Extraordinary GU-rich single-strand RNA identified from SARS coronavirus contributes an excessive innate immune response

Abstract

A dangerous cytokine storm occurs in the SARS involving in immune disorder, but many aspects of the pathogenetic mechanism remain obscure since its outbreak. To deeply reveal the interaction of host and SARS-CoV, based on the basic structural feature of pathogen-associated molecular pattern, we created a new bioinformatics method for searching potential pathogenic molecules and identified a set of SARS-CoV specific GU-rich ssRNA fragments with a high-density distribution in the genome. In vitro experiments, the result showed the representative SARS-CoV ssRNAs had powerful immunostimulatory activities to induce considerable level of pro-inflammatory cytokine TNF-a, IL-6 and IL-12 release via the TLR7 and TLR8, almost 2-fold higher than the strong stimulatory ssRNA40 that was found previously from other virus. Moreover, SARS-CoV ssRNA was able to cause acute lung injury in mice with a high mortality rate in vivo experiment. It suggests that SARS-CoV specific GU-rich ssRNA plays a very important role in the cytokine storm associated with a dysregulation of the innate immunity. This study not only presents new evidence about the immunopathologic damage caused by overactive inflammation during the SARS-CoV infection, but also provides a useful clue for a new therapeutic strategy.

Fig. 1

TNF-a release induced by the ssRNA mediated by TLR7. The RAW264.7 cells in the experiment groups were treated by 5 μg/ml ssRNA for 24 h.

Fig. 2

Cytokines release induced by the ssRNAs at different dose mediated by TLR8. (a) TNF-a production after 10 h stimulation; (b) IL-12 production after 24 h stimulation; (c) IL-6 production after 24 h stimulation.

Fig. 3

TNF-a level changes after TLR7 or TLR8 knockdown at 50% siRNA transfection rates. Column 1 in the TLR7 group, RAW264.7 cells treated only with 100 μ/ml DOTAP/HBS. Column 2 in the TLR7 group, RAW264.7 cells treated with 5 μg/ml ssRNA83 for 24 h. Column 3 in the TLR7 group, the 5 pmol/ml TLR7 siRNA transfected into RAW264.7 cells before treated with 5 μg/ml ssRNA83 for 24 h; Column 1 in the TLR8 group, THP1 cells treated only with 100 μ/ml DOTAP/HBS. Column 2 in the TLR8 group, THP1 cells treated with 5 μg/ml ssRNA120 for 24 h. Column 3 in the TLR8 group, the 5 pmol/ml TLR8 siRNA transfected into THP1 cells before treated with 5 μg/ml ssRNA120 for 24 h.

Fig. 4

Multiple cytokines release of hPBMC induced by ssRNA. hPBMC were treated by 5 μg/ml ssRNA for 24 h.

Fig. 5

Life table analysis of mice under attack of SARS-CoV specific ssRNA. 1. Control group (n = 10); 2. Treatment group with 1 mg/kg SARS ssRNA83 (n = 10).

Fig. 6

Lung histopathological changes of mice (H&E × 100). (A) Control group; (B) treatment group with 1 mg/kg SARS ssRNA83.

Fig. 7

Cytokines release under ssRNA83 treatment. Treatment group with 1 mg/kg SARS ssRNA83 for each mouse for 4 h.

Fig. S1

Comparison of the stimulatory activities by TLR8 between ssRNA120 and fakeRNA120.