Myristoylated rhinovirus VP4 protein activates TLR2-dependent pro-inflammatory gene expression.
Bentley, JK; Han, M; Jaipalli, S; Hinde, J; Lei, J; Ishikawa, T; Goldsmith, AM; Rajput, C; Hershenson, MB
2019-07-01
Abstract
Asthma exacerbations are often caused by rhinovirus (RV). We and others have shown that Toll-like receptor 2 (TLR2), a membrane surface receptor that recognizes bacterial lipopeptides and lipoteichoic acid, is required and sufficient for RV-induced proinflammatory responses in vitro and in vivo. We hypothesized that viral protein-4 (VP4), an internal capsid protein that is myristoylated upon viral replication and externalized upon viral binding, is a ligand for TLR2. Recombinant VP4 and myristoylated VP4 (MyrVP4) were purified by Ni-affinity chromatography. MyrVP4 was also purified from RV-A1B-infected HeLa cells by urea solubilization and anti-VP4 affinity chromatography. Finally, synthetic MyrVP4 was produced by chemical peptide synthesis. MyrVP4-TLR2 interactions were assessed by confocal fluorescence microscopy, fluorescence resonance energy transfer (FRET), and monitoring VP4-induced cytokine mRNA expression in the presence of anti-TLR2 and anti-VP4. MyrVP4 and TLR2 colocalized in TLR2-expressing HEK-293 cells, mouse bone marrow-derived macrophages, human bronchoalveolar macrophages, and human airway epithelial cells. Colocalization was absent in TLR2-null HEK-293 cells and blocked by anti-TLR2 and anti-VP4. Cy3-labeled MyrVP4 and Cy5-labeled anti-TLR2 showed an average fractional FRET efficiency of 0.24 ± 0.05, and Cy5-labeled anti-TLR2 increased and unlabeled MyrVP4 decreased FRET efficiency. MyrVP4-induced chemokine mRNA expression was higher than that elicited by VP4 alone and was attenuated by anti-TLR2 and anti-VP4. Cytokine expression was similarly increased by MyrVP4 purified from RV-infected HeLa cells and synthetic MyrVP4. We conclude that, during RV infection, MyrVP4 and TLR2 interact to generate a proinflammatory response.Publisher
American Physiological Society
ISSN
1040-0605 1522-1504
Deep Blue DOI
Other DOIs
PMID
30908938
Subjects
Toll-like receptor asthma cytokine macrophage rhinovirus Adolescent Amino Acid Sequence Animals Asthma Capsid Proteins Child Eosinophilia Epithelial Cells Female HEK293 Cells HeLa Cells Host-Pathogen Interactions Humans Macrophages Male Mice Mice, Inbred C57BL Myristic Acids Picornaviridae Infections Protein Binding Protein Processing, Post-Translational Rhinovirus Signal Transduction Toll-Like Receptor 2 Viral Proteins Virus Replication
Types
Article
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