A Genome-Wide Screen for Machinery Involved in Downregulation of MHC Class I by HIV-1 Nef

• Published in: PloS one Vol. 10; no. 10; p. e0140404
• Main Authors: Choma, Maja K, Lumb, Jennifer, Kozik, Patrycja, Robinson, Margaret S
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 14-10-2015; Public Library of Science (PLoS)
• Subjects: Acquired immune deficiency syndrome; AIDS; Blood banks; Carrier Proteins - genetics; Carrier Proteins - metabolism; Clathrin; Cloning; Dopamine Plasma Membrane Transport Proteins - genetics; Dopamine Plasma Membrane Transport Proteins - metabolism; Down-Regulation; Genes; Genetic aspects; Genetic screening; Genome-Wide Association Study; Genomes; Genotype & phenotype; HeLa Cells; Histocompatibility antigen HLA; HIV; HIV infections; HLA-A2 Antigen - genetics; HLA-A2 Antigen - metabolism; Human immunodeficiency virus; Humans; Infections; Intracellular Signaling Peptides and Proteins; Major histocompatibility complex; Medical research; Medical screening; Microtubule-Associated Proteins - genetics; Microtubule-Associated Proteins - metabolism; nef Gene Products, Human Immunodeficiency Virus - genetics; nef Gene Products, Human Immunodeficiency Virus - metabolism; Nef protein; Pathogenesis; Phenotypes; Physiological aspects; Pools; Potassium Channels - genetics; Potassium Channels - metabolism; Proteins; Reagents; siRNA; Transcription factors; Viruses; United Kingdom; Singapore; United Kingdom > UK; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0140404

The HIV-1-encoded protein, Nef, plays a key role in the development of AIDS. One of Nef's functions is to keep MHC class I off the surface of infected cells, a process that requires the host proteins clathrin and AP-1. To identify other proteins involved in this pathway, we carried out a genome-wide siRNA library screen on HeLa cells co-expressing HLA-A2 and an inducible form of Nef. Out of 21,121 siRNA pools, 100 were selected for further analysis, based on their ability to either inhibit or enhance downregulation of MHC-I by Nef. When cells were treated with the same siRNA pools as those used in the screen, 79% produced a similar phenotype. However, when the cells were treated with different siRNA reagents targeting the same genes, only 16% produced a similar phenotype. This indicates that most of the hits found in the original screen are likely to have been off-target, an important concern that is often not taken into account in siRNA screening studies. Nevertheless, we identified novel host factors involved in Nef-induced downregulation of MHC-I, including four genes, MIIP, CAMSAP3, SLC6A3, and KCTD19, where multiple reagents produced a strong inhibitory effect on Nef activity. Other hits slightly below our very high stringency cutoff point may also deserve further study. Thus, our dataset is a valuable resource for scientists investigating the pathogenesis of HIV.