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. 2010 Jul 1;185(1):424-32.
doi: 10.4049/jimmunol.0903291. Epub 2010 May 28.

A Common Polymorphism in the Caspase Recruitment Domain of RIG-I Modifies the Innate Immune Response of Human Dendritic Cells

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Free PMC article

A Common Polymorphism in the Caspase Recruitment Domain of RIG-I Modifies the Innate Immune Response of Human Dendritic Cells

Jianzhong Hu et al. J Immunol. .
Free PMC article

Abstract

Infection of human dendritic cells (DCs) by negative-strand RNA viruses, such as Newcastle disease virus, leads to the induction of the IFNbeta gene, IFNB1, through the activation of the RNA helicase RIG-I, which is encoded by DDX58. Expression levels of IFNB1 and DDX58 in infected DCs showed positive correlations at the population and the single-cell levels. DDX58 has a common and potentially functional single nucleotide polymorphism, rs10813831 (A/G), encoding an Arg7Cys amino acid change in the RIG-I protein caspase recruitment domain (CARD). Quantitative RT-PCR analysis on Newcastle disease virus-infected primary DCs from 130 individuals revealed a significant association of the Arg7Cys single nucleotide polymorphism with increased IFNB1 and DDX58 transcription. Allelic imbalance analysis ruled out allele-specific DDX58 message levels and suggested that the observed association between Arg7Cys and IFNB1 and DDX58 transcription originated from a functional change in RIG-I due to the amino acid substitution in the CARD. DDX58 transfection experiments in 293T cells confirmed a biological functional difference between RIG-I 7Cys and the more common RIG-I 7Arg. Taken together, these data indicate that the innate immune response to viral infection of human cells is modified by a functional polymorphism in the RIG-I CARD.

Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Expression covariation between IFNB1 and DDX58 in NDV-infected DCs. Ten hours after NDV infection at an MOI of 0.5, total RNA was extracted, and relative expression levels were determined for IFNB1 and DDX58 mRNA in each DC sample. The expression levels of DDX58 and IFNB1 are plotted on a logarithmic scale. These data showed a strong correlation between IFNB1 and DDX58 expression in this population (r2 = +0.53; p < 0.005).
FIGURE 2
FIGURE 2
Expression covariation between IFNB1 and DDX58 in single DCs. Ten hours after NDV infection at an MOI of 0.5, DDX58 and IFNB1 mRNA in each individual DC from a single donor was quantified by a nested real-time quantitative RT-PCR method described in Materials and Methods. The expression levels of DDX58 and IFNB1 are plotted on a logarithmic scale. Straight lines join duplicate measurements. A, The data showed that without Ab blockage, DDX58 induction was seen in the entire cell population, but with a narrower distribution than that seen for IFNB1 expression. B, With Ab blockage, a much stronger association was observed between DDX58 and IFNB1 expression (r2 = +0.60 versus +0.28).
FIGURE 3
FIGURE 3
DDX58 and IFNB1 mRNA expression in DCs derived from 130 individuals were examined 10 h after NDV infection. DDX58, IFNB1, and ACTB (internal control) mRNA were quantified by real-time PCR. The relative IFNB1 (left panel) and DDX58 (right panel) expression levels (normalized to ACTB) were converted to a logarithmic scale for comparison among genotypes (0 = GG, ArgArg homozygote; 1 = GA, ArgCys heterozygote; 2 = AA, CysCys homozygote) of the DDX58 Arg7Cys polymorphism. The box plots show median (solid bar) and interquartile ranges (boxes).
FIGURE 4
FIGURE 4
AI of DDX58 expression. DDX58 amplicons containing the 3′UTR polymorphism rs12006123 were amplified by PCR from preamplified total cDNAs to serve as templates for ASPCR. AI was determined by ASPCR. The box plots show median (solid bar) and interquartile ranges (boxes). The AI value of each sample without NDV infection (A) or with NDV infection (B) was compared for the three genotypes (0 = GG, 1 = GA, 2 = AA) of SNP rs12006123 and a genomic DNA control from 13 randomly selected heterozygous individuals (3 = genomic DNA). The heterozygotes in A and B were characterized by the Arg7Cys polymorphism genotype (0, 1, 2) in C and D. There was no AI associated with the Arg7Cys SNP in uninfected (C) or infected (D) DC samples (p = 0.63 without infection; p = 0.89 with infection).
FIGURE 5
FIGURE 5
RIG-I position 7 polymorphism phenotypes in transiently transfected 293T cells. A and B, Twelve hours after transfection with RIG-IArg7, RIG-I Cys7, or empty plasmids, 293T cells were infected with influenza A/PR/8/34 ΔNS1 at an MOI of 3 or with NDV at an MOI of 2. A, Relative IFNB1 induction was analyzed using an IFNβ-luciferase reporter assay to compare the two polymorphic forms of RIG-I. Experiments were performed in triplicate, and error bars show the SD variation within constructs. B, The mRNA expression levels of IFNB1 from 0–24 h after NDV infection were measured by real-time PCR. C, Comparison of the effect of Arg7 and Cys7 on the relative IFNB1 induction by the RIG-I CARD region (aa 1–211 from RIG-I). D and E, RIG-I Arg7 and Cys7 CARD region modifications detected by Western blot. D, Arrowheads denote the modified forms of the proteins. E, RIG-I Arg7 CARD shows significantly more polyubiquitination than the Cys7 CARD in 293T cells. HC, Ab H chain; Influenza PA, influenza polymerase acidic subunit A; LC, Ab L chain.
FIGURE 6
FIGURE 6
RIG-I, RIPLET, and TRIM25 expression in uninfected and NDV-infected 293T cells and human monocyte-derived DCs. Western blot showing the level of protein expression for RIG-I, two isoforms of RIPLET (gene RNF135) and TRIM25 (gene TRIM25) at 0, 6, and 12 h after NDV infection in 293T cells and human DCs. A, Time course after NDV infection for RIG-I, RIPLET, and TRIM25 expression in transfected 293T cells. Normalized to tubulin. B, Same time course for donor 1 DCs. Arrowheads denote TRIM25 and the two isoforms of RIPLET. C, Combined assay of transfected 293T cells and DCs from a second donor with (+) or without (−) NDV infection (10 h). C7, RIG-I Cys7 transfection; Ctr, no transfection; R7, RIG-I Arg7 transfection.

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