Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

Abstract

Human metapneumovirus (hMPV) is a leading cause of lower respiratory infection in pediatric populations globally. This study examined proteomic profile changes in A549 cells infected with hMPV and two attenuated mutants with deleted PDZ domain-binding motif(s) in the M2-2 protein. These motifs are involved in the interruption of antiviral signaling, namely the interaction between the TNF receptor associated factor (TRAF) and mitochondrial antiviral-signaling (MAVS) proteins. The aim of this study was to provide insight into the overall and novel impact of M2-2 motifs on cellular responses via an unbiased comparison. Tandem mass tagging, stable isotope labeling, and high-resolution mass spectrometry were used for quantitative proteomic analysis. Using quantitative proteomics and Venn analysis, 1248 common proteins were detected in all infected samples of both technical sets. Hierarchical clustering of the differentiated proteome displayed distinct proteomic signatures that were controlled by the motif(s). Bioinformatics and experimental analysis confirmed the differentiated proteomes, revealed novel cellular biological events, and implicated key pathways controlled by hMPV M2-2 PDZ domain-binding motif(s). This provides further insight for evaluating M2-2 mutants as potent vaccine candidates.

A tRNA-derived RNA Fragment Plays an Important Role in the Mechanism of Arsenite -induced Cellular Responses

“…Nuclear extracts of cells were prepared, as we previously described, and quantified with a protein quantification kit from Bio-Rad (Hercules, California). Proteins were fractionated by 10% SDS-PAGE denaturing gels, and transferred to polyvinylidene difluoride membranes, as we previously described 13 , 23 , 24 . Membranes were blocked with 5% milk in TBS-Tween 20 and incubated with the proper primary antibody to detect p65.…”

The role of M2‐2 PDZ‐binding motifs in pulmonary innate immune responses to human metapneumovirus

“…49,51 AECs, our proteomics data have revealed that there are many other changes than motif-controlled immunity, such as pathways involved in cell signaling, chromatin remodeling, and oxidative responses. 24 Overall, our finding demonstrates that M2-2 motif mutations are beneficial to induce a robust pulmonary innate immune response to HMPV infection.…”

“…Overall, the data were consistent with our previous findings in the suppressive role of two M2-2 PDZ-binding motifs in cellular innate immunity. 21,22 Attenuated replication and enhanced host innate immune responses to mutant infections also suggested that rHMPV lacking either motif could serve as live-attenuated vaccine candidates.…”

“…MATERIALS AND METHODS 2.1 | rHMPV preparation and titration WT-rHMPV or recombinant mutants with mutations in the motif 29-DEMI-32 (Mut-1) or the motif 39-KEALSDGI-46(Mut-2), were generated and propagated in LLC-MK2 cells (ATCC, Manassas, VA), as previously described 21,23,24. Viral titers of purified recombinant viruses or viruses from the infected mouse lungs were determined by immunostaining in LLC-MK2 cells, as described previously 21,25.…”

Cell-Mediated Responses to Human Metapneumovirus Infection

“…More specifically, M2-2 blocks the TLR7/9 dependent signaling pathway by suppressing IRF7 homodimerization through partial inhibition of IRF7 phosphorylation [135]. Two putative PDZ binding motifs in M2-2 play a major suppressive role in cellular innate immunity both in vitro and in vivo [136,137]. Infection of mice with reverse engineered hMPV virus with mutations in the M2-2 PDZ-binding motif induced higher DC maturation and recruitment of DCs and T cells in the lungs.…”