EGFR interacts with the fusion protein of respiratory syncytial virus strain 2-20 and mediates infection and mucin expression.

Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2-20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore, we hypothesized that the RSV 2-20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2-20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2-20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from "mucogenic" strains. RSV 2-20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease

MicroRNA-221 Modulates RSV Replication in Human Bronchial Epithelium by Targeting NGF Expression

Background: Early-life infection by respiratory syncytial virus (RSV) is associated with aberrant expression of the prototypical neurotrophin nerve growth factor (NGF) and its cognate receptors in human bronchial epithelium. However, the chain of events leading to this outcome, and its functional implications for the progression of the viral infection, has not been elucidated. This study sought to test the hypothesis that RSV infection modulates neurotrophic pathways in human airways by silencing the expression of specific microRNAs (miRNAs), and that this effect favors viral growth by interfering with programmed death of infected cells. Methodology: Human bronchial epithelial cells infected with green fluorescent protein-expressing RSV (rgRSV) were screened with multiplex qPCR arrays, and miRNAs significantly affected by the virus were analyzed for homology with mRNAs encoding neurotrophic factors or receptors. Mimic sequences of selected miRNAs were transfected into noninfected bronchial cells to confirm the role of each of them in regulating neurotrophins expression at the gene and protein level, and to study their influence on cell cycle and viral replication. Principal Findings: RSV caused downregulation of 24 miRNAs and upregulation of 2 (p,0.01). Homology analysis of microarray data revealed that 6 of those miRNAs exhibited a high degree of complementarity to NGF and/or one of its cognate receptors TrKA and p75 NTR. Among the selected miRNAs, miR-221 was significantly downregulated by RSV and it

Whole Genome Sequencing and Evolutionary Analysis of Human Respiratory Syncytial Virus A and B from Milwaukee, WI 1998-2010

BACKGROUND: Respiratory Syncytial Virus (RSV) is the leading cause of lower respiratory-tract infections in infants and young children worldwide. Despite this, only six complete genome sequences of original strains have been previously published, the most recent of which dates back 35 and 26 years for RSV group A and group B respectively. METHODOLOGY/PRINCIPAL FINDINGS: We present a semi-automated sequencing method allowing for the sequencing of four RSV whole genomes simultaneously. We were able to sequence the complete coding sequences of 13 RSV A and 4 RSV B strains from Milwaukee collected from 1998-2010. Another 12 RSV A and 5 RSV B strains sequenced in this study cover the majority of the genome. All RSV A and RSV B sequences were analyzed by neighbor-joining, maximum parsimony and Bayesian phylogeny methods. Genetic diversity was high among RSV A viruses in Milwaukee including the circulation of multiple genotypes (GA1, GA2, GA5, GA7) with GA2 persisting throughout the 13 years of the study. However, RSV B genomes showed little variation with all belonging to the BA genotype. For RSV A, the same evolutionary patterns and clades were seen consistently across the whole genome including all intergenic, coding, and non-coding regions sequences. CONCLUSIONS/SIGNIFICANCE: The sequencing strategy presented in this work allows for RSV A and B genomes to be sequenced simultaneously in two working days and with a low cost. We have significantly increased the amount of genomic data that is available for both RSV A and B, providing the basic molecular characteristics of RSV strains circulating in Milwaukee over the last 13 years. This information can be used for comparative analysis with strains circulating in other communities around the world which should also help with the development of new strategies for control of RSV, specifically vaccine development and improvement of RSV diagnostics

Mechanisms of Cellular Uptake with Chitosan/DNA Complex in Hepatoma Cell Line

Chitosan (CS) has a high potential for gene delivery into mammalian cells. However, its uptake mechanism is not well clarified. We investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine), caveolae-mediated endocytosis (genistein), macropinocytosis (LY 29004 and wortmannin), microtubuli polymerization (nocodazole) and of membrane cholesterol recycle (methyl-β-cyclodextrin) on the transfection efficiency with CS/pEGFP complexes and on the internalization of CS/rhodamine-labeled pEGFP complexes by hepatoma cell line (Huh 7 cells). The transfection was blocked by nocodazole, genistein, and methyl-β-cyclodextrin, respectively. CS/DNA complexes internalization was clearly inhibited by genistein. We conclude that the complexes uptake predominantly by caveolin-mediated pathways. In addition, fluorescence colocalization studies with acidotropic probes, LysoSensor dye, illustrated that CS/DNA complexes are targeted to lysosomes for the degradation after internalization.</jats:p

Identification of Deletion Mutant Respiratory Syncytial Virus Strains Lacking Most of the G Protein in Immunocompromised Children with Pneumonia in South Africa ▿

Respiratory syncytial virus (RSV) G protein deletion mutants replicate effectively in vitro but have not been detected in nature. Subtyping of RSV strains in hospitalized children in South Africa identified G protein PCR amplicons significantly reduced in size in 2 out of 209 clinical specimens screened over 4 years. Sequence analysis revealed subtype B strains lacking nearly the entire G protein ectodomain in one HIV-positive and one HIV-exposed child hospitalized with pneumonia. The association of clinical strains lacking most of the G protein with lower respiratory tract infection in immunocompromised children may have implications for RSV vaccine development

Residues of the Human Metapneumovirus Fusion (F) Protein Critical for Its Strain-Related Fusion Phenotype: Implications for the Virus Replication Cycle▿

The paramyxovirus F protein promotes fusion of the viral and cell membranes for virus entry, as well as cell-cell fusion for syncytium formation. Most paramyxovirus F proteins are triggered at neutral pH to initiate membrane fusion. Previous studies, however, demonstrated that human metapneumovirus (hMPV) F proteins are triggered at neutral or acidic pH in transfected cells, depending on the strain origin of the F sequences (S. Herfst et al., J. Virol. 82:8891–8895, 2008). We now report an extensive mutational analysis which identifies four variable residues (294, 296, 396, and 404) as the main determinants of the different syncytial phenotypes found among hMPV F proteins. These residues lie near two conserved histidines (H368 and H435) in a three-dimensional (3D) model of the pretriggered hMPV F trimer. Mutagenesis of H368 and H435 indicates that protonation of these histidines (particularly His435) is a key event to destabilize the hMPV F proteins that require low pH for cell-cell fusion. The syncytial phenotypes were reproduced in cells infected with the corresponding hMPV strains. However, the low-pH dependency for syncytium formation could not be related with a virus entry pathway dependent on an acidic environment. It is postulated that low pH may be acting for some hMPV strains as certain destabilizing mutations found in unusual strains of other paramyxoviruses. In any case, the results presented here and those reported by Schowalter et al. (J. Virol. 83:1511–1522, 2009) highlight the relevance of certain residues in the linker region and domain II of the pretriggered hMPV F protein for the process of membrane fusion