Systems Analysis of Protein Fatty Acylation in Herpes Simplex Virus-Infected Cells Using Chemical Proteomics

Abstract

Protein fatty acylation regulates diverse aspects of cellular function and organization and plays a key role in host immune responses to infection. Acylation also modulates the function and localization of virus-encoded proteins. Here, we employ chemical proteomics tools, bio-orthogonal probes, and capture reagents to study myristoylation and palmitoylation during infection with herpes simplex virus (HSV). Using in-gel fluorescence imaging and quantitative mass spectrometry, we demonstrate a generalized reduction in myristoylation of host proteins, whereas palmitoylation of host proteins, including regulators of interferon and tetraspanin family proteins, was selectively repressed. Furthermore, we found that a significant fraction of the viral proteome undergoes palmitoylation; we identified a number of virus membrane glycoproteins, structural proteins, and kinases. Taken together, our results provide broad oversight of protein acylation during HSV infection, a roadmap for similar analysis in other systems, and a resource with which to pursue specific analysis of systems and functions.

Graphical abstract

Figure 1

Enrichment Strategy Chemoproteomic Workflow (A), Structures of Lipid Probes (B), and Multi-functional Capture Reagent AzTB (C). Inside the cell and the test tube, colored shapes represent proteins, the triple bar represents alkyne; on the capture reagent (AzTB), red N₃ represents azide, the pink star represents TAMRA fluorophore, and the blue double pentagon represents biotin.

Figure 2

In-Gel Fluorescent Analysis of Protein Fatty Acylation During HSV Infection in RPE-1 Cells Cells were pulsed with YnPal or YnMyr (25 μM) for 6 hr and harvested at various times as shown. Proteins were isolated, processed for coupling to capture reagent AzTB, and analyzed for palmitoylation (A) or myristoylation (B). Arrows point to bands of increased fluorescence intensities (proteins with increased acylation levels) and arrowheads point to bands with decreased fluorescence intensities (proteins with decreased acylation levels).

Figure 3

Quantitative Proteomics Analysis of Host Protein Fatty Acylation During HSV Infection in RPE-1 Cells (A) SILAC-based quantitative proteomics workflow. (B) Virus-induced changes to protein palmitoylation (n = 4) plotted against statistical significance of the ratio measured. (C) Virus-induced changes to protein myristoylation (n = 4) plotted against statistical significance. Black, proteins with myristoylation requirement (N-terminal Gly); red, validated NMT substrates (Broncel et al., 2015; Thinon et al., 2014). In (B) and (C) each data point represents a protein or a protein group (Cox et al., 2011).

Figure 4

Infection-Induced Changes to Nascent Protein Abundances (A) SILAC-based proteomic workflow. (B) Experimental results from samples prior to affinity enrichment: Log2 nascent protein abundance ratio HSV/Mock (n = 3) plotted against significance of the change measured. Red, proteins with myristoylation requirement (N-terminal Gly); black, all other proteins quantified. Each data point represents a protein or a protein group (Cox et al., 2011).

Figure 5

SILAC-Based Identification of Fatty Acylated HSV-Encoded Proteins (A) SILAC-based experimental workflow. (B) Heatmap for YnPal/Pal (25 μM, 5–19 hpi) enrichment (n = 3) of proteins derived from HSV-1[17], HSV-1[KOS], and HSV-2[186]. Black to red gradation represents the degree (n = 3) of enrichment of proteins labeled with YnPal over Pal, expressed as a heatmap. Gray bars represent missing values (applied when quantification was missing for more than one out of three samples).