RNA editing enzyme adenosine deaminase is a restriction factor for controlling measles virus replication that also is required for embryogenesis

Abstract

Measles virus (MV), a member of the family Paramyxoviridae and an exclusively human pathogen, is among the most infectious viruses. A progressive fatal neurodegenerative complication, subacute sclerosing panencephalitis (SSPE), occurs during persistent MV infection of the CNS and is associated with biased hypermutations of the viral genome. The observed hypermutations of A-to-G are consistent with conversions catalyzed by the adenosine deaminase acting on RNA (ADAR1). To evaluate the role of ADAR1 in MV infection, we selectively disrupted expression of the IFN-inducible p150 ADAR1 isoform and found it caused embryonic lethality at embryo day (E) 11-E12. We therefore generated p150-deficient and WT mouse embryo fibroblast (MEF) cells stably expressing the MV receptor signaling lymphocyte activation molecule (SLAM or CD150). The p150(-/-) but not WT MEF cells displayed extensive syncytium formation and cytopathic effect (CPE) following infection with MV, consistent with an anti-MV role of the p150 isoform of ADAR1. MV titers were 3 to 4 log higher in p150(-/-) cells compared with WT cells at 21 h postinfection, and restoration of ADAR1 in p150(-/-) cells prevented MV cytopathology. In contrast to infection with MV, p150 disruption had no effect on vesicular stomatitis virus, reovirus, or lymphocytic choriomeningitis virus replication but protected against CPE resulting from infection with Newcastle disease virus, Sendai virus, canine distemper virus, and influenza A virus. Thus, ADAR1 is a restriction factor in the replication of paramyxoviruses and orthomyxoviruses.

Fig. 1.

Generation of the Adar1^p150 gene disruption. (A) Schematic illustrating the gene targeting strategy to disrupt expression of the p150 isoform of ADAR1 specifically. Genomic sequences flanking the Neo cassette and encompassing the IFN-inducible P_A promoter and associated exon 1A regions were inserted into the targeting vector to yield pKO-Adar1^p150-floxP, thus resulting in specific disruption of the corresponding region of the Adar1 locus on homologous recombination. (B) Representative genotyping results of embryos obtained at E14 and homozygous for the disrupted Adar1^p150 allele (lanes 2 and 3) and heterozygous for the Adar1^p150 disruption (lanes 4 and 5) or WT (lanes 6 and 7). The DNA size standard is shown in lane 1. (C) Frequencies of offspring and associated genotypes resulting from interbreeding of mice heterozygous for disruption of the p150 isoform of ADAR1. (D) Representative embryo morphologies seen for WT embryos or embryos heterozygous for the Adar1^p150 disruption (Upper) compared with embryos homozygous for the disrupted Adar1^p150 allele (Lower) at the indicated times of development. (E) RT-PCR analysis of RNA isolated from WT, p150^+/−, and p150^−/− MEF cells detecting IFN-inducible exon 1A-containing and constitutive exon 1B-containing transcripts encoding the p150 and p110 isoforms of ADAR1, respectively. GAPDH is shown as an internal control.

Fig. 2.

p150 isoform of ADAR1 plays an antiviral role in the replication of MV. (A) WT and p150^−/− MEF cells expressing either GFP as a control or hSLAM-GFP as indicated were infected with MV and observed for development of CPE by bright-field microscopy at the indicated times postinfection. (B) Restoration of mouse ADAR1 expression in p150^−/− MEF cells protects from development of full CPE as a result of infection with MV. WT and p150^−/− MEF cells expressing hSLAM-GFP and reconstituted with lentiviral vectors delivering mouse ADAR1 (p150^−/− Lenti-mADAR1) or with empty control lentiviral vectors (p150^−/− Lenti-empty) were left uninfected or infected with MV. Cells were observed for development of CPE by bright-field microscopy at 48 h postinfection.

Fig. 3.

p150 isoform of ADAR1 protects from infection with members of the Paramyxoviridae. (A) WT, parental p150^−/−, or reconstituted p150^−/− MEF cells as described in the legend for Fig. 2 were left uninfected or infected with NDV or SeV. Cells were observed for development of CPE by bright-field microscopy at 48 h postinfection. (B) Infection of MEF cells as described above with wild CDV, followed by bright-field microscopy at 67 h postinfection. The following MOIs were used for infections: NDV and SeV, MOI of 0.1 pfu/cell; CDV, MOI of 0.5 50% tissue culture infectious dose per cell.

Fig. 4.

p150 isoform of ADAR1 protects from infection with influenza A virus but not from infection with LCMV, VSV, or reovirus. (A) WT, parental p150^−/−, or reconstituted p150^−/− MEF cells as described in the legend for Fig. 2 were left uninfected or infected with the WSN strain of influenza A virus at an MOI of 1.0 pfu/cell and observed by bright-field microscopy at 48 h postinfection. (B) WT MEF or MEF cells heterozygous or homozygous for the p150 disruption were infected with the clone 13 strain of LCMV. Cells were fixed at 12 h postinfection, stained with antibody to LCMV nucleoprotein to detect the presence of viral antigen, and observed by fluorescence microscopy. Single-cycle yields of VSV (C) and reovirus (D) in untreated and IFN-treated WT, p150^+/−, and p150^−/− MEF cells are shown.