. Protein interaction studies indicate that many of our hits are enriched in endocytosis. To further our inquiry into these candidate hits, we are generating stable CRISPR-knockout cell lines in a T-cell model, SUP-T1, and using protein expression studies and microscopic visualization techniques to investigate the underlying molecular mechanisms exacerbating these phenotypes. Deciphering the relationships between HIV-1 and the membrane trafficking pathway using a revolutionary CRISPR-based genetic screening approach will not only improve our current understanding of viral pathogenesis, but may also translate into novel antiviral targets towards an HIV-1 cure"--