We screened peritoneal macrophages isolated from third generation (G3) C57BL/6J mice carrying ENU-induced mutations for susceptibility to ex vivo
infection by viruses (MCMV-GFP [27
] or the mouse-adapted influenza virus strain PR8) or a nonreplicating viral vector (hAd5-F16-GFP [16
]). The number of macrophages infected by MCMV or hAd5-F16 vector was monitored by flow cytometry to detect GFP-labeled cells; infection by PR8 was determined by the reactivity of macrophages to hemagglutinin (HA) antibody, which also was monitored by flow cytometry. After incubation with MCMV-GFP or PR8 for 24 h or with hAd5-F16-GFP for 72 h, the number of infected macrophages from C57BL/6J and G3 mice bearing ENU-induced mutations in homozygous and/or heterozygous form was analyzed.
To validate the effectiveness of the screen, we tested macrophages from mice with known mutations (A). Macrophages from homozygous Stat1domino
) mice were highly permissive to infection by all three viruses, confirming the known requirement for type I IFN signaling in viral control (17
). In contrast, normal percentages of macrophages from Tlr3−/−
), or Myd88poc/poc
) mice were infected by MCMV-GFP, hAd5-F16-GFP, or PR8, indicating that redundant mechanisms for sensing these viruses exist in macrophages, or that requirements for sensing these viruses differ between macrophages and other cell types. IFN-γ signaling also was dispensable for the control of MCMV-GFP, hAd5-F16-GFP, or PR8 by macrophages, likely because macrophages are not a significant source of IFN-γ.
Fig 1 Screen for increased susceptibility to infection by MCMV, human adenovirus, and influenza virus. Thioglycolate-elicited peritoneal macrophages were infected with MCMV-GFP (24 h at an MOI of 1), hAd5-F16-GFP (72 h at 104 particles/cell), or PR8 (24 h at (more ...)
Macrophages from a total of 4,500 G3 mice were infected with MCMV-GFP, hAd5-F16-GFP, and PR8 (B). Two strains, designated macro-1 and macro-2, produced macrophages that were infected by all three viruses with increased frequency relative to that of C57BL/6J macrophages (B). Both the macro-1 and macro-2 phenotypes were transmitted recessively. Macrophages from a third strain, atc, were infected with increased frequency by hAd5-F16-GFP and MCMV-GFP (B). The permissiveness of atc macrophages to hAd5-F16-GFP was similar to that of macrophages from Stat1dom/dom mice, whereas permissiveness to MCMV-GFP was intermediate between wild-type and Stat1dom/dom levels. Infection by PR8 was equally well controlled by atc and C57BL/6J macrophages. The atc phenotype also was recessive. As another measure of viral control, the amount of type I IFN produced by the infected macrophages was measured in the culture supernatant. In response to dsDNA, type I IFN production by macro-2 macrophages was reduced compared to that produced by wild-type cells (C). In contrast, hAd5-F16-GFP induced normal type I IFN production by atc macrophages (C).
Because of the importance of type I IFN signaling to the innate antiviral response, the genes encoding STAT1 (Stat1
), Isgf3g (Irf9
), JAK1 (Jak1
), Tyk2 (Tyk2
), and the two chains of the type I IFN receptor (Ifnar1
) were sequenced in macro-1
mice. An adenine-to-cytosine transversion in exon 8 of Ifnar1
, causing the missense mutation T341P in IFNAR1, was identified in macro-1
mice (A). An adenine-to-guanine transition in exon 2 of Ifnar2
, causing the replacement of the start methionine with valine, was found in macro-2
mice (B). The susceptibility of macro-1
macrophages to infection by MCMV, human adenovirus, and influenza is consistent with a deficiency of IFNAR signaling, as is the reduced dsDNA- and adenovirus-dependent type I IFN production, which is known to be regulated by a positive feedback loop in which IFN-β and IFN-α4 induce the expression of the transcription factors IRF7 and IRF8 (25
). The detection of IFNAR1 and IFNAR2 deficiencies validated the effectiveness of the screen.
Fig 2 Ifnar1 and Ifnar2 mutations in macro-1 and macro-2 mice. DNA sequence chromatograms showing the mutated nucleotides in Ifnar1 (adenine to cytosine at position 1115 of cDNA) (A) and Ifnar2 (adenine to guanine at position 221 of cDNA) (B). The lower panels (more ...)
phenotype was distinct from those of mice with Ifnar1
deficiency, and therefore the mutation was mapped by bulk segregation analysis (BSA). Animals with the atc
) were outcrossed to C57BL/10J mice, and affected F1 mice were backcrossed to atc
). Using 40 F2 mice with mutant phenotypes and 9 with normal phenotypes, BSA indicated the strongest linkage of the atc
mutation with a marker at position 103735349 on chromosome 2 (synthetic logarithm of odds [LOD], 6.0) (A). Among nucleotides covered at least once within the 38 Mb surrounding the marker with peak linkage (19 Mb upstream and downstream), 327 mutations were identified by SOLiD whole-genome sequencing, of which 281 were successfully reexamined by conventional capillary sequencing. A single mutation was validated at position 118226389, 14.5 Mb from the marker with peak linkage (B). The mutation corresponds to a thymine-to-cytosine transition of the sixth nucleotide of intron 2 of Eif2ak4
, encoding GCN2 (). The cDNA sequencing of the four largest Eif2ak4
transcripts demonstrated that the mutation invariably results in the skipping of exon 2, in some cases along with the skipping of other exons (D). The most abundant transcript lacked exons 2, 3, and 4. No GCN2 expression was detected in Eif2ak4atc/atc
macrophages by immunoblotting (E).
Fig 3 Mutation in Eif2ak4 in atc mice. (A) BSA mapping of the atc mutation using 40 mice with mutant phenotypes and 9 mice with normal phenotypes. LOD scores for 124 markers across the genome were calculated based on BSA. (B) DNA sequence chromatogram showing (more ...)
We evaluated the function of the GCN2atc
protein upon the stimulation of Eif2ak4atc/atc
mouse embryonic fibroblasts with UV radiation, a known activator of GCN2 (9
). As expected, UVB resulted in the increased phosphorylation of eIF2α in C57BL/6J fibroblasts but not in Eif2ak4atc/atc
fibroblasts (F), indicating that the atc
mutation causes the complete loss of GCN2 function, at least where eIF2α phosphorylation is concerned.
To determine whether eIF2α phosphorylation by GCN2 is part of the antiviral response to DNA viruses, we compared eIF2α phosphorylation (eIF2α-P) levels by immunoblotting in C57BL/6J and Eif2ak4atc/atc fibroblasts 0, 8, 16, 24, 32, and 40 h after infection with MCMV. C57BL/6J fibroblasts displayed minimal eIF2α-P at each time point up to 32 h but increased eIF2α-P 40 h after MCMV infection. In contrast, no eIF2α-P was detectable in Eif2ak4atc/atc fibroblasts at any time point (A). In a separate experiment, we measured viral titers at the same time intervals in the culture supernatant of wild-type and Eif2ak4atc/atc fibroblasts. Supernatant from Eif2ak4atc/atc fibroblasts displayed titers similar to those of wild-type cell supernatant at all time points examined (B). These findings suggest that eIF2α phosphorylation by GCN2 is not required to prevent virus replication and shedding for at least 32 h after infection; an effect on viral titer may lag behind the observation of defective eIF2α phosphorylation in Eif2ak4atc/atc cells, which first occurs 40 h postinfection.
Fig 4 Increased susceptibility to MCMV infection in Eif2ak4atc/atc mice. (A) Immunoblot analysis of eIF2α phosphorylation (eIF2a-P) on serine 51 in whole-cell lysates of fibroblasts from C57BL/6J or Eif2ak4atc/atc mice at the indicated times after infection (more ...)
We then tested whether the deficiency in eIF2α-P affects susceptibility to MCMV in vivo. C57BL/6J and Eif2ak4atc/atc mice were injected intraperitoneally with 2 × 105 PFU/mouse of MCMV and observed for sickness. During the 2-week period following infection, 11 of 68 Eif2ak4atc/atc mice died, whereas all 39 of the C57BL/6J controls remained healthy (P = 0.0041) (C). Thus, GCN2 deficiency increases susceptibility to MCMV infection in mice as it does in cultured macrophages.
The deaths of Eif2ak4atc/atc
mice in response to MCMV infection all occurred on or before 9 days postinfection, which is consistent with the hypothesis that susceptibility stems from a defect in the innate immune response. However, the expression of Eif2ak4
has been identified as part of a transcriptional signature highly correlated with the strength of the adaptive immune response, in particular the CD8+
T-cell response, to the yellow fever vaccine YF-17D (30
). We therefore tested the antibody responses of Eif2ak4atc/atc
mice but found that they mounted normal IgG responses to the T-dependent antigen β-galactosidase and normal IgM responses to the T-independent antigen NP-Ficoll (D). This finding, together with the fact that Rag1-deficient mice survive MCMV infection without incident for several weeks after inoculation, supports the interpretation that MCMV susceptibility in Eif2ak4atc/atc
mice is caused by an innate immune defect.