Proteomic understanding of SARS-CoV-2 infection and COVID-19: Biological, diagnostic, and therapeutic perspectives

The research community responded rapidly to the SARS-CoV-2 pandemic with a burst of proteomic studies to understand this new virus. We focus in this chapter on proteomic approaches and advances in the areas of the viral proteome and the viral-host protein-protein interactions exploited to facilitate the pathogenic life cycle of SARS-CoV2. We also outline the proteome and posttranslational protein modifications of infected cells and blood proteomics of COVID-19 patients in relation to pathogenesis, host response, and disease severity. Finally, tools and application for COVID-19 proteomics and implementations in diagnostics and therapies are surveyed before summarizing the present achievements and future perspectives. © 2023 Elsevier Inc. All rights reserved.

SARS-CoV-2 Papain-Like Protease: Structure, Function and Inhibition.

Emerging variants of SARS-CoV-2 and potential novel epidemic coronaviruses underline the importance of investigating various viral proteins as potential drug targets. The papain-like protease of coronaviruses has been less explored than other viral proteins; however, its substantive role in viral replication and impact on the host immune response make it a suitable target to study. This review article focuses on the structure and function of the papain-like protease (PLpro ) of SARS-CoV-2, including variants of concern, and compares it to those of other coronaviruses, such as SARS-CoV-1 and MERS-CoV. The protease's recognition motif is mirrored in ubiquitin and ISG15, which are involved in the antiviral immune response. Inhibitors, including GRL0617 derivatives, and their prospects as potential future antiviral agents are also discussed.

Protein Posttranslational Signatures Identified in COVID-19 Patient Plasma.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly contagious virus of the coronavirus family that causes coronavirus disease-19 (COVID-19) in humans and a number of animal species. COVID-19 has rapidly propagated in the world in the past 2 years, causing a global pandemic. Here, we performed proteomic analysis of plasma samples from COVID-19 patients compared to healthy control donors in an exploratory study to gain insights into protein-level changes in the patients caused by SARS-CoV-2 infection and to identify potential proteomic and posttranslational signatures of this disease. Our results suggest a global change in protein processing and regulation that occurs in response to SARS-CoV-2, and the existence of a posttranslational COVID-19 signature that includes an elevation in threonine phosphorylation, a change in glycosylation, and a decrease in arginylation, an emerging posttranslational modification not previously implicated in infectious disease. This study provides a resource for COVID-19 researchers and, longer term, and will inform our understanding of this disease and its treatment.

Variable posttranslational modifications of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), started in 2019 in China and quickly spread into a global pandemic. Nucleocapsid protein (N protein) is highly conserved and is the most abundant protein in coronaviruses and is thus a potential target for both vaccine and point-of-care diagnostics. N Protein has been suggested in the literature as having posttranslational modifications (PTMs), and accurately defining these PTMs is critical for its potential use in medicine. Reports of phosphorylation of N protein have failed to provide detailed site-specific information. We have performed comprehensive glycomics, glycoproteomics and proteomics experiments on two different N protein preparations. Both were expressed in HEK293 cells; one was in-house expressed and purified without a signal peptide (SP) sequence, and the other was commercially produced with a SP channeling it through the secretory pathway. Our results show completely different PTMs on the two N protein preparations. The commercial product contained extensive N- and O-linked glycosylation as well as O-phosphorylation on site Thr393. Conversely, the native N Protein model had O-phosphorylation at Ser176 and no glycosylation, highlighting the importance of knowing the provenance of any commercial protein to be used for scientific or clinical studies. Recent studies have indicated that N protein can serve as an important diagnostic marker for COVID-19 and as a major immunogen by priming protective immune responses. Thus, detailed structural characterization of N protein may provide useful insights for understanding the roles of PTMs on viral pathogenesis, vaccine design and development of point-of-care diagnostics.

Simultaneous enrichment and separation of neutral and sialyl glycopeptides of SARS-CoV-2 spike protein enabled by dual-functionalized Ti-IMAC material.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a serious threat to human health all over the world. The development of effective vaccines has been focusing on the spike (S) glycoprotein, which mediates viral invasion to human cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor. In this work, we perform analytical characterization of N- and O-linked glycosylation of the SARS-CoV-2 S glycoprotein. We explore the novel use of dual-functionalized titanium (IV)-immobilized metal affinity chromatography (Ti-IMAC) material for simultaneous enrichment and separation of neutral and sialyl glycopeptides of a recombinant SARS-CoV-2 S glycoprotein from HEK293 cells. This strategy helps eliminate signal suppression from neutral glycopeptides for the detection of sialyl glycopeptides and improves the glycoform coverage of the S protein. We profiled 19 of its 22 potential N-glycosylated sites with 398 unique glycoforms using the dual-functional Ti-IMAC approach, which exhibited improvement of coverage by 1.6-fold compared to the conventional hydrophilic interaction chromatography (HILIC) glycopeptide enrichment method. We also identified O-linked glycosylation site that was not found using the conventional HILIC approach. In addition, we reported on the identification of mannose-6-phosphate (M6P) glycosylation, which substantially expands the current knowledge of the spike protein's glycosylation landscape and enables future investigation into the influence of M6P glycosylation of the spike protein on its cell entry.

VPTMdb: a viral posttranslational modification database.

In viruses, posttranslational modifications (PTMs) are essential for their life cycle. Recognizing viral PTMs is very important for a better understanding of the mechanism of viral infections and finding potential drug targets. However, few studies have investigated the roles of viral PTMs in virus-human interactions using comprehensive viral PTM datasets. To fill this gap, we developed the first comprehensive viral posttranslational modification database (VPTMdb) for collecting systematic information of PTMs in human viruses and infected host cells. The VPTMdb contains 1240 unique viral PTM sites with 8 modification types from 43 viruses (818 experimentally verified PTM sites manually extracted from 150 publications and 422 PTMs extracted from SwissProt) as well as 13 650 infected cells' PTMs extracted from seven global proteomics experiments in six human viruses. The investigation of viral PTM sequences motifs showed that most viral PTMs have the consensus motifs with human proteins in phosphorylation and five cellular kinase families phosphorylate more than 10 viral species. The analysis of protein disordered regions presented that more than 50% glycosylation sites of double-strand DNA viruses are in the disordered regions, whereas single-strand RNA and retroviruses prefer ordered regions. Domain-domain interaction analysis indicating potential roles of viral PTMs play in infections. The findings should make an important contribution to the field of virus-human interaction. Moreover, we created a novel sequence-based classifier named VPTMpre to help users predict viral protein phosphorylation sites. VPTMdb online web server (http://vptmdb.com:8787/VPTMdb/) was implemented for users to download viral PTM data and predict phosphorylation sites of interest.

Transcriptional and Non-Transcriptional Activation, Posttranslational Modifications, and Antiviral Functions of Interferon Regulatory Factor 3 and Viral Antagonism by the SARS-Coronavirus.

The immune system defends against invading pathogens through the rapid activation of innate immune signaling pathways. Interferon regulatory factor 3 (IRF3) is a key transcription factor activated in response to virus infection and is largely responsible for establishing an antiviral state in the infected host. Studies in Irf3-/- mice have demonstrated the absence of IRF3 imparts a high degree of susceptibility to a wide range of viral infections. Virus infection causes the activation of IRF3 to transcribe type-I interferon (e.g., IFNß), which is responsible for inducing the interferon-stimulated genes (ISGs), which act at specific stages to limit virus replication. In addition to its transcriptional function, IRF3 is also activated to trigger apoptosis of virus-infected cells, as a mechanism to restrict virus spread within the host, in a pathway called RIG-I-like receptor-induced IRF3 mediated pathway of apoptosis (RIPA). These dual functions of IRF3 work in concert to mediate protective immunity against virus infection. These two pathways are activated differentially by the posttranslational modifications (PTMs) of IRF3. Moreover, PTMs regulate not only IRF3 activation and function, but also protein stability. Consequently, many viruses utilize viral proteins or hijack cellular enzymes to inhibit IRF3 functions. This review will describe the PTMs that regulate IRF3's RIPA and transcriptional activities and use coronavirus as a model virus capable of antagonizing IRF3-mediated innate immune responses. A thorough understanding of the cellular control of IRF3 and the mechanisms that viruses use to subvert this system is critical for developing novel therapies for virus-induced pathologies.

Vimentin as a Multifaceted Player and Potential Therapeutic Target in Viral Infections.

Vimentin is an intermediate filament protein that plays key roles in integration of cytoskeletal functions, and therefore in basic cellular processes such as cell division and migration. Consequently, vimentin has complex implications in pathophysiology. Vimentin is required for a proper immune response, but it can also act as an autoantigen in autoimmune diseases or as a damage signal. Although vimentin is a predominantly cytoplasmic protein, it can also appear at extracellular locations, either in a secreted form or at the surface of numerous cell types, often in relation to cell activation, inflammation, injury or senescence. Cell surface targeting of vimentin appears to associate with the occurrence of certain posttranslational modifications, such as phosphorylation and/or oxidative damage. At the cell surface, vimentin can act as a receptor for bacterial and viral pathogens. Indeed, vimentin has been shown to play important roles in virus attachment and entry of severe acute respiratory syndrome-related coronavirus (SARS-CoV), dengue and encephalitis viruses, among others. Moreover, the presence of vimentin in specific virus-targeted cells and its induction by proinflammatory cytokines and tissue damage contribute to its implication in viral infection. Here, we recapitulate some of the pathophysiological implications of vimentin, including the involvement of cell surface vimentin in interaction with pathogens, with a special focus on its role as a cellular receptor or co-receptor for viruses. In addition, we provide a perspective on approaches to target vimentin, including antibodies or chemical agents that could modulate these interactions to potentially interfere with viral pathogenesis, which could be useful when multi-target antiviral strategies are needed.