Proteomic Organellar Mapping Reveals Modulation of Peroxisomes by HIV-1

Abstract

As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new, unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses we found that classifier performance in this system was organelle-dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine (SVM) learning for mitochondrial and ER proteins, but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, BANDLE, on SVM-classified data. Comparative BANDLE analysis of cells induced to express the wild-type viral genome vs. cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as TCR signaling components and coatomer complex. Lastly, we found that SVM classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.