Structural basis for human respiratory syncytial virus NS1-mediated modulation of host responses

Abstract

Human respiratory syncytial virus (hRSV) is a major cause of morbidity and mortality in the paediatric, elderly and immune-compromised populations^1,2. A gap in our understanding of hRSV disease pathology is the interplay between virally encoded immune antagonists and host components that limit hRSV replication. hRSV encodes for non-structural (NS) proteins that are important immune antagonists^3-6; however, the role of these proteins in viral pathogenesis is incompletely understood. Here, we report the crystal structure of hRSV NS1 protein, which suggests that NS1 is a structural paralogue of hRSV matrix (M) protein. Comparative analysis of the shared structural fold with M revealed regions unique to NS1. Studies on NS1 wild type or mutant alone or in recombinant RSVs demonstrate that structural regions unique to NS1 contribute to modulation of host responses, including inhibition of type I interferon responses, suppression of dendritic cell maturation and promotion of inflammatory responses. Transcriptional profiles of A549 cells infected with recombinant RSVs show significant differences in multiple host pathways, suggesting that NS1 may have a greater role in regulating host responses than previously appreciated. These results provide a framework to target NS1 for therapeutic development to limit hRSV-associated morbidity and mortality.

Figure 1. Sequence alignment of NS1 proteins from the genus Orthopneumovirinae

Sequences NS1 from human (hNS1; P04544), bovine (bNS1; Q65694), and NS2 from pneumonia virus of mice (mNS2; Q6PWL3) were aligned by ClustalW. Secondary structural elements for hRSV NS1 are shown above. Residues conserved across all family members are highlighted red, partially conserved are red with a blue box, and not conserved are not highlighted.

Figure 2. hRSV NS1 is a structural paralog of hRSV matrix (M) protein

a, The crystallographic asymmetric unit contains two molecules of hRSV NS1 (molecule A, magenta; molecule B, light blue). b, Structural alignment of hRSV NS1 (magenta) with hRSV M NTD (gray, PDB 2VQP). RMSD is 3.78 Å over 96 residues. Cartoon representation of hRSV c, NS1 and d, M N-terminal domain (NTD) highlighting similar secondary structural elements using color. Secondary structural elements not common between NS1 and M are colored in white. Electrostatic surface representation and topology diagrams of hRSV e–f, NS1 and g–h, M NTD, respectively. d, f top, hRSV NS1 and e, f bottom, hRSV M NTD. For e and g red, white, and blue represent negative, neutral, and positive electrostatic potential, respectively (−5 to +5 kBTe-1). For f and h, coloring scheme is the same in c–d.

Figure 3. hRSV NS1 mutations impact NS1 function

a, Accessible surface area (ASA) analysis of NS1 residues 1-118 and 119-139 using Areaimol with a search probe radius used of 1.4Å. Increase in ASA of NS1 residues 1-118 in the absence of the C-terminal α3 helix is plotted (left). ASA for NS1 residues 1-118 within chain A and B of the crystal structure was calculated in the presence and absence of the helix α3 (119-139). The average of the ASA values of chains A and B were considered. The increase in ASA was calculated by subtracting the average ASA in the absence of helix α3 from that in the presence of α3 helix. For residues 119 to 139 within helix α3 (right), the increase in ASA was calculated in the presence and absence of the rest of the polypeptide (1-118). The dotted line represents average plus one standard deviation of ΔASA. b, Results based on atomistic simulations using the ABSINTH implicit solvation model and forcefield paradigm show overall helical propensity calculated as a probability of finding helical stretches within the α3 sequence with at least L consecutive residues in such a stretch. The light grey colors refer to the peptide in isolation and the dark gray colors are for α3 in the context of the remainder of NS1. c, Simulation results for the per-residue helical propensity in the isolated α3 peptide calculated as a probability of finding helical stretches within the sequence with at least L consecutive residues (see methods) in a stretch. d, Cartoon representation of hRSV NS1 structure highlighting the residues mutated in this study (yellow). e, CD wavelength scans and f, thermal denaturation of NS1 constructs. NS1 WT (black), NS1 F17A (red), NS1 F56A (green), and NS1 Y125A (blue) at 10 μM. g, IFN-b reporter activity upon Sendai virus (SeV) infection in 293T cells (bolded underline) for vector only (E), wildtype and mutants NS1 proteins. Ebola VP35 is used as the positive control. The firefly values are normalized with renilla reporter values. Fold induction is determined by setting mock treated vector values as 1. The experiment is representative of three independent experiments. Each bar represents mean of three replicates with error bars indicating standard deviation. The p-values are determined by one way Anova followed by tukey’s test, **** p<0.0001. The expression of each construct used in the luciferase assay is shown by the western blot using anti-flag antibody.

Figure 4. NS1 unique regions are important for modulating host responses

Human monocyte-derived dendritic cells (MDDCs) were transduced with either only empty lentiviral vectors (empty) or lentivirus vectors that express either EBOV-VP35, hRSV-NS1, hRSV-NS1-(1-118), hRSV-NS1-L132A/L133A. RNA was isolated from transduced MDDCs that were either mock-infected or infected with SeV. RNA was extracted at the indicated hours post-infection; and a, IFN-β, b, TNF-α, c, ISG54, and d, ISG56 mRNA levels were quantified RT-qPCR, normalizing their levels to that of β-actin mRNA. The graph indicates the fold-change relative to the mock-infected samples. For a–d, symbols are: ○, empty vector; ▲, hRSV NS1 1-118; ▼, hRSV NS1 L132A/L133A; ■, hRSV NS1 WT; and ●, EBOV VP35. hRSV NS1 inhibits upregulation of DC maturation markers. Transduced MDDCs were infected with SeV for 20h, harvested and stained for expression of CD40, CD80, CD83 and CD86. Fold change in mean fluorescence intensity (MFI) for the indicated proteins in transduced MDDCs where fold increase indicates comparisons of SeV-infected to uninfected MDDCs. Error bars indicate standard deviations from three independent experiments (*p< 0.05 relative to empty vector-transduced MDDCs at the same time point). e, CD40, f, CD80, g, CD83, and h, CD86 upon SeV infection. i, Growth curves of Vero cells (left) and A549 cells (right) infected with hRSVs encoding wildtype or mutant NS1 proteins. Virus RNA was isolated from medium at indicated time points and quantified by qPCR. Values presented as fold-change relative to 2h time point. Results are expressed as mean ± s.e.m. from three biological replicates. , NS1 wildtype; , NS1 1-118; , NS1 Y125A; and , NS1 L132A/L133A. j, Heatmap representing genes with number of matching reads greater than five in A549 cells 96 hpi post infection with hRSV NS1 wildtype or mutant proteins. Colors represent expression values; red indicates higher expression and blue indicates lower expression within a gene expression profile. k, Heatmaps representing the members of the top set of genes in IFN induction (left panel), IFN response (middle panel), and antioxidative response (right panel) signaling pathways in A549 cells 96 hpi with hRSV NS1 wildtype or mutant proteins.