Ifit1 inhibits Japanese encephalitis virus replication through binding to 5' capped 2'-O unmethylated RNA

Abstract

The interferon-inducible protein with tetratricopeptide (IFIT) family proteins inhibit replication of some viruses by recognizing several types of RNAs, including 5'-triphosphate RNA and 5' capped 2'-O unmethylated mRNA. However, it remains unclear how IFITs inhibit replication of some viruses through recognition of RNA. Here, we analyzed the mechanisms by which Ifit1 exerts antiviral responses. Replication of a Japanese encephalitis virus (JEV) 2'-O methyltransferase (MTase) mutant was markedly enhanced in mouse embryonic fibroblasts and macrophages lacking Ifit1. Ifit1 bound 5'-triphosphate RNA but more preferentially associated with 5' capped 2'-O unmethylated mRNA. Ifit1 inhibited the translation of mRNA and thereby restricted the replication of JEV mutated in 2'-O MTase. Thus, Ifit1 inhibits replication of MTase-defective JEV by inhibiting mRNA translation through direct binding to mRNA 5' structures.

Fig 1

Generation of an MTase-defective JEV mutant. (A) Sequence homology between NS5 proteins of JEV (AT31 strain, GenBank accession number AB196926) and WNV (00-3356 strain, GenBank accession number EF530047). Arrowheads, MTase catalytic K-D-K-E tetrad; *, consensus sequences between the two proteins. (B) 2′-O MTase activity of JEV WT and JEV K61A mutant recombinant NS5 proteins by thin-layer chromatography assays. The substrate m7GpppA-RNA (³²P-labeled JEV 5′-terminal 200 nucleotides) was methylated in vitro with the respective recombinant NS5 proteins or vaccinia virus 2′-O MTase, digested with P1 nuclease, and developed on PEI-cellulose plates. The positions of 2′-O methylated (m7GpppAm) and unmethylated (m7GpppA) RNA are indicated. Data are representative of four independent experiments.

Fig 2

Generation of Ifit1^−/− mice. (A) Schematic representation of the Ifit1 gene-targeting strategies. Solid boxes, coding regions of the Ifit1 gene; open boxes, untranslated regions; Neo and HSV tk, a neomycin-resistance gene cassette and a herpes simplex virus thymidine kinase gene, respectively. The positions of the probe and restriction enzyme site for Southern blotting are shown. (B) Genomic DNA was isolated from the tails of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) Ifit1 mutant mice. A Southern blot analysis performed after digestion of the genomic DNA with BamHI shows the correct targeting of the locus. (C) Peritoneal exudative macrophages were harvested from wild-type (+/+) or Ifit1-deficient (−/−) mice. Total RNA (10 μg) was blotted onto a nylon membrane, and Ifit1 and β-actin mRNA expression was detected by Northern blot analysis with the respective cDNA probes. LPS lanes, cells stimulated with 100 ng/ml of lipopolysaccharide for 4 h to induce endogenous Ifit1 expression; Med lanes, cells treated with medium alone.

Fig 3

Ifit1^−/− MEFs and macrophages fail to restrict the replication of the 2′-O MTase mutant JEV. (A, B) Culture supernatants of wild-type and Ifit1^−/− MEFs (A) and macrophages (B) infected with JEV WT and the JEV K61A mutant (MOIs, 0.1 for MEFs and 0.5 for macrophages) were harvested at the indicated days postinfection. The virus titers in 1-ml supernatant aliquots were determined by focus-forming assays on Vero cells and expressed as the log₁₀ number of FFU/ml. Data are shown as means ± SDs of quadruplicate samples generated from four independent experiments with statistical significance. ND, not detected. *, P < 0.05. (C, D) Accumulation of JEV WT (C) and the JEV K61A mutant (D) RNA in wild-type and Ifit1^−/− MEFs at 4 days postinfection determined by quantitative real-time RT-PCR. JEV NS5 RNA levels were normalized to the level of host GAPDH and are expressed as the fold change in Ifit1^−/− cells versus wild-type cells (value for wild type = 1). Data are representative of three independent experiments with statistical significance. *, P < 0.05. (E) Culture supernatants of vector-transduced (+vector) and Flag-tagged Ifit1 gene-transduced (+Ifit1) Ifit1^−/− MEFs infected with the JEV K61A mutant (MOI, 0.1) were harvested at 3 days postinfection. The virus titers in 1-ml supernatant aliquots were determined by focus-forming assays on Vero cells and expressed as the log₁₀ number of FFU/ml. Expression of Ifit1 and β-actin determined by immunoblotting with anti-Flag or anti-β-actin antibodies is shown at the bottom. Data are representative of three independent experiments. *, P < 0.05. (F) Wild-type and Ifit1^−/− MEFs were infected with the JEV K61A mutant (MOI, 0.1). At 4 days postinfection, cells were harvested and analyzed for Ifnb expression by quantitative RT-PCR. Ifnb RNA levels were expressed relative to those of GAPDH. ND, not detected. Data are shown as means ± SDs and are representative of data from three independent experiments.

Fig 4

Ifit1 preferentially binds to virus RNA lacking 2′-O methylation. (A) Electrophoretic mobility shift of biotin-labeled RNA (JEV 5′-terminal 200 nucleotides) with recombinant Ifit1. The presence or absence of a 5′ cap and 2′-O Me of the JEV 5′-terminal 200 nucleotides is indicated. Unlabeled 5′-PPP RNA was used as a competitor. The loss of the band indicates binding of RNA and Ifit1 (top). The band intensities (in percent) calculated by ImageJ are shown at the bottom. Data are representative (top) and means ± SDs (bottom) of five independent experiments. *, P < 0.05. (B) Lysates from HEK293T cells transfected with HA-tagged Ifit1 were incubated with 2.5 pmol of biotin-labeled RNA. The presence or absence of a 5′ cap and 2′-O Me of the JEV 5′-terminal 200 nucleotides is indicated. 5′ OH RNA was produced by incubating in vitro-transcribed RNA with CIAP. RNA was incubated with streptavidin beads, and the precipitates were separated by SDS-PAGE and immunoblotted with an anti-HA antibody (top). Med and Input, samples from whole-cell lysates of empty vector- and Ifit1-transfected 293T cells, respectively. The percent band intensities calculated by ImageJ are shown at the bottom. Data are representative (top) and means ± SDs (bottom) of three independent experiments. *, P < 0.05. (C) MEFs stably expressing Ifit1 (Ifit1^+/+ + Ifit1) or Ifit1^−/− MEFs were infected with JEV WT or the JEV K61A mutant at an MOI of 1.0. The cells were harvested after 24 h, and JEV RNA/Ifit1-binding complexes were immunoprecipitated with a mouse anti-Flag antibody or mouse IgG. The immunoprecipitated RNA was analyzed by nested RT-PCR using primers that detect the JEV NS1 gene. Each value was normalized by the value for the input (indicated in percent). Data are means ± SDs of three independent experiments. *, P < 0.05.

Fig 5

Ifit1 selectively inhibits the translation of mRNA lacking 2′-O methylation. (A) The luciferase RNA amounts at 6 h after RNA transfection were determined by quantitative real-time RT-PCR. The relative luciferase mRNA amounts, calculated as the amount of each transfected mRNA (luc2) divided by the level of GAPDH mRNA expression, are shown. The presence or absence of a 5′ cap and 2′-O Me of the introduced luciferase RNA is indicated. Data are shown as means ± SDs and are representative of three independent experiments. (B, C) Wild-type and Ifit1^−/− MEFs pretreated with type I IFN (B) or untreated (C) were transiently transfected with luciferase mRNA. Luciferase activities were measured at 6 h after the transfection and are shown as relative light units (RLU). The presence or absence of a 5′ cap and 2′-O Me of the introduced luciferase RNA is indicated. Data are shown as means ± SDs of triplicate samples of the representative results. Similar results were obtained in three independent experiments. *, P < 0.05.