The Central Conserved Region (CCR) of Respiratory Syncytial Virus (RSV) G Protein Modulates Host miRNA Expression and Alters the Cellular Response to Infection

Abstract

Respiratory Syncytial Virus (RSV) infects respiratory epithelial cells and deregulates host gene expression by many mechanisms including expression of RSV G protein (RSV G). RSV G protein encodes a central conserved region (CCR) containing a CX3C motif that functions as a fractalkine mimic. Disruption of the CX3C motif (a.a. 182-186) located in the CCR of the G protein has been shown to affect G protein function in vitro and the severity of RSV disease pathogenesis in vivo. We show that infection of polarized Calu3 respiratory cells with recombinant RSV having point mutations in Cys173 and 176 (C173/176S) (rA2-GC12), or Cys186 (C186S) (rA2-GC4) is associated with a decline in the integrity of polarized Calu-3 cultures and decreased virus production. This is accompanied with downregulation of miRNAs let-7f and miR-24 and upregulation of interferon lambda (IFNλ), a primary antiviral cytokine for RSV in rA2-GC12/rA2-GC4 infected cells. These results suggest that residues in the cysteine noose region of RSV G protein can modulate IFN λ expression accompanied by downregulation of miRNAs, and are important for RSV G protein function and targeting.

Keywords: CX3CR1; IFNλ; RSV; microRNAs; respiratory syncytial virus.

Figure 1

rA2-GC4 infection results in a decline of polarized Calu-3 cells. Data represent the median resistance measurement, Ω × cm², of polarized liquid-covered culture (LCC) Calu-3 cells infected apically with RSVA2, rA2-GC12 or rA2-GC4 at a multiplicity of infection (MOI) of 1 for varying time points. The data presented is representative of one of three independent experiments. * represents significance (p ≤ 0.05) relative to mock as determined by one-way ANOVA and Bonferroni post hoc comparison of all data at α = 0.05).

Figure 2

Mutations in the CX3C motif affect folding patterns of the cysteine noose. Peptide sequences corresponding to the RSV G protein central conserved region (CCR) were analyzed for folding pattern using Pepfold [57]. Arrows indicate positions of cysteine in the RSV A2 backbone and the changes in the rA2-GC12 and rA2-GC4 peptides.

Figure 3

rA2-GC4 and rA2-GC12 viruses replicate poorly in polarized liquid covered culture (LCC)-Calu3 cells. Data represent relative levels of RSV M mRNA from RSV/A2, rA2-GC12 or rA2-GC4 virus infected LCC-Calu3 wells (n = 3) ± SEM in PFU/mL as determined by qRT-PCR at indicated time points. * indicates statistical significance (p ≤ 0.05) as determined by one-way ANOVA followed by the post hoc Bonferroni test at a significance level (α = 0.05).

Figure 4

The central conserved region (CCR) of RSV G protein alters IFNλ expression. Polarized LCC Calu-3 cells were infected with RSV/A2, rA2-GC12 or rA2-GC4 virus (MOI = 1.0). Total RNA was isolated from mock and infected cells at 12 h and 24 h pi. Expression of IFNα1, IFN λ, SOCS-1 and SOC-3 were determined by gene specific primer-probe combinations using a one-step qRT-PCR assay at 12 h (a) or 24 h (b) pi Data represent ± SEM of three independent experiments. ** indicates statistical significance (p < 0.01) and **** indicates statistical significance (p < 0.001) as determined by two-way ANOVA and the Bonferroni post hoc test.

Figure 5

The central conserved region (CCR) of RSV G protein alters miRNA expression. Total RNA from polarized LCC Calu-3 cells mock infected, or infected apically with RSV A2, rA2-GC12 or rA2-GC4 at MOI = 1 was isolated using Trizol as per manufacturer’s protocol. Expression of let-7f (a) and miR-24 (b) was determined with RT-qPCR using a miRNA specific forward oligo and a universal oligo relative to 18S as housekeeping gene. Data represent ±SEM three independent experiments. * indicates p ≤ 0.05 relative to values from RSV-A2.