ABSTRACT
Respiratory syncytial virus (RSV) contains a conserved CX3C motif on the ectodomain of the G-protein. The motif has been indicated as facilitating attachment of the virus to the host initiating infection via the human CX3CR1 receptor. The natural CX3CR1 ligand, CX3CL1, has been shown to induce signaling pathways resulting in transcriptional changes in the host cells. We hypothesize that binding of RSV to CX3CR1 via CX3C leads to transcriptional changes in host epithelial cells. Using transcriptomic analysis, the effect of CX3CR1 engagement by RSV was investigated. Normal human bronchial epithelial (NHBE) cells were infected with RSV virus containing either wildtype G-protein, or a mutant virus containing a CX4C mutation in the G-protein. RNA sequencing was performed on mock and 4-days-post-infected cultures. NHBE cultures were also treated with purified recombinant wild-type A2 G-protein. Here we report that RSV infection resulted in significant changes in the levels 766 transcripts. Many nuclear associated proteins were upregulated in the WT group, including nucleolin. Alternatively, cilia-associated genes, including CC2D2A and CFAP221 (PCDP1), were downregulated. The addition of recombinant G-protein to the culture lead to the suppression of cilia-related genes while also inducing nucleolin. Mutation of the CX3C motif (CX4C) reversed these effects on transcription decreasing nucleolin induction and lessening the suppression of cilia-related transcripts in culture. Furthermore, immunohistochemical staining demonstrated decreases in in ciliated cells and altered morphology. Therefore, it appears that engagement of CX3CR1 leads to induction of genes necessary for RSV entry as well as dysregulation of genes associated with cilia function.ImportanceRespiratory Syncytial Virus (RSV) has an enormous impact on infants and the elderly including increased fatality rates and potential for causing lifelong lung problems. Humans become infected with RSV through the inhalation of viral particles exhaled from an infected individual. These virus particles contain specific proteins that the virus uses to attach to human ciliated lung epithelial cells, initiating infection. Two viral proteins, G-protein and F-protein, have been shown to bind to human CX3CR1and nucleolin, respectively. Here we show that the G-protein induces nucleolin and suppresses gene transcripts specific to ciliated cells. Furthermore, we show that mutation of the CX3C-motif on the G-protein, CX4C, reverses these transcriptional changes.
ABSTRACT
RATIONALE: Emerging data suggest an important role for T lymphocytes in the pathogenesis of chronic lung disease in preterm infants. Comprehensive assessment of the lymphocyte transcriptome may identify biomarkers and mechanisms of disease. METHODS: Small volume peripheral blood samples were collected from premature infants enrolled with consent in the Prematurity and Respiratory Outcomes Program (PROP), at the time of discharge from the hospital. Blood samples were collected at two sites and shipped to a central laboratory for processing. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation and separated into individual lymphocyte cell types by fluorescence-activated cell sorting. Gating strategies were optimized to ensure reproducible recovery of highly purified lymphocyte populations over a multi-year recruitment period. RNA was isolated from sorted cells and characterized by high-throughput sequencing (RNASeq). RESULTS: Blood volumes averaged 2.5ml, and sufficient PBMCs were collected from 165 of the 246 samples obtained (67%) from the 277 recruited subjects to complete sorting and RNASeq analysis on the resulting sorted cells. The number of total lymphocytes per ml of blood in the neonatal subjects was approximately 4 million/ml. Total lymphocyte frequencies recovered following sort varied widely among subjects, as did the frequency of individual lymphocyte and NK cell sub-populations. RNA yield from sorted cells varied according to cell type, but RNA of sufficient quantity and quality was recovered to enable RNASeq. SUMMARY: Our results describe a validated procedure for the generation of genome-wide expression data from isolated lymphocyte sub-populations obtained from newborn blood.
