Quantitative Temporal Viromics of an Inducible HIV-1 Model Yields Insight to Global Host Targets and Phospho-Dynamics Associated with Protein Vpr

Abstract

The mechanisms by which human immunodeficiency virus (HIV) circumvents and coopts cellular machinery to replicate and persist in cells are not fully understood. HIV accessory proteins play key roles in the HIV life cycle by altering host pathways that are often dependent on post-translational modifications (PTMs). Thus, the identification of HIV accessory protein host targets and their PTM status is critical to fully understand how HIV invades, avoids detection and replicates to spread infection. To date, a comprehensive characterization of HIV accessory protein host targets and modulation of their PTM status does not exist. The significant gap in knowledge regarding the identity and PTMs of HIV host targets is due, in part, to technological limitations. Here, we applied current mass spectrometry techniques to define mechanisms of viral protein action by identifying host proteins whose abundance is affected by the accessory protein Vpr and the corresponding modulation of down-stream signaling pathways, specifically those regulated by phosphorylation. By utilizing a novel, inducible HIV-1 CD4+ T-cell model system expressing either the wild type or a vpr-negative viral genome, we overcame challenges associated with synchronization and infection-levels present in other models. We report identification and abundance dynamics of over 7000 proteins and 28,000 phospho-peptides. Consistent with Vpr's ability to impair cell-cycle progression, we observed Vpr-mediated modulation of spindle and centromere proteins, as well as Aurora kinase A and cyclin-dependent kinase 4 (CDK4). Unexpectedly, we observed evidence of Vpr-mediated modulation of the activity of serine/arginine-rich protein-specific kinases (SRPKs), suggesting a possible role for Vpr in the regulation of RNA splicing. This study presents a new experimental system and provides a data-resource that lays the foundation for validating host proteins and phosphorylation-pathways affected by HIV-1 and its accessory protein Vpr.

Fig. 1.

- Characterization of the Jurkat WT and ΔVPR cell lines following induction of HIV with doxycycline treatment. A, Time course of HIV-1 Gag/p24 expression in WT and ΔVPR cells. Conditions are color coded as above for 0, 3, 6, 12, and 24 h postdoxycycline induction of HIV expression. Histograms show FITC (intracellular Gag/p24) intensity for live gated WT (left) and ΔVPR (left) cells. B, The cell cycle block (or not) in these cells. WT (left) and ΔVPR (right) cells were labeled with PI and gated on live single cells. Histograms show PI-labeled DNA content with the lower peak representing cells in G0/G1 and the higher peak representing cells at G2/M phase.

Fig. 2.

- Quantitative Temporal Viromics. A, Wild-type and vpr-deleted HIV expressing cell lines were analyzed in biological duplicate. Cells were lysed and digested prior to labeling with TMT reagents for quantitative proteomic and phospho-proteomic profiling. Proteins are identified at the MS2 level and quantified at the MS3 level. B, Temporal protein profiles show a time-dependent increase in detected viral proteins. This increase is reproducible between wild-type (WT) and vpr-deleted cell lines (ΔVPR). C, Venn Diagram showing overlap of proteins identified and quantified between WT (green) and ΔVPR cell lines (blue). The 5793 proteins in common were used for subsequent analyses.

Fig. 3.

- Proteomic Profiling of Wild-Type HIV expressing cells reveals enriched clusters of protein expression. A, Temporal protein profiles recapitulate findings from other CD4+ T-cell models. B, K-means clustering of proteins allows Gene Ontology enrichment analysis of profiles of enriched clusters of protein functions. Average cluster profiles are depicted beside and on top of the gene and GO based heat maps respectively. Heat map of proteins is relative z-score per row with black representing minimum and red maximum. Heat map of Gene ontology analysis reflects significant enrichment factors and is on a global scale. Black is not present, yellow low enrichment and red high enrichment. Terms on the right of the plot are colored dependent upon the colored bar on the left of the protein heat map corresponding to the adjacent k-means cluster.

Fig. 4.

- Proteomic Profiling of ΔVPR cells reveals potential host targets of Vpr. A, K-means clustering of proteins allows Gene ontology enrichment analysis of profiles of enriched clusters of protein functions. Average cluster profiles are depicted beside and on top of the gene and GO based heat maps respectively. Heat map of proteins is relative z-score per row with black representing minimum and red maximum. Heat map of Gene ontology analysis reflects significant enrichment factors and is on a global scale. Black is not present, yellow low enrichment and red high enrichment. Terms on the right of the plot are colored dependent upon the colored bar on the left of the protein heat map corresponding to the adjacent k-means cluster. B, Volcano plot of vpr-deletion (ΔVPR) relative to wild-type (WT) protein level changes at 24 h post viral induction. X-axis shows log2 fold change and y axis (-)Log of Student's t test based p value. Yellow dots are significantly enriched proteins (p < 0.05) and red dots are significantly enriched and greater than two standard deviations from the median. C, STRING-db analysis of proteins significantly enriched (red dots) in ΔVPR relative to WT HIV. Red nodes correlate to red dots, and ubiquitin is highlighted in yellow.

Fig. 5.

- Validation of quantitative mass spectrometry findings via Western blotting. A, Western blotting analysis of HIV and host proteins in WT and ΔVPR Jurkat tetHIV inducible cell lines. B, Validation of tetHIV model using a SupT1 infection model. Similar protein expression patterns are observed, but there is a lag time from the infection process which required a later time point (48 h). C, Average mass spectrometry derived values for blots in A and B. Vpr was not detected in our proteomics experiments or the CEM-T4 experiments performed by Greenwood et al. Values are shown relative to the zero-time point, which was normalized to 1.

Fig. 6.

- Phospho-proteomic profiling of ΔVPR cells reveals potential host targets of Vpr. A, Venn Diagram showing overlap of phospho-peptides identified and quantified between wild-type (green) and ΔVPR cell lines (blue). The 6,304 phospho-peptides in common were used for subsequent analyses. B, Volcano plot of ΔVPR relative to wild-type (WT) phospho-peptide level changes at 3 h post viral induction. X-axis shows log2 fold change and y axis (-)Log of Student's t test based p value. Yellow dots are significantly enriched proteins (p < 0.05) and blue and red dots are significantly enriched and greater than two standard deviations from the median up-(blue) or down-regulated (red) relative to ΔVPR. C, Volcano plot of ΔVPR relative to WT phospho-peptide level changes at 24 h post viral induction. D, STRING-db analysis of phospho-proteins significantly enriched (red and blue dots) in ΔVPR relative to WT HIV at 3 h post induction. E, STRING-db analysis of phospho-proteins significantly enriched (red and blue dots) in ΔVPR relative to WT HIV at 24 h post induction. Ubiquitin is highlighted in yellow in STRING plots.

Fig. 7.

- Phospho-proteomics reveals Vpr-modulated kinase motifs. A, Comparison of Inducible HIV with data from the Wojcechowskyj et al. data set shows a 1081 protein overlap for proteins with identified phosphorylation sites. B, Within the 1081 proteins, 1477 phospho-sites were identified as being the same between the two data sets. Previously annotated motifs show an enrichment for cell cycle associated domains. C, Motif analysis of phospho-peptides shows differential WT-leaning motifs (Higher in WT than ΔVPR) and ΔVPR-Leaning motifs (Higher in ΔVPR than WT). Full motif analysis can be found in supplemental Figs. S9–S14 and supplemental File S13.