| Viruses are obligate intracellular parasites that rely on cellular molecules in order to enter and replicate in cells. Evidence continues to accumulate suggesting that viruses actively promote their replication through manipulation of signal transduction pathways, i.e. reprogramming the target cell. However, we know very little of the extent to which viruses reprogram cells during entry and what consequence this may have on virus replication. Since cellular signaling is an attractive target for drug development, monitoring virus-induced signaling may also provide novel antiviral targets. Human immunodeficiency virus-1 (HIV-1), the causative agent of AIDS, enters target cells by specifically engaging a primary receptor, CD4, and then either CCR5 or CXCR4. In addition to mediating fusion with the host cell, engagement of these receptors activates signal transduction pathways that can reprogram the cell for optimal viral replication. The extent to which HIV-1 reprograms the cell during entry is unknown and expanding our knowledge of HIV-dependent signaling may provide additional therapeutic options. We took advantage of mass spectrometry (MS)-based quantitative phosphoproteomics technology, which has emerged as an invaluable tool for examining novel cellular signaling events, in primary cells to examine phosphorylation-dependent cellular reprogramming by HIV-1. We initiated these studies by examining signaling induced by CXCL12, the natural ligand for CXCR4, one of the coreceptors for HIV-1in a cell line. We next measured signaling events induced by HIV-1 particles in primary CD4 T cells. Extensive bioinformatic and biochemical validation show that MS-derived phosphosite changes likely reflect actual CXCL12- and HIV-responsive phosphosites. Functional experiments employing RNAi also show that many CXCL12- and HIV-responsive phosphoproteins regulate HIV-1 replication in an entry dependent manner. Detailed mechanistic experiments with one HIV-responsive phosphosprotein, SRm300, show that this protein regulates HIV-1 gene expression and splicing in multiple cell types, suggesting that HIV-1 may reprogram the cellular splicing machinery in order to facilitate efficient replication. Since many pathogens manipulate host cellular signaling during infection, these studies provide important proof of principle that phosphoproteomics is a viable screening technology for uncovering not only novel viral, but also pathogen host factors. |
