A nonsense mutation in the gene encoding 2'-5'-oligoadenylate synthetase/L1 isoform is associated with West Nile virus susceptibility in laboratory mice

Abstract

A mouse model has been established to investigate the genetic determinism of host susceptibility to West Nile (WN) virus, a member of the genus flavivirus and family Flaviviridae. Whereas WN virus causes encephalitis and death in most laboratory inbred mouse strains after peripheral inoculation, most strains derived from recently trapped wild mice are completely resistant. The phenotype of resistance/susceptibility is determined by a major locus, Wnv, mapping to chromosome 5 within the 0.4-cM-wide interval defined by markers D5Mit408 and D5Mit242. We constructed a high resolution composite/consensus map of the interval by merging the data from the mouse T31 Radiation Hybrid map and those from the homologous region of human chromosome 12q, and found the cluster of genes encoding 2'-5'-oligoadenylate synthetases (2'-5'-OAS) to be the most prominent candidate. This cluster encodes a multimember family of IFN-inducible proteins that is known to play an important role in the established endogenous antiviral pathway. Comparing the cDNA sequences of 2'-5'-OAS L1, L2, and L3 isoforms, between susceptible and resistant strains, we identified a STOP codon in exon 4 of the gene encoding the L1 isoform in susceptible strains that can lead to a truncated form with amputation of one domain, whereas all resistant mice tested so far have a normal copy of this gene. The observation that WN virus sensitivity of susceptible mice was completely correlated with the occurrence of a point mutation in 2'-5'-OAS L1 suggests that this isoform may play a critical role in WN pathogenesis.

Fig 1.

Analysis of 74 haplotypes from backcross mice that died after experimental infection with WN virus allowed localization of the genetic determinant for resistance (symbol Wnv) in a 2.7-cM-long segment of mouse chromosome 5. Open rectangles symbolize homozygosity for the microsatellite marker in question, filled rectangles symbolize heterozygosity. Genetic distances between molecular markers are given with 95% confidence intervals. They were calculated by using the whole backcross progeny (203 mice).

Fig 2.

Distribution of viral antigen-positive cells within WN virus-infected brain. BALB/c (a) and BALB/c-MBT (N₇) congenic (b) mice were i.c. inoculated with 1,000 FFU of IS-98-ST1. Day 5 of injection, mouse brains were prepared for frozen sectioning and assayed for the presence of WN antigens by indirect fluorescence-antibody assay. Tissue sections were immunostained with anti-WN polyclonal antibody. Magnification, ≈×40.

Fig 3.

(a) Mice from (BALB/c × MBT)F₁ × MBT or (C57BL/6 × MAI)F₁ × MAI backcross progenies, with a Chr 5 recombinant in the interval where the Wnv locus maps (flanked by microsatellite markers D5Mit431 and D5Mit321), were identified and used for the generation of a second backcross generation (N₃) by crossing with their susceptible parental strain (BALB/c or C57BL/6). Ten of twelve mice of this N₃ generation were bred, then i.p. challenged with 1,000 FFU of IS-98-ST1 virus. Matching the haplotype constitutions to the phenotype of resistance/susceptibility (Res./Sus.) allowed reduction of the critical region to the interval flanked by markers D5Mit431 and D5Mit242 (0.4 cM). (b) Several genes mapping to the region flanked by markers D5Mit431 and D5Mit242, harboring the Wnv locus, were identified in the mouse genetic map established from the T31 Radiation Hybrid (RH) panel. Markers and Aldh2, which are mapped both in mouse and man, allowed in turn the establishment of an exhaustive list of the gene in the region (Table 3).

Fig 4.

The C-T transition in the Arg codon at position 757 results in the occurrence of an opal (stop) codon in exon 4 of the gene encoding L1 isoform of 2′-5′-OAS in laboratory mice. This mutation results in the truncation of the L1 isoform, with amputation of one of its presumptive functional domains (GenBank accession nos: BALB/c, ; MBT/Pas, ). The structure of the gene encoding 2′-5′-OAS L1 isoform, with its six exons, appears at the top of Fig. 4. Scale for exons is 10 times larger than for introns. Susceptible strains, listed on the left side, are all classical laboratory inbred strains whereas most resistant strains, listed on the right side, are inbred strains derived from recently trapped wild specimens. Strain PL/J is the only exception known so far of a resistant classical laboratory inbred strain.