Japanese Encephalitis Virus NS1' Protein Antagonizes Interferon Beta Production

Abstract

Japanese encephalitis virus (JEV) is a mosquito-borne virus and the major cause of viral encephalitis in Asia. NS1', a 52-amino acid C-terminal extension of NS1, is generated with a -1 programmed ribosomal frameshift and is only present in members of the Japanese encephalitis serogroup of flaviviruses. Previous studies demonstrated that NS1' plays a vital role in virulence, but the mechanism is unclear. In this study, an NS1' defected (rG66A) virus was generated. We found that rG66A virus was less virulent than its parent virus (pSA14) in wild-type mice. However, similar mortality caused by the two viruses was observed in an IFNAR knockout mouse model. Moreover, we found that rG66A virus induced a greater type I interferon (IFN) response than that by pSA14, and JEV NS1' significantly inhibited the production of IFN-β and IFN-stimulated genes. Taken together, our results reveal that NS1' plays a vital role in blocking type I IFN production to help JEV evade antiviral immunity and benefit viral replication.

Keywords: Immune evasion; Japanese encephalitis virus (JEV); NS1′; Type I interferon (IFN-I).

Fig. 1

Recovery and characterization of the rG66A mutant virus. (A) Schematic depiction of the translational pathway of non-PRF and PRF flavivirus polyprotein. The sequence of flammulated letters indicates the slippery hepta-nucleotide frameshift motif and in flammulated oval is the silent nucleotide mutation (G → A) in pSA14 virus contributing downstream pseudoknot interactions. Flammulated arrow indicates where a -1 programmed ribosomal frameshift in pSA14 virus occurs. The frameshift polyprotein was aborted by a termination codon at 52 amino acids after C-terminal extension of NS1. Numbers in the frame represent nucleotides and amino acids in NS2A gene of JEV SA14. (B) BHK-21 cells were infected or mock-infected with pSA14 or rG66A viruses. NS1 and NS1′ proteins were detected by western blotting with anti-NS1 monoclonal antibody (2B8) at 48 h post-infection.

Fig. 2

Comparison of neurovirulence of pSA14 and rG66A virus in mice. A, B Groups of 5-week-old C57BL/6 J mice (n = 10 per group) were infected with 2 × 10² PFU through the intracranial route. C, D Groups of 5-week-old C57BL/6 J mice (n = 10 per group) were infected with 1 × 10⁶ PFU through the intraperitoneal route. E, G Groups of 5-week-old IFNAR^−/− C57BL/6 J mice (n = 8 per group) were infected with 1 × 10⁶ PFU through the intraperitoneal route. Survival curves are presented (A, C, and E). Presentation of clinical signs of disease on indicated days are presented (B, D, and G). Five-week-old C57BL/6 J (F) mice or IFNAR^−/− C57BL/6 J (H) mice were infected intraperitoneally with 1 × 10⁶ PFU of WT-pSA14 and rG66A mutant virus. At day 5 following infection, the blood, spleen, and brain were harvested, weighed, homogenized, and assayed for JEV titers via plaque assay. *P < 0.05; **P < 0.01; ***P < 0.001. Error bars represent SEM. ns, no significance. All data are pooled from two independent experiments.

Fig. 3

NS2A G66A mutation enhances induction of IFN-β and ISGs expression in cells. A, B Kinetics of replication of WT-pSA14 or rG66A mutant virus in TM4 (A) or A549 (B) cells. Cells were infected at an MOI of 1, and the viral titer was measured at 12, 24, 36, and 48 h post-infection using plaque assay of harvested cell supernatant. Growth kinetics from a typical experiment are shown. C, D Replication kinetics of WT-pSA14 or rG66A mutant virus in TM4 (C) or A549 (D) cells treated with IFNAR pAb. TM4 and A549 cells were treated with pAb followed by infection of pSA14 or rG66A at an MOI of 1. Viral titer was measured at 12, 24, 36, and 48 h post-infection using plaque assay with harvested cell supernatant. Growth kinetics from a typical experiment are shown. (E) Replication kinetics of WT-pSA14 and rG66A mutant virus in Vero cells. Cells were infected with pSA14 or rG66A at an MOI of 1, and the viral titer was measured at 12, 24, 36, and 48 h post-infection using plaque assay with harvested culture fluids. Growth kinetics from a typical experiment is shown. F, G TM4 (F) and A549 (G) cells were infected with WT-pSA14 or rG66A mutant virus (MOI = 1). mRNA levels of IFN-β, ISG15, OAS-1, and Mx-1 were determined by qRT-PCR at 24 h post-infection. *P < 0.05, **P < 0.01, ***P < 0.001. All data are pooled from two or three independent experiments.The results were normalized to β-actin expression in each sample. Con means control.

Fig. 4

JEV NS1′ reduces poly(I:C) triggered IFN-β and ISGs. A HeLa cells were transfected with empty vector (EV) or plasmid encoding NS1 or NS1′. Expression of JEV NS1/NS1′ protein was determined by immunoblotting at 24 h post-transfection with an anti-NS1 antibody (2B8), and GAPDH served as a reference control. B HeLa cells were transfected with an EV or plasmid encoding NS1 or NS1′, followed by treatment with or without poly(I:C). After 24 h of incubation, the mRNA levels of IFN-β, ISG15, OAS-1, and Mx-1 were determined by qRT-PCR. C Kinetics of replication of WT-pSA14 or rG66A mutant virus in HeLa cells. Cells were transfected with an empty vector or expression vector encoding NS1 or NS1′ for 12 h and then infected with WT-pSA14 or rG66A mutant virus at an MOI of 1 for 24 h. Growth kinetics from a typical experiment are shown. D HeLa cells were transfected with EV, or plasmid encoding NS1 or NS1′, followed by infection with WT-pSA14 or rG66A mutant virus at an MOI of 1. mRNA levels of IFN-β, ISG15, OAS-1, and Mx-1 were determined by qRT-PCR at 24 h post-infection. *P < 0.05, **P < 0.01, ***P < 0.001. ns, no significance. All data are pooled from two or three independent experiments. The results were normalized to β-actin expression in each sample.