Autophagy regulation during HIV infection

National Institute Of Environmental Health Sciences

Investigators

Abstract

During the fiscal year, we built the experimental foundation to interrogate Nef–PI3K regulation of autophagy and progressed from resource setup to cellular readouts. In particular, we obtained key plasmids, purified Nef, validated its interaction with PI3K subunits, positioning the complexes for structural work. Additionally, we created knock-in cell lines to perform endogenous-level immunoprecipitation experiments. Unbiased lipidomics of Nef-expressing cells revealed significant phospholipid perturbations, consistent with altered vesicle trafficking and autophagy flux. We also generated stable Nef-positive cell lines to support correlative imaging and functional assays. Research tools were human cell lines (HeLa for autophagy modeling; U937 and Jurkat as T-cell models). Methods and significant materials included cryo-EM single-particle analysis, correlative light and electron microscopy (CLEM), cryo-focused ion beam–scanning EM (cryo-FIB-SEM), co-immunoprecipitation, autophagy-flux assays, fluorescence/confocal microscopy, and lipidomics; these were executed using our in-house cryo-EM facility and institutional mass spectrometry support. Collectively, these results confirm Nef-linked remodeling of membrane lipid homeostasis, establish tractable cell systems for structure–function mapping, and de-risk the next phase aimed at high-resolution structures and targeted functional tests.