Identification and characterization of a loss-of-function human MPYS variant - PubMed (original) (raw)

Identification and characterization of a loss-of-function human MPYS variant

L Jin et al. Genes Immun. 2011 Jun.

Abstract

MPYS, also known as STING and MITA, is an interferon (IFN)β stimulator essential for host defense against RNA, DNA viruses and intracellular bacteria. MPYS also facilitates the adjuvant activity of DNA vaccines. Here, we report identification of a distinct human MPYS haplotype that contains three non-synonymous single nucleotide polymorphisms (SNPs), R71H-G230A-R293Q (thus, named the HAQ haplotype). We estimate, in two cohorts (1,074 individuals), that ∼3% of Americans are homozygous for this HAQ haplotype. HAQ MPYS exhibits a > 90% loss in the ability to stimulate IFNβ production. Furthermore, fibroblasts and macrophage cells expressing HAQ are defective in Listeria monocytogenes infection-induced IFNβ production. Lastly, we find that the loss of IFNβ activity is due primarily to the R71H and R293Q SNPs in HAQ. We hypothesize that individuals carrying HAQ may exhibit heightened susceptibility to viral infection and respond poorly to DNA vaccines.

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Conflict of interest statement

Conflict of Interests

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. Identification of a R71H (rs11554776)-G230A (rs78233829)-R293Q (rs7380824) MPYS SNPs haplotype (HAQ) in human population

a. PCR sequencing of mpys exons from human samples that are homozygous (HAQ/HAQ) (upper three panels) and heterozygous (H232/HAQ) (lower three panels) for the HAQ haplotype. Red letters indicate the altered amino acids by SNPs. b. A family consists of homozygous (HAQ/HAQ) and heterozygous (H232/HAQ) of HAQ MPYS. c. Alignment of HAQ with WT MPYS. Altered amino acids due to SNPs are indicated by arrows.

Figure 2

Figure 2. HAQ of MPYS is severely defective in IFNβ stimulation

a. 293MT cells (~1×105) were transfected with indicated plasmid (100ng) along with IFNβ-luciferase reporters. After 24 hrs, the luciferase activity was measured as described in Methods. Error bars represent SD of a duplicate. P value was calculated by student T-test (one-tailed). b. 293MT cells were transfected with indicated plasmids. The blot was probed with anti-MPYS or p-IRF3 Ab (4D4G). c. 293MT cells were transfected with indicated plasmids as before. The supernatant was collected and IFNβ proteins were measured as described in Methods. Error bars represent SD of a duplicate. ND: not detected.

Figure 3

Figure 3. The WT/HAQ genotype is also defective in induction of IFNβ

a. 293MT cells were transfected with 100ng of each indicated plasmid (e.g 100ng each of WT and HAQ for the WT/HAQ) along with IFNβ-luciferase reporters. After 24 hrs, the luciferase activity was measured as before. Error bars represent SD of a duplicate. b. 293MT cells were transfected with indicated plasmids as before. The supernatant was collected and IFNβ proteins were measured as described in Methods. Error bars represent SD of a duplicate. ND: not detected. c. 293MT cells were transfected with indicated plasmids. The blot was probed as before. Highlighted are the genotypes that are defective in IFNβ stimulation.

Figure 4

Figure 4. Listeria monocytogenes induction of IFNβ production is impaired in cells expressing HAQ

a. 293MT cells stably expressing Vec, WT or HAQ MPYS, were lysed in RIPA buffer. MPYS was detected as before. b. 293MT cells stably expressing Vec, WT or HAQ MPYS, were infected with Listeria monocytogenes as described in Methods. The IFNβ protein was measured as before. Error bars represent SD of a duplicate. ND: not detected. c. BMM cells reconstituted with MPYS, HAQ or Vec control were fixed, permeabilized and stained with α-MPYS rabbit Ab followed by anti-rabbit-Alexa647 as in Methods. d. BMM cells were infected with Listeria monocytogenes as describe in Methods. IFNβ production was measured after 20 hrs by ELISA.

Figure 5

Figure 5. R293Q and R71H SNPs are responsible for the IFNβ stimulation defect in HAQ of MPYS

a. 293MT cells were transfected with indicated plasmid (100ng) along with IFNβ-luciferase reporters. Luciferase activity was measured after 24 hrs as before. Error bars represent SD of a duplicate. b. 293MT cells were transfected with indicated plasmids. Phosphorylated IRF3 was probed as before. c. 293MT cells were transfected with indicated plasmids as before. The supernatant was collected and IFNβ proteins were measured as before. Error bars represent SD of a duplicate. ND: not detected. d&e. Alignment of MPYS sequence from 7 different species. The locations of motifs and SNPs were indicated. f. 293MT cells were transfected with indicated plasmid (100ng) along with IFNβ-luciferase reporters. Luciferase activity was measured after 24 hrs as before. Error bars represent SD of a duplicate. g. 293MT cells were transfected with indicated plasmids. Phosphorylated IRF3 was probed as before. h. 293MT cells were transfected with indicated plasmids as before. The supernatant was collected and IFNβ proteins were measured as before. Error bars represent SD of a duplicate. ND: not detected.

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