Pepscan mapping of viral hemorrhagic septicemia virus glycoprotein G major lineal determinants implicated in triggering host cell antiviral responses mediated by type I interferon

Abstract

Surface glycoproteins of enveloped virus are potent elicitors of type I interferon (IFN)-mediated antiviral responses in a way that may be independent of the well-studied genome-mediated route. However, the viral glycoprotein determinants responsible for initiating the IFN response remain unidentified. In this study, we have used a collection of 60 synthetic 20-mer overlapping peptides (pepscan) spanning the full length of glycoprotein G (gpG) of viral hemorrhagic septicemia virus (VHSV) to investigate what regions of this protein are implicated in triggering the type I IFN-associated immune responses. Briefly, two regions with ability to increase severalfold the basal expression level of the IFN-stimulated mx gene and to restrict the spread of virus among responder cells were mapped to amino acid residues 280 to 310 and 340 to 370 of the gpG protein of VHSV. In addition, the results obtained suggest that an interaction between VHSV gpG and integrins might trigger the host IFN-mediated antiviral response after VHSV infection. Since it is known that type I IFN plays an important role in determining/modulating the protective-antigen-specific immune responses, the identification of viral glycoprotein determinants directly implicated in the type I IFN induction might be of special interest for designing new adjuvants and/or more-efficient and cost-effective viral vaccines as well as for improving our knowledge on how to stimulate the innate immune system.

FIG. 1.

Expression of VHSV gpG on the surfaces of EPC-gpG cells. Fresh EPC cell monolayers were cotransfected with the pAE6-gpG and pAE6-pac (puromycin resistance gene) plasmid constructs. After an incubation period in the presence of puromycin, the remaining cells were screened for the presence of surface VHSV gpG (cells expressing VHSV gpG on the membrane) by immunofluorescence (IF) using a cocktail of anti-G MAbs and fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG Ab. Shown are EPC-gpG cell micrographs at fluorescent (field 1) and visible (field 2) light fields; field 3 is a merged image of fields 1 and 2.

FIG. 2.

mx3 transcript (A) and protein (B) expression in RTG-2 cells coincubated with paraformaldehyde-fixed EPC-gpG cells. Fresh RTG-2 cells and paraformaldehyde-fixed EPC-gpG cells were cocultured at different ratios, and expression of mx3 was assessed 24 h later both at transcriptional and protein levels by RT-qPCR and IF, respectively. (A) Fold changes in mx3 gene expression assayed by RT-qPCR. Fold increases were calculated relative to the level for nontreated RTG-2 cells. Data are the mean fold changes ± SD for three independent experiments, each performed in duplicate. Asterisks indicate significant differences in mx3 gene induction relative to the level for control cells at P values of <0.05 (*) and P values of <0.01 (**). ▪, cells cocultured with paraformaldehyde-fixed EPC-gpG cells; □, cells cocultured with paraformaldehyde-fixed EPC cells. (B) RTG-2 cells cocultured with paraformaldehyde-fixed EPC-gpG (lower panel) or EPC cells (upper panel) at a 1/40 ratio and stained with an anti-Mx3 protein serum. Shown are RTG-2 cell micrographs at fluorescent (field 1) and visible (field 2) light fields; field 3 is a merged image of fields 1 and 2.

FIG. 3.

Fold changes in the mx3 gene expression in response to VHSV gpG synthetic 20-mer peptides assayed by real-time RT-qPCR (A) and schematic overview of VHSV gpG features plotted on a linear diagram (B). (A) Fresh RTG-2 cells, grown in 48-well plates, were incubated with 3 μg/ml of each of the peptides from the pepscan of 20-mer peptides. After 48 h of incubation at 20°C, total RNA was extracted and the expression of transcripts of mx3 was estimated by RT-qPCR. Fold increases were calculated relative to the level for nontreated RTG-2 cells. Data are the mean fold changes ± SD for three independent experiments, each performed in duplicate. Gray bars indicate pepscan peptides that induced significant changes in mx3 gene expression relative to the level for control cells at P values of <0.05 (*) and P values of <0.01 (**). (B) Sequence of the VHSV gpG protein colored by domains as their homologous counterparts of VSV gpG (63, 64). Red, domain I, or the lateral domain; blue, domain II, or the trimerization domain; orange, domain III, or the PH domain; yellow, domain IV, or the fusion domain. The filled diamond indicates the positions of the N-linked glycosylation sites (Asn30, Asn378, Asn389, and Asn438). SP, signal peptide (amino acids 1 to 20); TM, transmembrane region (amino acids 472 to 492); ReBs, putative receptor binding site (amino acids 268 to 282). Horizontal bars represent the mx3-inducing regions defined by the pepscan peptides p60, p70 and p80 (yellow), p290 and p300 (light green), p350 and p360 (magenta), and p470 and p480 (dark green).

FIG. 4.

Fold changes in the mx3 gene expression in response to VHSV gpG regions selected from the pepscan (mx3-inducing peptides) assayed by RT-qPCR. RTG-2 cells were treated with different concentrations (from 1 to 12 μg/ml) of each of the mx3-inducing peptides (p9, p31, and p33 [A] and p30 and p33 [B]) or of WF-Ple for 48 h at 20°C. Total RNA was then extracted and mx3 gene expression estimated by RT-qPCR. Fold increases were calculated relative to the level for nontreated RTG-2 cells. Data are the mean fold changes ± SD for three independent experiments, each performed in duplicate. As shown in panel A, p33 significantly increased the mx3 gene expression at peptide concentrations of 3, 6, and 12 μg/ml while p9 and WF-Ple did not, and p31 caused significantly increased expression levels at the highest concentrations tested (6 and 12 μg/ml). Likewise, both p30 and p33 (B) significantly increased the mx3 gene expression at peptide concentrations of 3, 6, and 12 μg/ml. *, P < 0.05; **, P < 0.01. Significant differences between the mean fold changes in mx3 gene expression induced by p33 and p32 at 3, 6, and 12 μg/ml were also found by Tukey's test (P < 0.05). ▾, p9; ▴, p30; ♦, p31; ▪, p32; •, p33; X, WF-Ple.

FIG. 5.

Resistance to VHSV infection of RTG-2 cells treated with the mx3-inducing peptides. RTG-2 cells were treated with increasing concentrations (3, 6, and 12 μg/ml) of p31 (A), p33 (B), p30 (C), p32 (D), or WF-Ple (E) and then infected with VHSV (MOI of 2 × 10⁻³) for 2 h at 14°C. After the unbound virus was washed, the infected cell monolayers were incubated for 24 h at 14°C and VHSV infectivity was estimated by RT-qPCR. Black triangles represent increasing peptide concentrations. The results are expressed as percentages of infectivity and calculated by the formula (VHSV infectivity in treated cells/VHSV infectivity in nontreated cells) × 100. Data are the mean percentages of infectivity ± SD for three independent experiments, each performed in triplicate. All mx3-inducing peptides significantly reduced the VHSV infectivity at all of the concentrations tested but 3 μg/ml for p30 and p33. *, P < 0.05; **, P < 0.01. Tukey's test (P < 0.05) also showed significant differences between the mean percentage of infectivity of the cells treated with p33 and that of the cells treated with p32 both at 6 and 12 μg/ml.

FIG. 6.

Resistance to VHSV infection of RTG-2 cells treated with conditioned medium (CM) prepared from RTG-2 cells treated with the mx3-inducing peptides. RTG-2 cells were incubated for 24 h with increasing dilutions (1/5, 1/25, and 1/125) of CM prepared from cells treated with poly(I:C) (A), p31 (B), or p33 (C) and then infected with VHSV. Twenty-four hours after infection, VHSV infectivity was estimated by RT-qPCR. Black triangles represent increasing CM dilutions. The results are expressed as percentages of infectivity and calculated by the formula (VHSV infectivity in CM-treated cells/VHSV infectivity in nontreated cells) × 100. Data are the mean percentages of infectivity ± SD for two independent experiments, each performed in triplicate. Asterisks indicate significant differences in VHSV infectivity relative to the level for control cells at P values of <0.05 (*) and P values of <0.01 (**).

FIG. 7.

gpG major mx3-inducing regions mapped onto the surface of the prefusion conformation of the VHSV gpG trimer (viewed looking down toward the membrane). Homology modeling for prediction of the 3D structure of VHSV gpG was carried out as indicated in Materials and Methods, using the prefusion conformation of VSV gpG (PDB file code 2J6J) as a template structure. Green, p31 (residues 280 to 310); magenta, p33 (residues 340 to 370); purple, RGD motif (residues 356 to 358).