Viral pathogenesis results from changes in host cells due to virus usurpation of the host cell and the innate cellular responses to thwart infection. We measured global changes in protein expression and localization in HIV-1 infected T-cells using subcellular fractionation and the Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) proteomic platform. Eight biological replicates were performed in two independent experimental series. In silico merging of both experiments identified 287 proteins with altered expression (p < .05) between control and infected cells- 172 in the cytoplasm, 84 in the membrane, and 31 in nuclei. 170 of the proteins are components of the NIH HIV interaction database. Multiple Reaction Monitoring and traditional immunoblotting validated the altered expression of several factors during infection. Numerous factors were found to affect HIV infection in gain- and loss-of-expression infection assays, including the intermediate filament vimentin which was found to be required for efficient infection.
The accelerated development of technology over the last three decades has driven biological sciences to high-throughput profiling experiments, now broadly referred to as systems biology. The unprecedented improvement of analytical instrumentation has opened new avenues for more complex experimental designs and expands the knowledge in genomics, proteomics, and other omics fields. Despite the collective efforts of hundreds of researchers, gleaning all the expected information from omics experiments is still quite far. This paper summarizes what has been learned from high-throughput proteomics studies thus far, and what is believed should be done to reveal even more valuable information from such studies. It is drawn from the background in using proteomics to study human immunodeficiency virus 1 infection of macrophages and/or T cells, but it is believed that some conclusions will be more broadly applicable.
Macrophages comprise a major component of the human innate immune system that is involved in maintaining homeostasis and responding to infections or other insults. Besides cytokines and chemokines, macrophages presumably influence the surrounding environment by secreting various types of metabolites. Characterization of secreted metabolites under normal and pathological conditions is critical for understanding the complex innate immune system. To investigate the secreted metabolome, we developed a novel workflow consisting of one Reverse Phase (RP) C18 column linked in tandem with a Cogent cholesterol modified RP C18. This system was used to compare the secreted metabolomes of human monocyte derived macrophages (hMDM) under normal conditions to those exposed to methamphetamine (Meth). This new experimental approach allowed us to measure 92 metabolites, identify 11 of them as differentially expressed, separate and identify three hydroxymethamphetamine (OHMA) isomers, and identify a new, yet unknown metabolite with m/z of 192. This study is the first of its kind to address the secreted metabolomic response of hMDM to an insult by Meth. Besides the discovery of novel metabolites secreted by macrophages, we provide a novel methodology to investigate metabolomic profiling.
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