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Myristoylated rhinovirus VP4 protein activates TLR2-dependent proinflammatory gene expression.

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.

SUBMITTER: Bentley JK

PROVIDER: S-EPMC6689742 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Myristoylated rhinovirus VP4 protein activates TLR2-dependent proinflammatory gene expression.

Bentley J Kelley JK Han Mingyuan M Jaipalli Suraj S Hinde Joanna L JL Lei Jing J Ishikawa Tomoko T Goldsmith Adam M AM Rajput Charu C Hershenson Marc B MB

American journal of physiology. Lung cellular and molecular physiology 20190325 1

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 myristoy ...[more]

PMID: 30908938

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Project description:IntroductionApical periodontitis (AP) is a common oral disease caused by the inflammatory destruction of the periapical tissues due to the infection of the root canal system of the tooth. It also contributes to systemic bacterial translocation, where peripheric mononuclear blood cells (PBMCs) can act as carriers. Toll-like receptor (TLR) 2 mediates the response to infection and activates inflammatory responses. DNA methylation can be induced by bacteria and contributes to the modulation of this response. Despite the evidence that supports the participation of PBMCs in immune-inflammatory disorders, the inflammatory profile and epigenetic regulatory mechanisms of PBMCs in AP individuals are unknown.AimTo determine TLR2 gene methylation and inflammatory profiles of PBMCs in AP.MethodsCross-sectional exploratory study. Otherwise, healthy individuals with AP (n=27) and controls (n=30) were included. PMBCs were isolated by a Ficoll gradient, cultured for 24 hours, and both RNA and DNA were extracted. DNA was bisulfite-treated, and specific sites at the promoter region of the TLR2 gene were amplified by qPCR using validated primers. To verify its amplification, agarose gels were performed. Then, the PCR product was sequenced. mRNA expression of TLR2 was determined by qPCR. The soluble levels of 105 inflammatory mediators were first explored with Proteome Profiler Human Cytokine Array Kit. Consequently, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, IL-6Rα, IL-1β, and IL-12p70 levels were measured by Multiplex assay.ResultsPBMCs from individuals with AP demonstrated a proinflammatory profile showing higher soluble levels of TNF-α, IL-6, and IL-1β compared to controls (p<0.05). Higher TLR2 expression and higher global methylation pattern of the promoter region of the gene were found in AP compared to controls (p<0.05). The CpGs single-sites at positions -166 and -146 were completely methylated, while the site -102 was totally unmethylated, independently of the presence of AP. DNA methylation of CpG single-sites in positions -77 and +24 was positively associated with TLR2 expression.ConclusionsPBMCs from AP subjects show a hyperinflammatory phenotype and TLR2 upregulation in association with single CpG-sites' methylation from the TLR2 gene promoter, thereby contributing to a sustained systemic inflammatory load in individuals with periapical endodontic diseases.

Project description:BackgroundDespite the clinical burden attributable to rhinovirus (RV) infections, the RV transmission dynamics and the impact of interventions on viral transmission remain elusive.MethodsA total of 3,935 nasopharyngeal specimens were examined, from which the VP4/VP2 gene was sequenced and genotyped. RV transmission clusters were reconstructed using the genetic threshold of 0.005 substitutions/site, estimated from the global VP4/VP2 sequences. A transmission cluster is characterized by the presence of at least two individuals (represent by nodes), whose viral sequences are genetically linked (represent by undirected edges) at the estimated genetic distance threshold supported by bootstrap value of ≥ 90%. To assess the impact of facemask, pleconaril and social distancing on RV transmission clusters, trials were simulated for interventions with varying efficacy and were evaluated based on the reduction in the number of infected patients (nodes) and the reduction in the number of nodes-connecting edges. The putative impact of intervention strategies on RV transmission clusters was evaluated through 10,000 simulations.ResultsA substantial clustering of 168 RV transmission clusters of varying sizes were observed. This suggests that RV disease burden observed in the population was largely due to multiple sub-epidemics, predominantly driven by RV-A, followed by RV-C and -B. No misclassification of RV species and types were observed, suggesting the specificity and sensitivity of the analysis. Through 10,000 simulations, it was shown that social distancing may be effective in decelerating RV transmission, by removing more than 95% of nodes and edges within the RV transmission clusters. However, facemask removed less than 8% and 66% of nodes and edges, respectively, conferring moderate advantage in limiting RV transmission.ConclusionHere, we presented a network-based approach of which the degree of RV spread that fuel disease transmission in the region was mapped for the first time. The utilization of RV transmission clusters in assessing the putative impact of interventions on disease transmission at the population level was demonstrated.

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Myristoylated rhinovirus VP4 protein activates TLR2-dependent proinflammatory gene expression.

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Myristoylated rhinovirus VP4 protein activates TLR2-dependent proinflammatory gene expression.

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