Dataset Information

Comparative Analysis of T Cell Spatial Proteomics and the Influence of HIV Expression

ABSTRACT: As systems biology approaches to virology have become more tractable, it has become possible to analyze highly studied viruses such as HIV in new, unbiased ways, including spatial proteomics. We have employed here a differential centrifugation protocol to fractionate an inducible model of HIV-expression in Jurkat T cells for proteomic analysis by mass spectrometry. 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 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 WT and ΔNef models also identified known Nef-dependent interactors such as TCR signaling and coatomer complex. Lastly, we found that SVM classification showed higher consistency and was less sensitive to HIV-dependent noise in our data. These findings illustrate important considerations for future studies of the spatial proteome following viral infection or expression where their generalizability can be further assessed.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Alicia Richards

LAB HEAD: Dr. Nevan Krogan

PROVIDER: PXD029956 | Pride | 2022-01-16

REPOSITORIES: Pride

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Publications

Comparative Analysis of T-Cell Spatial Proteomics and the Influence of HIV Expression.

Oom Aaron L AL Stoneham Charlotte A CA Lewinski Mary K MK Richards Alicia A Wozniak Jacob M JM Shams-Ud-Doha Km K Gonzalez David J DJ Krogan Nevan J NJ Guatelli John J

Molecular & cellular proteomics : MCP 20220108 3

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 s ...[more]

PMID: 35017099

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Project description:Antiretroviral therapy (ART) has the potency of suppressing systemic viral replication and spread. Still, persistent human immunodeficiency virus type-1 (HIV) survive for decades during treatment in latently infected cells, making up the latent viral reservoir. This reservoir is believed to reside in subtypes of memory T cells and cell from monocytic lineages, but some functional and naïve T cells have also shown to carry latent HIV. Eradication of latently infected cells, in terms of a sterilizing cure, have proven to be a hurdle since there is a heterogeneity in mechanisms governing latency and integration site into the genome. Over the course of suppressive therapy, persisting HIV, sustained by low levels of viral replication, homeostatic proliferation, and cell to cell transmission, is a driver of chronic immune activation in the body. For the immune system to elicit an appropriate inflammatory response it is dependent on secretion of inflammatory molecules, such as chemokines and cytokines. Most chemokines are pro-inflammatory factors that facilitate leukocyte recruitment to site of inflammation where they can exert their regulatory role in response to pathological and physiological stressors e.g., infection, inflammation, and tissue damage. Alterations in chemokine receptor expression and chemokine levels modulates the activity of the immune response and in HIV infection contribute to chronic inflammation and mortality. Therefore, people living with HIV on suppressive therapy (PLWH) are at higher risk of age associated co-morbidities due to elevated immune activation and chronic inflammation induced by ART toxicities, microbiome dysbiosis leading to microbial translocation and dysregulated immune cell activation and function. As a result, the causative HIV pathogenesis and immune dysfunction further chronic immune activation. This result in a vicious cycle as immune activation is driver of HIV disease progression and inflammaeging leading to earlier onset of age-related diseases. An alternative approach in cure strategies is a functional cure for HIV aiming at suppressing viral replication without ART. A model for functional cure studies are elite controllers (EC). EC constitute a small fraction of HIV+ individuals (<0.5%) exhibiting the unique characteristic of natural control of viral replication in absence of ART. However, there is a population-based heterogeneity of how these individuals naturally supress viral replication. This heterogeneity is caused by viral genetic factors, variability of integration site in the human genome, together with host response against the pathogen and immunological factors. In terms of immune cell activation, studies have shown how EC maintain lower levels of inflammatory markers compared to PLWH. Contradictory, some studies have shown elevated inflammatory markers in EC which could be attributed to the heterogeneity of the EC group or as others have shown it is a consequence of loss of spontaneous control of HIV. Therefore, as no consensus exist further studies comparing variability of immune function between EC and ART can aid in understanding the mechanisms of natural control of HIV. In our recent study (Sperk et al 2021, iScience) we hypothesised that modulated CCR6/CCL20 chemokine axis and CCR2-CCL7-CCL2 signalling can play a protective role in the EC phenotype. Downregulation of CCR2 and CCR6 receptors in lymphocytes and higher plasma abundance of CCL4, CCL7 and CCL20 in EC compared to the HIV-negative controls can provide natural resistance to HIV-infection. In the present study, we further extended our analysis to evaluate the expression profile dynamics of key chemokine receptors, CCR2, CCR3, CCR5 and CCR6, in successfully treated PLWH. We compared well treated PLWH with HIV-1 elite controllers and its association with HIV-1 persistence. Furthermore, cell populations of interest were isolated for liquid chromatography mass spectrometry (LC-MS/MS) based untargeted proteomics analysis to evaluate specific characteristics regulating these cell populations and their role in HIV persistence. Our study provides important understanding of the chemokine receptor dynamics and its role in HIV persistence that differentiate elite controllers from long term treated PLWH.

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Project description:HIV-related fatigue is multi-causal in origin and potentially related to mitochondrial dysfunction caused by toxicity from nucleoside reverse transcriptase inhibitor (NRTI) antiretroviral therapy. CD14+ cells are undifferentiated macrophages, vulnerable to HIV infection, and easily accessible for gene expression experiments in a purified cell population. We utilized a novel mitochondrially-specific gene expression microarray to assess mitochondrial and nuclear genes in CD14+ cells of low- and high-fatigued, NRTI-treated HIV/AIDS patients (n=5 each). Novel Bayesian and liquid association network methods identified 33 genes predictive of low versus high fatigue and 32 genes predictive of healthy versus HIV infection. Sulfotransferase 2B1 (SULT2B1) is relevant to both the cholesterol and testosterone pathway, and like several inner mitochondrial membrane genes also identified, predictive of fatigue status, while outer mitochondrial membrane genes were predictive of HIV status. A surprising finding was that adenylate cyclase 2 (ADCY2) was a predictor of both HIV and fatigue; it had the highest Kendallâs Tau association value in the HIV group, but in reverse, the lowest Tau value in the fatigue group. Assaying CD14+ cells may provide an alternative to muscle biopsy and a minimally invasive procedure to evaluate patient mitochondrial function, and Bayesian and network tools are useful to identify the link between subjective symptom perceptions and underlying biologically pathways. Fatigue status in HIV patients treated with NRTIs was found to be linked to RNA expression differences related to mitochondrial function. Additional studies are needed to confirm the relevance of our findings in CD14+ cells in other tissues (e. g. skeletal muscle) and to understand the significance of key genes such as SULT2B and ADCY2 in fatigue and HIV disease. The design was a comparison of HIV high fatigue to HIV low fatigue. Low fatigue was determined as 3-7 on the Revised Piper Fatigue Score and High fatigue was a score of 7 or greater. Five HIV negative control samples were used to compare normal fatigue and non-disease status.

Dataset Information

Comparative Analysis of T Cell Spatial Proteomics and the Influence of HIV Expression

Publications

Comparative Analysis of T-Cell Spatial Proteomics and the Influence of HIV Expression.

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