Identification and characterization of GLDC as host susceptibility gene to severe influenza

Abstract

Glycine decarboxylase (GLDC) was prioritized as a candidate susceptibility gene to severe influenza in humans. The higher expression of GLDC derived from genetic variations may confer a higher risk to H7N9 and severe H1N1 infection. We sought to characterize GLDC as functional susceptibility gene that GLDC may intrinsically regulate antiviral response, thereby impacting viral replication and disease outcome. We demonstrated that GLDC inhibitor AOAA and siRNA depletion boosted IFNβ- and IFN-stimulated genes (ISGs) in combination with PolyI:C stimulation. GLDC inhibition and depletion significantly amplified antiviral response of type I IFNs and ISGs upon viral infection and suppressed the replication of H1N1 and H7N9 viruses. Consistently, GLDC overexpression significantly promoted viral replication due to the attenuated antiviral responses. Moreover, GLDC inhibition in H1N1-infected BALB/c mice recapitulated the amplified antiviral response and suppressed viral growth. AOAA provided potent protection to the infected mice from lethal infection, comparable to a standard antiviral against influenza viruses. Collectively, GLDC regulates cellular antiviral response and orchestrates viral growth. GLDC is a functional susceptibility gene to severe influenza in humans.

Keywords: GLDC; antiviral response; genetic susceptibility; severe influenza; viral replication.

Figure 1. Genotype‐expression correlation of association SNP

1. A, B

   The genotype‐expression correlation of rs1755609 and GLDC in lymphoblast cell lines (LCLs, A) and human lung tissues (B). The box denotes the interquartile range; thick line within the box is the median; diamond represents the mean; whiskers are minimum and maximum and open dot is outlier. The genotype‐expression correlation pattern in LCLs from 77 Chinese and 72 Japanese are plotted in the left and right, respectively. The lung eQTL data from three centers are presented. Linear regression is used for data analysis.

2. C

   The high linkage disequilibrium (LD) pattern in three SNPs, including two association SNPs rs2438409 and rs1755609 (boxed), in multiple populations. The LD pattern is plotted using genotyping data of 77 Chinese Han in Beijing (CHB), 72 Japanese (JPT), and 73 Caucasian of European ancestry living in Utah USA (CEU) from 1000 Genomes Project. The boxes are colored according to r ² measure on a white and gray scale. The numbers inside the boxes are r ² measure. The gray box without number indicates the highest r ² of 1.0.

Figure EV1. Dose‐dependent inhibition of cellular thymidine in the AOAA‐treated A549 cells

A549 cells were treated with the indicated concentrations of AOAA. After 48 h, cells were harvested for the detection of the relative amount of thymidine with LC‐MS/MS. Data shown are representative of two independent experiments, n = 5. Graphs show mean ± SD. Unpaired t‐test is used for data analysis.

Figure 2. GLDC inhibition and depletion activated type I IFNs and ISGs

1. A

   A549 cells co‐transfected with pIFNβ‐Luc and TK‐Nano‐Luc were further transfected with Poly I:C (0 and 50 μg/ml) and simultaneously treated with AOAA (0 and 50 μM). The cells were lysed for luciferase assay after 16 h. A549‐Dual and A549‐Dual KO‐RIG‐I cells carrying ISRE reporter gene were transfected and treated with the indicated concentrations of Poly I:C and AOAA respectively in triplicate. After 16 h, the cell‐free supernatants were used for luciferase assay. The data present the normalized luciferase activity relative to mock Poly I:C transfection and mock AOAA treatment. Data shown are representative of three independent experiments, n = 3.

2. B

   A549 cells transfected with GLDC siRNA (siGLDC) or scrambled siRNA (siCtrl) were further transfected with Poly I:C (50 μg/ml). After 24 h, the cell lysates and media were harvested for detection of mRNA expression of GLDC and IFNα by RT–qPCR, and measurement of IFNα production by ELISA, respectively. Data shown are representative of three independent experiments, n = 3.

Data information: Unpaired t‐test is used for data analysis. Graphs show mean ± SD.

Figure 3. GLDC inhibition suppressed viral replication

1. A, B

   A549 cells were inoculated with H1N1 (A) and H7N9 (B) viruses at an MOI of 0.01. The cells were treated with AOAA (0, 10, 50 μM) in triplicate prior to and after the inoculation. At the indicated hours post‐inoculation, cell lysates and cell‐free media (supernatant) were harvested for the detection of viral load with RT–qPCR assay, and viral titration with plaque assay, respectively. Data shown are representative of at least three independent experiments, n = 3. Graphs show mean ± SD. Unpaired t‐test is used for data analysis.

Figure 4. The amplified antiviral response contributed to AOAA‐mediated inhibition of viral replication

1. A, B

   A549 cells were inoculated with H1N1 (A) and H7N9 (B) viruses. The cells were treated with AOAA (0, 10, 50 μM) prior to and after the inoculation. At the indicated hours post‐inoculation, cell lysates were harvested for the detection of expression level of antiviral molecules with RT–qPCR assay. Data shown are representative of at least three independent experiments, n = 3. Graphs show mean ± SD. Unpaired t‐test is used for data analysis.

Figure EV2. AOAA induced the heightened ISGs at 48 h after H7N9 infection and cell viability assay of AOAA

1. A

   A549 cells treated with AOAA (0, 10, 50 μM) were then inoculated H7N9 virus before and after a MOI of 0.01 inoculation. At 48 hpi, the cells were harvested for detecting expression levels of ISGs. Data shown are representative of three experiments, n = 3. Graphs show mean ± SD. Unpaired t‐test is used for data analysis.

2. B

   The effect of AOAA on cell viability was assessed in A549 cells. A549 cells were incubated with the indicated concentration of AOAA at 37°C for 48 h. The cell‐free culture medium was applied to MTT assay. Data shown are representative of two experiments, n = 3. Graphs show mean ± SD. Unpaired t‐test is used for data analysis.

Figure EV3. The effect of brequinar on antiviral immunity and viral replication

After 1‐h pretreatment of 10 μM brequinar (Tocris Biosciences, Cat No. 6196) or DMSO, A549 cells were inoculated with H7N9 virus with a MOI of 0.01 then were maintained in DMEM with 2 μg/ml TPCK‐trypsin, 3% BSA and 10 μM brequinar or DMSO. At the indicated time points, cell lysates and cell‐free media were collected for detection of viral load, cellular gene expression, and viral titration. Data shown are representative of two experiments, n = 3. Graphs show mean ± SD. Unpaired t‐test is used for data analysis.

Figure 5. GLDC depletion and overexpression affected viral replication through the altered antiviral response

1. A–D

   A549 cells were transfected with GLDC siRNA (siGLDC) or scrambled siRNA (siCtrl) (A and B), or transfected with pcDNA3.1‐GLDC or pcDNA3.1‐His A vector (C and D). The transfected cells were inoculated with H7N9 virus at an MOI of 0.01. At 48 hpi, cell lysates and culture media (supernatant) of the siRNA‐transfected cells were harvested for detection of viral loads and viral titers (A). The cell lysates were collected at 24 hpi to measure the expression levels of antiviral genes (B). At the indicated hpi, cell lysates and supernatants of the plasmid‐transfected cells were harvested for viral load detection (C). The cell lysates collected at 24 hpi were used to measure the expression levels of antiviral genes (D). Data shown are representative of three experiments, n = 3. Graphs show mean ± SD. Unpaired t‐test is used for data analysis.

2. E

   The effective depletion of GLDC by siRNA was shown by RT–qPCR assay (left panel) and Western blot (48 hpi, upper right panel). GLDC overexpression at the indicated hpi was verified by Western blot (lower right panel). Data shown in left panel are representative of at least three experiments, n = 3. Graphs show mean ± SD. Unpaired t‐test is used for data analysis.

3. F

   A549 cells transfected with GLDC plasmid or vector were inoculated with MERS‐CoV in triplicate. Cell‐free culture media were harvested for detection of viral load at the indicated time points. Data shown are representative of two independent experiments, n = 3. Graphs show mean ± SD. Unpaired t‐test is used for data analysis.

Source data are available online for this figure.

Figure EV4. GLDC overexpression promoted replication of H1N1 via the attenuated antiviral response

A549 cells transfected with pcDNA3.1‐GLDC or pcDNA3.1‐His A vector were inoculated with H1N1 virus at an MOI of 0.01. At the indicated hpi, cell lysates and cell‐free supernatants were harvested for viral load detection. The cell lysates collected at 12 hpi were used to measure the expression levels of antiviral genes. Data shown are representative of three experiments, n = 3. Graphs show mean ± SD. Unpaired t‐test is used for data analysis.

Figure 6. GLDC inhibition reduced viral load and protected the mice from lethal infection of H1N1 virus

1. A

   Regime of the mouse experiment.

2. B

   Survival rates of AOAA‐, zanamivir‐, and PBS‐treated mice after infection (n = 9). Mantel–Cox test is used for data analysis.

3. C

   Viral loads and viral titers in the lung homogenates of the indicated mice (n = 3). Unpaired t‐test is used for data analysis. Graphs show mean ± SD.

4. D

   The normalized expression levels of IFNα in lung tissues of the indicated mice (n = 3) and fold change of ISGs at day 5 post‐infection. Unpaired t‐test is used for data analysis. Graphs show mean ± SD.

5. E

   Representative images of virus‐infected cells in the indicated mice. Mouse lung tissues are applied to immunofluorescence staining using the antibody against influenza virus NP (green) and confocal imaging.

Figure EV5. Mouse body weight change during infection and evaluation of AOAA toxicity in vivo

1. A, B

   The body weight change of AOAA‐, zanamivir‐, and PBS‐treated mice (n = 9) after virus inoculation.

2. C

   Body weight change of the mice intranasally administered with AOAA (n = 3, 10 mg/kg body weight) and PBS (n = 3).

Data information: Unpaired t‐test is used for data analysis. Graphs show mean ± SD.