Affiliation:
1. Department of Chemistry, University of Wisconsin, Madison, United States
2. McArdle Laboratory for Cancer Research and Institute for Molecular Virology, University of Wisconsin, Madison, United States
Abstract
HIV-1 generates unspliced (US), partially spliced (PS), and completely spliced (CS) classes of RNAs, each playing distinct roles in viral replication. Elucidating their host protein ‘interactomes’ is crucial to understanding virus-host interplay. Here, we present HyPR-MSSV for isolation of US, PS, and CS transcripts from a single population of infected CD4+ T-cells and mass spectrometric identification of their in vivo protein interactomes. Analysis revealed 212 proteins differentially associated with the unique RNA classes, including preferential association of regulators of RNA stability with US and PS transcripts and, unexpectedly, mitochondria-linked proteins with US transcripts. Remarkably, >80 of these factors screened by siRNA knockdown impacted HIV-1 gene expression. Fluorescence microscopy confirmed several to co-localize with HIV-1 US RNA and exhibit changes in abundance and/or localization over the course of infection. This study validates HyPR-MSSV for discovery of viral splice variant protein interactomes and provides an unprecedented resource of factors and pathways likely important to HIV-1 replication.
Funder
National Institutes of Health
National Science Foundation
University of Wisconsin-Madison
Greater Milwaukee Foundation
Office of the Vice Chancellor for Research and Graduate Education, University of Wisconsin-Madison
Publisher
eLife Sciences Publications, Ltd
Subject
General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience
Cited by
17 articles.
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