Interferon-induced Ifit2/ISG54 protects mice from lethal VSV neuropathogenesis

Abstract

Interferon protects mice from vesicular stomatitis virus (VSV) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (ISG) mediate the antiviral effect. A prominent family of ISGs is the interferon-induced with tetratricopeptide repeats (Ifit) genes comprising three members in mice, Ifit1/ISG56, Ifit2/ISG54 and Ifit3/ISG49. Intranasal infection with a low dose of VSV is not lethal to wild-type mice and all three Ifit genes are induced in the central nervous system of the infected mice. We tested their potential contributions to the observed protection of wild-type mice from VSV pathogenesis, by taking advantage of the newly generated knockout mice lacking either Ifit2 or Ifit1. We observed that in Ifit2 knockout (Ifit2(-/-)) mice, intranasal VSV infection was uniformly lethal and death was preceded by neurological signs, such as ataxia and hind limb paralysis. In contrast, wild-type and Ifit1(-/-) mice were highly protected and survived without developing such disease. However, when VSV was injected intracranially, virus replication and survival were not significantly different between wild-type and Ifit2(-/-) mice. When administered intranasally, VSV entered the central nervous system through the olfactory bulbs, where it replicated equivalently in wild-type and Ifit2(-/-) mice and induced interferon-β. However, as the infection spread to other regions of the brain, VSV titers rose several hundred folds higher in Ifit2(-/-) mice as compared to wild-type mice. This was not caused by a broadened cell tropism in the brains of Ifit2(-/-) mice, where VSV still replicated selectively in neurons. Surprisingly, this advantage for VSV replication in the brains of Ifit2(-/-) mice was not observed in other organs, such as lung and liver. Pathogenesis by another neurotropic RNA virus, encephalomyocarditis virus, was not enhanced in the brains of Ifit2(-/-) mice. Our study provides a clear demonstration of tissue-, virus- and ISG-specific antiviral action of interferon.

Figure 1. Generation of Ifit2/ISG54 and Ifit1/ISG56 knockout mice.

A, gene targeting strategy for genomic deletion of complete protein-encoding regions of Ifit2 = ISG54 or Ifit1 = ISG56 in embryonic stem cells; TAG/TGA, stop codons; grey boxes = exons. B, genotyping of deficiency for Ifit2 or Ifit1 by PCR on mouse tail DNA. C, IFN-β-induced protein expression of Ifit2/P54, Ifit1/P56 and Ifit3/P49 in Ifit2 ^−/− or Ifit1^−/− primary MEFs.

Figure 2. Ifit2 protects mice from lethal intranasal VSV infection.

A, survival of Ifit2^−/−, IFNAR ^−/− and wt mice after intranasal infection with 4×10² pfu of VSV Indiana. B, survival of Ifit1^−/− and Ifit2-heterozygous (Ifit2^+/−) mice after infection with 4×10² pfu of VSV; experiments in A and B shared wt mice (n = number of animals used). C, survival of Ifit2^−/−, Ifit1^−/− and wt mice after intranasal infection with a higher dose of VSV (4×10⁶ pfu). D, survival of Ifit2^−/−, Ifit1^−/−, IFNAR ^−/− and wt mice after infection with 5×10² pfu of EMCV. In A–D, data are cumulative from at least two independent experiments (exceptions: Figure 2B, Ifit2 ^+/− mice and Figure 2D, Ifit1 ^−/− mice infected in a single experiment). Statistical significance of survival differences relative to wt mice is indicated by p-values; n.s., not significant; i.n., intranasal.

Figure 3. Ifit2 does not inhibit VSV entry and replication in olfactory bulbs.

A, schematic entry route of VSV into the central nervous system of wt mice after intranasal infection, and VSV spread within brain, as reported in the literature. OB, olfactory bulbs; CX, cortex; MB, midbrain; CB, cerebellum; BS, brain stem; SC, spinal cord. B, VSV P protein in OB of VSV-infected wt, Ifit2 ^−/− and IFNAR ^−/− mice at 2 d.p.i., detected by immunohistofluorescence. C, VSV RNA levels in OB of uninfected or VSV-infected wt, Ifit2 ^−/− and IFNAR ^−/− mice at 1, 2 or 6 d.p.i., plotted as mean+SD on log scale; ND, none detected. D, infectious VSV titers in wt and Ifit2 ^−/− OB at 2 and 6 d.p.i.; plotted as pfu/g with mean on log scale; dashed line depicts threshold of detection. In C and D, n = 4–8 mice per infected group accumulated from three independent experiments; in B, n = 2 mice from two independent experiments. All infections were 4×10² pfu of VSV administered intranasally. Asterisks indicate statistical significance: ** p = 0.006, * p<0.05; n.s.: not significant.

Figure 4. Ifit2 suppresses VSV replication in the brain after intranasal infection.

A, infectious VSV titers in wt and Ifit2 ^−/− brains at 2 and 6 days after intranasal infection, plotted as pfu/g with mean on log scale; dashed line depicts threshold of detection. B, VSV RNA levels in brains of uninfected or VSV-infected wt, Ifit2 ^−/− and IFNAR ^−/− mice at 2 or 6 d.p.i., plotted as mean+SD on log scale. C, VSV RNA levels in different regions of the brains of uninfected or VSV-infected wt and Ifit2 ^−/− mice at 6 d.p.i., plotted as mean+SD on log scale. D, VSV P protein in midbrain neurons of Ifit2 ^−/− mice at 6 d.p.i.; detection by immunohistofluorescence-labeling of VSV-P (red) and neuron (NeuN) or astrocyte (GFAP) markers (green); in A and B: n = 4–8 mice per infected group accumulated from three independent experiments; in C: n = 4 mice per infected group; in D: n = 2 mice per infected group; all infections in A–D were intranasal with 4×10² pfu of VSV. ND, none detected. Brains in A and B were separated from OBs assayed in Figure 3D and 3C, respectively. Asterisks indicate statistical significance: *** p≤0.0009; n.s.: not significant.

Figure 5. Ifit2 and Ifit1 are induced in VSV-infected regions of OB and brain.

A/B, D/E, Ifit2, Ifit1 and IFN-β mRNA levels in OB (A, B) and separated brains (D, E) of uninfected or intranasally VSV-infected wt, Ifit2 ^−/− and IFNAR ^−/− mice at 2 or 6 d.p.i., plotted as mean+SD. The same OB and brains were also assayed in Figure 3C and 4B for VSV RNA levels. In A/B/D/E: n = 4–8 mice per infected group accumulated from three independent experiments; ND, none detected. C, Ifit2 ( = P54) protein in wt OB sections, uninfected or 2 d.p.i., with parallel detection of VSV P protein in adjacent sections, detected by immunohistochemistry; arrowheads indicate Ifit2-positive, VSV-negative cells surrounding the glomeruli; n = 2 mice. All infections were intranasal with 4×10² pfu of VSV. Asterisks indicate statistical significance: ** p<0.005, *** p<0.0005; n.s.: not significant.

Figure 6. Wt mice are as susceptible as Ifit2^−/− mice to intracranial VSV infection.

A, survival of wt and Ifit2^−/− mice after intracranial injection of 10 pfu of VSV; cumulative data from three independent experiments. B, infectious VSV titers in wt and Ifit2 ^−/− brains at 24 and 48 hours after intracranial injection of 10 pfu of VSV; plotted as pfu/g with mean on log scale; dashed line depicts threshold of detection; n = 7 mice per infected group from two independent experiments; n.s.: not significant. C, Ifit2, Ifit1, IFN-β and VSV-N RNA levels in brains of intracranially VSV-infected or PBS-injected wt and Ifit2 ^−/− mice at 24 hours p.i., plotted as mean+SD, VSV RNA levels plotted on log scale; n = 3 mice per infected group; ND, none detected.

Figure 7. Unlike the brain, other organs of Ifit2^−/− mice are not more susceptible to intranasal VSV infection.

A–C, infectious VSV titers in organs of wt, Ifit2 ^−/− or IFNAR ^−/− mice (n = 4–10 mice per group accumulated from three independent experiments) at 2, 4, 5 and 6 days after intranasal infection; livers (A), lungs (B) and brains (C, incl. OB) of the same mice were assayed and plotted as pfu/g with mean; not all available livers were titered. Dashed line depicts threshold of detection. D/E, Ifit2, Ifit1, IFN-β mRNA levels in livers (D) and lungs (E) of uninfected or VSV-infected wt mice at 2 d.p.i., plotted as mean+SD; n = 4 mice per infected group; ND, none detected. All infections were intranasal with 4×10² pfu of VSV. Asterisks indicate statistical significance: * p<0.05, ** p<0.005.