News

Cora N. Betsinger and Ileana M. Cristea, Princeton University, Department of Molecular Biology, Princeton, New Jersey, USA

A mission of the HUPO Biology/Disease-driven Human Proteome Project (B/D-HPP) is to explore how the human proteome can provide a lens for understanding human disease. The Human Infectious Diseases team (HID-HPP) of the B/D-HPP, is specifically devoted to the study of human diseases caused by infectious pathogens (https://www.hupo.org/Infectious-Disease-Initiative). One objective of HID-HPP is to develop, make broadly available, and apply proteomic methods to understand the biology and pathogenicity of viruses. For example, members of the HID-HPP have applied a range of proteomic methods to define alterations in the cellular proteome, protein interactome, and protein posttranslational modifications during infection with diverse viral pathogens¹, such as herpesviruses and influenza A^2–6. Given the ongoing global pandemic derived from infection with the novel SARS-CoV-2 virus⁷, here we highlight the demonstrated and promised power of proteomics to provide urgently needed insight into the biology and pathogenicity of this coronavirus and to uncover therapeutic targets.

Upon the emergence of a new viral pathogen in the human population, some of the first steps undertaken are to isolate the virus from patient samples and sequence the viral genome. This is critical for the taxonomic classification of the virus, determination of its phylogenetic relationship to other viruses, and identification of zoonotic host species. However, genetic analysis cannot fully address many aspects of virus biology, including the identity and function of virus proteins, how the virus interacts with host cells during its entry and replication, and what changes infection elicits at the cell and system level. Over the past twenty years, three of the emergent viruses that have resulted in widespread human disease and fatality have been members of the Coronaviridae family. SARS-CoV was identified as the causative agent of the 2003 severe acute respiratory syndrome (SARS) outbreak, which had a fatality rate of 10%⁸. MERS-CoV emerged ten years later, in 2013, and had a case fatality rate of more than 30%⁸. The most recent emergent coronavirus is SARS-CoV-2, the agent responsible for the current global COVID-19 disease pandemic that has resulted in over 2.8 million infections and 193,710 deaths to date⁷.

The application of proteomic techniques to the study of these different types of coronaviruses has allowed for a more complete characterization of each virus and its pathogenesis. Proteomic methods were successfully applied to the study of SARS-CoV immediately following the 2003 SARS outbreak and contributed significantly to our understanding of SARS-CoV structure, replication, and pathology, as well as identified potential therapeutic targets. Mass spectrometry-based methods were initially used to characterize the structure and components of SARS infectious virus particles^9–11. These studies confirmed virus protein sequences predicted by nucleotide sequencing, identified antigenic virus proteins, located glycosylation sites decorating the virus spike protein necessary for entry into host cells, and revealed host proteins which were incorporated into the virus particles during assembly. An affinity purification mass spectrometry analysis of the coronavirus spike protein led to the identification of angiotensin-converting enzyme 2 (ACE2) as the cell surface receptor for SARS-CoV¹². As the same host receptor is also targeted by the novel SARS-CoV-2, these findings led to the recent testing of the clinically approved compound, camostat mesylate, as a mean to block CoV-2 infection¹³.

Other research teams applied proteomic methods to investigate changes in the cellular proteome during SARS-CoV infection^14–16. These studies revealed host processes that are dysregulated during infection for the benefit of virus replication. For instance, the host protein BCL2-associated athanogene 3 (BAG3) was identified as upregulated during SARS-CoV replication¹⁶. Knockdown of BAG3 suppressed SARS-CoV replication and protein synthesis, demonstrating its pro-viral function during infection and identifying it as a potential therapeutic target. Another group used mass spectrometry to identify two phosphorylation sites on the virus nucleocapsid (N) protein, which regulates viral RNA transcription and replication¹⁷. As phosphorylation impacts the ability of N to bind RNA, this finding could aid in the development of antivirals regulating the phosphorylation status of N. Mass spectrometry was also used in the search for biomarkers of SARS-CoV infection in human plasma samples^18–21. These studies provided insight into the pathogenesis of SARS and revealed diagnostic markers, as well as markers correlated with disease progression, prognosis, and viral load. The aim of these studies was to develop a SARS-specific fingerprint that could differentiate SARS patients from non-SARS patients early during infection and predict the expected progression and severity of disease for each individual, allowing for personalized treatment and appropriate resource allocation.

Considering the current SARS-CoV-2 pandemic, proteomic techniques will be highly beneficial for investigating the efficacy of antiviral therapies, identifying new therapeutic targets, and developing fast and effective early diagnostic tests for coronavirus infection. For instance, monitoring virus and host protein levels following treatment with trial antivirals would demonstrate drug efficacy and reveal off-target effects. Proteomics could also be used to identify candidates for the rational design of antivirals targeting pro-viral host processes, which are often more effective long-term treatment options due to the propensity of RNA viruses to mutate. Quantification of temporal changes in host protein levels throughout the time-course of coronavirus infection would illuminate proteins and cellular processes that are dysregulated by coronavirus as potential therapeutic targets. Furthermore, a range of proteomic methods are available for studying host-viral protein-protein and protein-nucleic acid interactions, promising to provide insight into interactions that could be disrupted to restore host defense and inhibit virus replication. Such methods include affinity purification, crosslinking, proximity labeling, and thermal proximity coaggregation. Proteomic techniques could also be used to overcome what has been a major challenge during the current pandemic, i.e., the development of a fast, effective, and reliable diagnostic test for early detection of coronavirus infection. Targeted mass spectrometry could be used to identify diagnostic and prognostic markers of SARS-CoV-2 infection in patient serum samples, similar to investigations done during the 2003 SARS-CoV outbreak^18–21. This potential for the implementation of diverse proteomic methods for studying SARS-CoV-2 can already be seen in the impressive number of recent manuscripts either published or in prepublication format on bioRxiv.

The desire of the international scientific community to rapidly respond to the new SARS-CoV-2 pandemic has been evident on all fronts of science, including within the proteomics field. This is exemplified by efforts from the Human Infectious Diseases team (HID-HPP) of the B/D-HPP, as well as the timely organization of the COVID-19 Mass Spectrometry Coalition (covid19-msc.org), spearheaded by Dr. Perdita Barran (University of Manchester). This coalition now involves a continuously growing number of HUPO and HPP scientists, including Drs. Fernando Corrales, Edward Emmott, Andrea Sinz, Catherine Costello, Gilberto B Domont, Stephen Pennington, Yu-Ju Chen, John Yates, and our group to name just a few. Through the combined experience and expertise of scientists globally, we will continue to illuminate the underlying biology and pathogenicity of SARS-CoV-2 and contribute this knowledge toward the development of antiviral treatment options.

References:

1. Greco, T. M., Diner, B. A. & Cristea, I. M. The Impact of Mass Spectrometry–Based Proteomics on Fundamental Discoveries in Virology. Annu. Rev. Virol. (2014) doi:10.1146/annurev-virology-031413-085527.

2. Emmott, E. et al. Quantitative proteomics using SILAC coupled to LC-MS/MS reveals changes in the nucleolar proteome in influenza A virus-infected cells. J. Proteome Res. 9, 5335–5345 (2010).

3. Dove, B. K. et al. A quantitative proteomic analysis of lung epithelial (A549) cells infected with 2009 pandemic influenza A virus using stable isotope labelling with amino acids in cell culture. Proteomics (2012) doi:10.1002/pmic.201100470.

4. Murray, L. A., Sheng, X. & Cristea, I. M. Orchestration of protein acetylation as a toggle for cellular defense and virus replication. Nat. Commun. (2018) doi:10.1038/s41467-018-07179-w.

5. Lum, K. K. et al. Interactome and Proteome Dynamics Uncover Immune Modulatory Associations of the Pathogen Sensing Factor cGAS. Cell Syst. (2018) doi:10.1016/j.cels.2018.10.010.

6. Hashimoto, Y., Sheng, X., Murray-Nerger, L. A. & Cristea, I. M. Temporal dynamics of protein complex formation and dissociation during human cytomegalovirus infection. Nat. Commun. (2020) doi:10.1038/s41467-020-14586-5.

7. Practice, B. B. Coronavirus disease 2019. World Heal. Organ. 2019, 2633 (2020).

8. Ng, L. F. P. & Hiscox, J. A. Coronaviruses in animals and humans. The BMJ (2020) doi:10.1136/bmj.m634.

9. Krokhin, O. et al. Mass spectrometric characterization of proteins from the SARS virus: a preliminary report. Mol. Cell. Proteomics (2003) doi:10.1074/mcp.M300048-MCP200.

10. Ying, W. et al. Proteomic analysis on structural proteins of Severe Acute Respiratory Syndrome coronavirus. in Proteomics (2004). doi:10.1002/pmic.200300676.

11. Neuman, B. W. et al. Proteomics Analysis Unravels the Functional Repertoire of Coronavirus Nonstructural Protein 3. J. Virol. (2008) doi:10.1128/jvi.02631-07.

12. Li, W. et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature (2003) doi:10.1038/nature02145.

13. Hoffmann, M. et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell (2020) doi:10.1016/j.cell.2020.02.052.

14. Zeng, R. et al. Proteomic analysis of SARS associated coronavirus using two-dimensional liquid chromatography mass spectrometry and one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by mass spectroemtric analysis. J. Proteome Res. (2004) doi:10.1021/pr034111j.

15. Jiang, X. S. et al. Quantitative analysis of Severe Acute Respiratory Syndrome (SARS)-associated coronavirus-infected cells using proteomic approaches: Implications for cellular responses to virus infection. Mol. Cell. Proteomics 4, 902–913 (2005) doi: 10.1074/mcp.M400112-MCP200

16. Zhang, L., Zhang, Z. P., Zhang, X. E., Lin, F. S. & Ge, F. Quantitative Proteomics Analysis Reveals BAG3 as a Potential Target To Suppress Severe Acute Respiratory Syndrome Coronavirus Replication. J. Virol. (2010) doi:10.1128/jvi.00213-10.

17. Lin, L. et al. Identification of phosphorylation sites in the nucleocapsid protein (N protein) of SARS-coronavirus. Int. J. Mass Spectrom. (2007) doi:10.1016/j.ijms.2007.05.009.

18. Chen, J. H. et al. Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. (2004) doi:10.1073/pnas.0407992101.

19. Poon, T. C. W. et al. Serial analysis of plasma proteomic signatures in pediatric patients with severe acute respiratory syndrome and correlation with viral load. Clin. Chem. (2004) doi:10.1373/clinchem.2004.035352.

20. Kang, X. et al. Proteomic fingerprints for potential application to early diagnosis of severe acute respiratory syndrome. Clin. Chem. (2005) doi:10.1373/clinchem.2004.032458.

21. Pang, R. T. K. et al. Serum proteomic fingerprints of adult patients with severe acute respiratory syndrome. Clin. Chem. (2006) doi:10.1373/clinchem.2005.061689.