32 research outputs found
RSV infection of both multi-ciliated and airway basal cells.
<p>Apical exposure of HBEC to RSV-A2-GFP led to the infection of multi-ciliated cells. GFP<sup>+</sup> (infected) cells were observed throughout the epithelium. These were identified as an acetylated α-tubulin<sup>+</sup>, ciliated cell (#, orange) by confocal imaging (A–B). Under these conditions, the sub-apical p63<sup>+</sup> basal cells (red) did not become infected by RSV (B). Removal of the surface of the epithelium immediately before exposure to RSV resulted in numerous p63<sup>+</sup>GFP<sup>+</sup> (*) cells 16 h later (C–D), consistent with infection of the airway basal cell. When well-differentiated cells were subjected to a mechanical scratch injury (E) followed by RSV infection multiple infected cells were clearly visible within the wound (F). A majority of the infected cells were determined as basal cells (p63<sup>+</sup>) by confocal microscopy (G–I). Presented are representative images from three independent experiments in two different donors. White bar indicates 1,000 µm in A, C, 50 um in B, D, 500 µm in E–F and 20 µm in G–I.</p
Respiratory Syncytial Virus Can Infect Basal Cells and Alter Human Airway Epithelial Differentiation
<div><p>Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality worldwide, causing severe respiratory illness in infants and immune compromised patients. The ciliated cells of the human airway epithelium have been considered to be the exclusive target of RSV, although recent data have suggested that basal cells, the progenitors for the conducting airway epithelium, may also become infected <i>in vivo</i>. Using either mechanical or chemical injury models, we have demonstrated a robust RSV infection of p63<sup>+</sup> basal cells in air-liquid interface (ALI) cultures of human bronchial epithelial cells. In addition, proliferating basal cells in 2D culture were also susceptible to RSV infection. We therefore tested the hypothesis that RSV infection of this progenitor cell would influence the differentiation status of the airway epithelium. RSV infection of basal cells on the day of seeding (MOI≤0.0001), resulted in the formation of an epithelium that showed a profound loss of ciliated cells and gain of secretory cells as assessed by acetylated α-tubulin and MUC5AC/MUC5B immunostaining, respectively. The mechanism driving the switch in epithelial phenotype is in part driven by the induced type I and type III interferon response that we demonstrate is triggered early following RSV infection. Neutralization of this response attenuates the RSV-induced loss of ciliated cells. Together, these data show that through infection of proliferating airway basal cells, RSV has the potential to influence the cellular composition of the airway epithelium. The resulting phenotype might be expected to contribute towards both the severity of acute infection, as well as to the longer-term consequences of viral exacerbations in patients with pre-existing respiratory diseases.</p></div
RSV infection of basal cells influences differentiation.
<p>The effects of RSV-A2-GFP infection of basal cells (100 pfu/insert, 3 h after seeding) were investigated over the duration of epithelial growth and differentiation. Infection of the epithelium was monitored by live cell imaging of GFP<sup>+</sup> objects every 2–3 days (A; dotted line) whilst viral release was determined by plaque assay at day 3, 6, 13 and 21 (A; filled bars). Mean data ± SEM from 3 independent studies are shown. Representative planar and transverse views by confocal microscopy showing RSV-A2-GFP infected cells at day 6 (B), infected non-ciliated cells at day 13 (C) and infected ciliated cells at day 20 (D). GFP was used to visualize RSV-infection (green), acetylated α-tubulin stain for cilia (orange) and p63 for basal cells (red). Scale bars indicate 50 µm.</p
Proposed model for the impact of RSV infection of the airway basal cell.
<p>The multi-potent airway basal cell plays a central role in epithelial repair following injury, resulting in an epithelium composed of both ciliated and goblet cells (A). In patients with an impaired epithelial barrier, resulting from either the natural history of an RSV infection (epithelial sloughing) or because of a pre-existing respiratory disease, the airway basal cell can now become exposed to inhaled RSV particles (B). Infection of the basal cells by RSV results in the release of IL-28A, IL-28B, IL-29 and IFN-β that influences basal cell differentiation towards a hypersecretory phenotype i.e. gain of goblet cells and loss of ciliated cells that has the potential to contribute towards a disease exacerbation.</p
Epithelial composition following RSV infection.
<p>The effects of RSV-A2-GFP infection of basal cells on epithelial differentiation were assessed by quantitative immunofluorescence. Representative, images of HBEC cultures (21 days after seeding/infection) that were either uninfected (A) or infected with 1, 10, 100, or 1,000 pfu of RSV-A2-GFP (B–E, respectively). MUC5AC<sup>+</sup> goblet cells and acetylated a-tubulin<sup>+</sup> ciliated cells are pseudo-colored green and orange respectively. Staining for each of the cell types was quantified by image analysis (F). An alternative RSV strain, RSV-A2-MOT0972, was also assessed for effects on epithelial differentiation by quantitative immunofluoresence (G) as described above. For each study, mean data ± SEM from 6–9 inserts over three independent experiments are shown. Statistical significance was determined with a one-way ANOVA with post-hoc Dunnetts test compared to untreated control cells. * indicates p<0.05, **p<0.01 and ***p<0.001.</p
The +3 initiation site is utilized during RSV infection.
<p>(A) Primer extension analysis of Tr sense RNA generated during RSV infection. Two primers were utilized hybridizing to positions 13–35 or 32–55 relative to the 5′ terminus of RSV genome RNA (left and right panels, respectively). Lanes 3 and 4 show cDNAs generated from RNA isolated from mock or RSV infected cells, respectively. The sizes of the products were determined by co-migration of <sup>32</sup>P end-labeled DNA oligonucleotides consisting of Tr sequence 3–35 or 1–35 (left panel, lanes 1 and 2, respectively), or 3–55 or 1–55 (right panel, lanes 1 and 2, respectively) to indicate the lengths of products initiated at +3 or +1. It should be noted that lanes 1–4 of the left panel are all from the same gel, but lanes 1 and 2 required a longer exposure to be detected. (B) Northern blot analysis of small genome sense RNA transcripts generated from the TrC promoter. Lanes 1 and 2 contain RNA isolated from mock or RSV infected cells, respectively. The blot was hybridized with a locked nucleic acid DNA oligonucleotide probe designed to anneal to nts 5–32 relative to the 5′ end of the RSV Tr sequence. (C) Alignment of the sequences from the 3′ terminus of the RSV TrC promoter and the ten nt L gene start (GS) signal. Identical nts are underlined and dashes indicate nts at the −1 and −2 positions relative to the L GS sequence, which are not part of the signal.</p
The isolated RSV RdRp adds nts to the 3′ end of the TrC template RNA.
<p>(A) A GTP label is incorporated into products of 26–28 nts in length. Wt or mutant (L<sub>N812A</sub>) RdRp was incubated with 0.2 µM TrC RNA template, or its complement Tr 1–25, as indicated, in a reaction containing 200 µM of each NTP and [α-<sup>32</sup>P]GTP. (B) GTP incorporation into the 26 nt product is independent of RNA synthesis. Reactions were performed as described for panel A, except that in lanes 3–5, the only NTP in the reaction was [α-<sup>32</sup>P]GTP. Lane 2 is a control containing all four NTPs and [α-<sup>32</sup>P]GTP. (C) Generation of the 26–28 nt products is dependent on the TrC RNA template containing a 3′-hydroxyl group. TrC RNA templates containing either a 3′-hydroxyl (OH; lane 2) or a 3′-puromycin (PMN; lanes 3 and 4) group were tested at a concentration of 2 µM in reactions containing 1 mM of each NTP and [α-<sup>32</sup>P]GTP. In each panel, lane 1 shows the molecular weight ladder.</p
Sequence analysis of the 3′ termini of RSV antigenome and genome RNA isolated from RSV infected cells.
<p>(A) Putative structures formed by the terminal sequences of the TrC and Le promoter regions. Nts 1–25 of the TrC and Le promoter sequences are shown (left and right panels, respectively), with potential secondary structures indicated. In the case of the TrC sequence, the nts added to the 3′ end of the TrC RNA are underlined. (B) Sequence analysis of the antigenome and genome termini. The traces show the sequence of the population of cDNAs representing the antigenome and genome terminal sequences (left and right panels, respectively). In each case, the upper panel shows the sequence of RNA tailed with ATP, and the lower panel shows the sequence of RNA tailed with CTP. Note that any 3′ nt addition matching the base used to tail the RNA would not be detected. (C) Representative traces of different cDNA clone sequences obtained that represent antigenome termini. The relative frequency of each clone of the 19 clones sequenced is indicated. Two clone traces that were obtained are not shown; these contained a deletion of position 1U (or substitution with an A) with no nt additions, and the sequence 3′ <u>CCG</u>CGCUCUUU, in which position 1 appears to have been substituted with a C, and a GCC sequence (underlined) has been added. In panels B and C, all sequences are presented as RNA and positions +1U, +5C, and +10U of the TrC or Le promoter are indicated. The A or C residues at the right hand side of each trace represent the sequence added by the E. coli poly A polymerase, and the additional nts lying between nt +1U of the promoter and the A or C tail are underlined.</p
RNA products are generated from the +3 site on the TrC template.
<p>(A and B). Effect of omitting UTP from the RNA synthesis reaction. RNA synthesis reactions were performed with all four NTPs (lane 1) or with UTP omitted (lane 2). Reactions contained 2 µM TrC template RNA, wt RdRp, and 1 mM each NTP including either [α-<sup>32</sup>P]ATP (A), or [α-<sup>32</sup>P]GTP (B). (C) The 21 nt product is initiated with GTP. RNA synthesis reactions were performed with either [α-<sup>32</sup>P]GTP (lane 1) or [γ-<sup>32</sup>P]GTP (lanes 2 and 3) as a label. The reactions contained 2 µM TrC template RNA, 10 µM cold GTP and 1 mM ATP, CTP and UTP, and either wt (lanes 1 and 2) or mutant (lane 3) RdRp. (D) [γ-<sup>32</sup>P]GTP is incorporated into 11 and 13 nt products if UTP is omitted from the reaction. RNA synthesis reactions were performed with either [α-<sup>32</sup>P]GTP (lane 1) or [γ-<sup>32</sup>P]GTP (lanes 2 and 3) as a label. The reactions included 2 µM TrC template RNA, 50 µM cold GTP and 1 mM ATP, and CTP and either wt (lanes 1 and 2) or mutant (lane 3) RdRp. Note that the 25 nt bands in panel D, lanes 2 and 3 could be due to kinase activity (either in the RSV RdRp or a contaminant of the preparation) phosphorylating the TrC template RNA. The long products detected with [α-<sup>32</sup>P]GTP in lanes 1 of panels C and D might be due to extensive 3′ nt addition, or repeated stuttering of the RdRp on the U tracts in the template.</p
Analysis of the role of internal sequences of the TrC RNA in 3′ nt addition.
<p>(A) Schematic diagram showing the two putative hairpin loop structures formed by the TrC RNA. Nts 1, 14 and 16, which were subjected to mutagenesis are underlined. (B) Effect of mutation of nt 1, or nts 14 and 16 of the TrC RNA on 3′ nt addition. Reactions were performed containing 25 nt TrC RNA that was of wt sequence (lanes 1 and 4), or containing a 1U/A substitution (lanes 2 and 5), or substitution of nts 14A and 16A with U residues (lanes 3 and 6). Reactions were performed using 0.2 µM RNA and 500 µM of each NTP. Lanes 1–3 show RNAs labeled with [α-<sup>32</sup>P]GTP, and lanes 4–6 show RNAs labeled with [α-<sup>32</sup>P]ATP.</p