A proteomic and molecular approach to decipher the repertoire of protein modifications during influenza A virus infection
Influenza virus is an important human respiratory pathogen that continues to have a major impact on global public health. The ever-evolving nature of influenza virus has restricted the development of a universal vaccine as well as the efficacy of currently available vaccines. In the absence of a universal vaccine, antiviral drugs are serving as the first line of defense against novel influenza virus strains. However, the emergence of drug-resistant variants of influenza virus has made the available drugs almost ineffective. Therefore, there is an undeniable need for developing universal and long-lasting antiviral strategies. Exploring new approaches such as targeting critical factors for influenza virus infection might lead to the development of novel antiviral strategies. There is growing evidence that influenza exploits host protein modification machinery to proliferate and escape the host innate immune response. Therefore, a detailed profiling of the protein modification events during influenza virus infection is required. Protein modifications occurring co- or post-translationally regulate the structure and function of a protein in various ways. Here, we used global mass spectrometry approach in association with bioinformatics analysis to identify and characterize the modifications of viral and host proteins in uninfected and IAV-infected human lung epithelial cells. To the best of our knowledge, this is the first time such a comprehensive approach has been employed in influenza virus proteomics. We identified 8 viral proteins: matrix protein (M1), nucleoprotein (NP), two non-structural proteins (NS1, NS2) and haemagglutinin (HA), modified by methylation, acetylation, allysine and/or ubiquitination. Three viral proteins, M1, NP and PA showed multiple modifications on the same amino acid residue, indicating potential crosstalk among protein modifications. The majority of modified amino acids exhibited sequence homology across IAV subtypes and strains, suggesting the fundamental importance of these modifications in IAV biology. Furthermore, the location of modified amino acids in structural context suggested the functional significance of modified sites in multiple steps of IAV life cycle. In addition, a total of 116 methylated, 103 acetylated, 29 allysine modified, 2 ubiquitinated and 5 acylated host proteins were found to be uniquely modified in IAV-infected cells. Characterization of these modified host proteins by protein functional association analysis revealed their involvement in metabolic pathways, mRNA splicing and cytoskeleton organization which are manipulated by IAV during infection. The functional enrichment analysis revealed the modified cellular proteins to be involved in multiple virus-related processes. Acetylation was the second most abundant modification identified in our study. Host histone deacetylases (HDACs) and histone acetyltransferases (HATs) are the central regulators of acetylation; where HATs add and HDACs remove the acetyl group from a target protein. Our lab has discovered and been studying the anti-IAV properties of HDACs. Therefore, we endeavoured to identify the potential involvement of HATs in IAV infection. An initial RNA interference screening with 18 HATs revealed that the depletion of 6 HATs reduced IAV progeny release. Among them, N-alpha-acetyltransferase 60 (Naa60), an N-terminal acetyltransferase that possesses the unique capacity of acetylating proteins both co- and post-translationally, was chosen for further investigation. By employing the RNA interference and overexpression strategies, this study demonstrated that Naa60 plays a pro-viral role and is a component of host antiviral response. The silencing of Naa60 expression impaired IAV progeny release by 50% and conversely, the ectopic Naa60 expression augmented IAV progeny release by 2.3 fold. Mechanistically, the IAV-induced expression of interferon (IFN) α was increased and decreased with the depletion and ectopic expression of Naa60, respectively. Furthermore, the knockdown of Naa60 resulted in increased phosphorylation of transcription factor, STAT1 as well as the expression of certain interferon-stimulate genes (ISGs) such as viperin and IFITM3, in IAV-infected cells. Conversely, the ectopic expression of Naa60 correlated with the above data and reduced IAV-induced IFNα and ISGs expression. Finally, the variations in the relative abundances of N-terminally acetylated peptides from viral M1, NP and multiple host proteins in the absence of Naa60 indicated that the acetyltransferase activity of Naa60 has implications in IAV infection. This PhD study provides a basic framework for future research on the functional significance of protein modifications in the IAV life cycle. Protein modifications with trackable functional roles might serve as a target for novel antiviral strategies. Additionally, the proviral properties and involvement of Naa60 in IAV-induced host innate antiviral response identified in this study represents an exciting prospect for future investigation into targeting Naa60 as a drug target to combat IAV infection.
Advisor: Husain, Matloob; Kleffmann, Torsten
Degree Name: Doctor of Philosophy
Degree Discipline: Department of Microbiology and Immunology
Publisher: University of Otago
Keywords: Influenza; IAV; PTM; Naa60; Viperin; Proviral; HAT; Interferons; Viral-Proteomics
Research Type: Thesis