Proteome-wide characterization of PTMs reveals host cell responses to viral infection and identifies putative antiviral drug targets

Abstract

Post-translational modifications (PTMs) are biochemical modifications that can significantly alter protein structure, function, stability, localization, and interactions with other molecules, thereby activating or inactivating intracellular processes. A growing body of research has begun to highlight the role of PTMs, including phosphorylation, ubiquitination, acetylation, and redox modifications, during virus-host interactions. Collectively, these PTMs regulate key steps in mounting the host immune response and control critical host pathways required for productive viral replication. This has led to the conception of antiviral therapeutics that focus on controlling host protein PTMs, potentially offering pathogen-agnostic treatment options and revolutionizing our capacity to prevent virus transmission. On the other hand, viruses can hijack the host cellular PTM machinery to modify viral proteins in promoting viral replication and evading immune surveillance. PTM regulation during virus-host interactions is complex and poorly mapped, and the development of effective PTM-targeted antiviral drugs will require a more comprehensive understanding of the cellular pathways essential for virus replication. In this review, we discuss the roles of PTMs in virus infection and how technological advances in mass spectrometry-based proteomics can capture systems-level PTM changes during viral infection. Additionally, we explore how such knowledge is leveraged to identify PTM-targeted candidates for developing antiviral drugs. Looking ahead, studies focusing on the discovery and functional elucidation of PTMs, either on the host or viral proteins, will not only deepen our understanding of molecular pathology but also pave the way for developing better drugs to fight emerging viruses.

Keywords: PTMs; acetylation; antiviral drug; phosphorylation; proteome; redox; ubiquitination; viral infection.

Figure 1

Protein kinases and phosphorylation in mediating host-virus interaction. (a) Control of translation initiation via the regulation of PKR activity; (b) CK2 phosphorylates multiple host cell substrates (e.g., CMTR1) and viral proteins (e.g., N) and is a drug target to control viral infection. (c) Viral infection actives NF-κB signaling via phosphorylation. APOPE3, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3; CK2, casein kinase II; CMTR1, cap methyltransferase 1; dsRNA, double-stranded RNA; eIF2α, eukaryotic translation initiation factor 2; IκB, inhibitor of kappa B; IKK, IκB kinase; N, viral nucleocapsid protein; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PKR, dsRNA-dependent protein kinase. Green circle with white letter P denotes phosphorylation. This and all the following figures were created using BioRender (https://www.biorender.com/).

Figure 2

Ubiquitination and acetylation. (a) Ubiquitination of RIG-I and MAVS activates innate immunity. (b) Acetylation of cGAS promotes its binding with viral DNA and stimulates the expression of ISRE genes under viral infection (e.g., herpes simplex virus 1); (c) Acetylation of viral NP by different host cell acetyltransferases at distinct sites may lead to increase or decrease in viral polymerase activity. cGAS, mitochondrial antiviral-signaling protein; CPB, cAMP-response element–binding protein; GCN5, acetyltransferases general control non-repressed 5 protein; ISRE, interferon-stimulated response element; KAT5, lysine acetyltransferase 5; MAVS, mitochondrial antiviral-signaling protein; NP, non-structural protein; PCAF, P300/CBP-associated factor. RIG-I, retinoic acid-inducible gene I; RNF115, RING finger protein 115. Orange circle with black letter U denotes ubiquitination. Purple circle with white letter A denotes acetylation.

Figure 3

Virus-induced ROS generation and redox modifications. (a) Virus interacts with mitochondria and NOX to generate cellular ROS; (b) Oxidative folding of Influenza virus A viral HA protein by host PDIs. (c) Reversible glutathionylation of M^pro. GRX, glutaredoxin; HA, hemagglutinin; M^pro, main protease; NOX, NADPH oxidase; PDI, protein disulfide isomerase; PHB1, mitochondria-bound prohibitin 1; ROS, reactive oxygen species; -S-S-, disulfide bond; VDAC3, voltage-dependent anion channel. 3D structures were obtained from the protein data bank (PDB) with the following accession numbers: 3AL4 for HA monomer, 3LZG for HA trimer, and 2BX4 for M^Pro. Red circle denotes glutathionylation.

Figure 4

MS-based proteomics approaches for studying PTMs. (a) A general workflow for PTM proteomics consisting of cell/tissue lysis, protein extraction and digestion, optional labeling, enrichment, and LC-MS/MS analysis. (b) Enrichment strategies for phosphorylation (IMAC), ubiquitination (affinity enrichment via K-GG antibody), and reversible thiol oxidation (thiol affinity enrichment). (c) Quantification approaches in PTM proteomics including isobaric labeling (TMT), SILAC, and label-free methods. DIA, data independent acquisition; DTT, dithiothreitol; IMAC, immobilized metal ion affinity chromatography; MS1, mass spectra of peptide precursors; MS2, mass spectra of peptide fragments; TMT, tandem mass tags. * in panel b denotes TMT labeling is optional.

Figure 5

Use PTMomics to understand host responses to viral infection and to develop novel antiviral therapies. (a) A typical experimental setup and data analysis pipeline for PTMomics studies. (b) Common cellular pathways that respond to viral infection. (c) Novel antiviral drugs that target protein PTMs. CK2, casein kinase II; HDAC9, histone deacetylase 9; GRK2, G protein-coupled receptor kinase 2; GSK3, glycogen synthase kinase-3; PLK3, polo-like kinase 3; SIRT2, sirtuin 2; USP13, ubiquitin-specific protease 13.