Conserved cysteine residues within the attachment G glycoprotein of respiratory syncytial virus play a critical role in the enhancement of cytotoxic T-lymphocyte responses

Abstract

The cytotoxic T-lymphocyte (CTL) response plays an important role in the control of respiratory syncytial virus (RSV) replication and the establishment of a Th1-CD4+ T cell response against the virus. Despite lacking Major Histocompatibility Complex I (MHC I)-restricted epitopes, the attachment G glycoprotein of RSV enhances CTL activity toward other RSV antigens, and this effect depends on its conserved central region. Here, we report that RSV-G can also improve CTL activity toward antigens from unrelated pathogens such as influenza, and that a mutant form of RSV-G lacking four conserved cysteine residues at positions 173, 176, 182, and 186 fails to enhance CTL responses. Our results indicate that these conserved residues are essential for the wide-spectrum pro-CTL activity displayed by the protein.

Fig. 1

Schematic representation of the membrane-bound form of the RSV G glycoprotein. The cytosolic (CT), transmembrane (TM), and extracellular domains of the protein are depicted by white, black, and gray rectangles, respectively. The sequence of G between amino acids 164 and 189, representing the conserved central region is labeled on top. The disulfide bonds that form the cystine noose domain between conserved cysteines 173 and 186, and between cysteines 176 and 182 are indicated with dotted lines. The CX3C motif between residues 182 and 186 is denoted with a black bar at the bottom. N amino terminus and C carboxy terminus

Fig. 2

Expression of exogenous G protein during RSV infections enhances CTL responses in a manner dependent on cysteines 173, 176, 182 and 186. a BALB/c mice were co-inoculated intranasally with 10⁶ pfu of WT RSV A (RSV) and 5 μg of one of the following plasmids: pCDNA 3.1 (an empty control plasmid), Gwt (a plasmid carrying a WT G gene), or C4A (a plasmid carrying a mutant form of the G gene with a quadruple alanine substitution at positions 173, 176, 182, and 186). Three days post-inoculation the animals received a booster of 5 μg of the indicated plasmids and 6 days later their lungs were extracted to isolate PMC. A control group of mice (−) was infected intranasally with 10⁶ pfu of RSV in the absence of plasmid and sacrificed at day 9 post-infection for lung extraction and PMC isolation. Isolated cells were tested in an ELISPOT assay using A-20 presenting cells loaded with the M2^82–90 peptide (see “Materials and methods” section) and the frequency of M2^82–90-specific IFN-^ÓELI-SPOTS was determined (P = 0.0449; Kruskal–Wallis test). b Mice were co-inoculated with 10⁶ pfu of rRSVΔG_172–187, a recombinant RSV virus lacking the coding region for residues 172 to 187 of the conserved cystine noose domain of G, and 5 μg of the indicated plasmids (either pCDNA 3.1, Gwt or C4A). Three days later the animals received a booster with the same plasmids and 6 days after the booster, PMCs were isolated and tested by ELISPOT assay. A control group of mice infected intranasally with 10⁶ pfu of rRSVΔG_172–187 in the absence of plasmid and sacrificed at day 9 post-infection (−) was included in these experiments. The frequencies of M2^82–90-specific IFN-γ ELISPOTS in the different experimental groups are shown (P = 0.0237; Kruskal–Wallis test). Data for both panels are presented as the mean ;SEM (error bars) and are representative of three experiments using three mice per infection per experiment. c Detection of membrane-bound G proteins by immunofluorescence. Human 293 cells were transiently transfected with either pCDNA 3.1, Gwt or C4A. Twenty-four hours after transfection cells were fixed with paraformaldehyde (but not permeabilized) and stained with anti-RSV antibody followed by Alexa fluor 568-conjugated secondary antibody. Immunofluorescence signals for each indicated sample are shown. White arrows indicate transfected cells expressing the G glycoprotein. Original magnification 20×. d DNA vaccine-mediated protein expression in lungs of BALB/c mice. Immunoblot analysis of lung extracts from mice inoculated intrana-sally with either pCDNA 3.1, Gwt or C4A was performed using an anti-RSV polyclonal antibody

Fig. 3

RSV lung titers and pulmonary histopathology. a Mice were co-inoculated intranasally with 10⁶ pfu of rRSVΔG_172–187 and 5 μg of the indicated plasmids (i.e. pCDNA 3.1, Gwt, or C4A). Three days post-inoculation the animals received a booster of 5 μg of the indicated plasmids, and were later sacrificed at 1, 4, or 6 days after the booster to obtain lung tissue. RSV titration was performed as described in “Materials and methods” section. RSV titers are shown as log of plaque forming units per gram of lung tissue (pfu/g) and represent the mean ;SEM of three experiments using five mice per infection per experiment. b Pulmonary histopathology in mice during peak inflammation at 7 days post co-inoculation with rRSVΔG_172–187 and the indicated plasmids; (−) indicates mocked infected and no plasmid. Lung sections stained with hematoxylin and eosin are shown (original magnification 10×)

Fig. 4

RSV-G enhances CTL activity against flu antigens during H1N1 infections. a C57BL/6 mice were co-inoculated intranasally with 10⁶ pfu of H1N1 and 10 μg of either pCDNA 3.1 or Gwt. Three days post-inoculation, the animals received a booster of 10 μg of the indicated plasmids and 6 days later their lungs were extracted to isolate PMC. Isolated cells were tested in an ELISPOT assay using A-20 presenting cells loaded with either influenza NP^366–374 or PA^224–233 peptides (see “Materials and methods” section) and the frequency of IFN-^©ELISPOTS was determined. Assays were performed in triplicates. b Expression of granzyme B by PA^224–233 peptide-specific CD8+ pulmonary CTLs. Lung mononuclear cells from mice co-inoculated intranasally with 10⁶ pfu of H1N1 and 10 μg of either pCDNA 3.1 (left panel) or Gwt (right panel) were activated by A-20 presenting cells previously loaded with influenza PA^224–233 peptides. Cells were then fixed and doubly labeled with FITC-conjugated anti-CD8α antibody and phycoerythrin (PE)-conjugated anti-granzyme B antibody. Flow cytometry analysis was performed on a total of 30,000 cells per sample