Poly(dA:dT) Suppresses HSV-2 Infection of Human Cervical Epithelial Cells Through RIG-I Activation

Abstract

Epithelial cells of the female reproductive tract (FRT) participate in the initial innate immunity against viral infections. Poly(dA:dT) is a synthetic analog of B form double-stranded (ds) DNA which can activate the interferon (IFN) signaling pathway-mediated antiviral immunity through DNA-dependent RNA Polymerase III. Here we investigated whether poly(dA:dT) could inhibit herpes simplex virus type 2 (HSV-2) infection of human cervical epithelial cells (End1/E6E7). We demonstrated that poly(dA:dT) treatment of End1/E6E7 cells could significantly inhibit HSV-2 infection. Mechanistically, poly(dA:dT) treatment of the cells induced the expression of the intracellular IFNs and the multiple antiviral IFN-stimulated genes (ISGs), including IFN-stimulated gene 15 (ISG15), IFN-stimulated gene 56 (ISG56), 2'-5'-oligoadenylate synthetase 1 (OAS1), 2'-5'-oligoadenylate synthetase 2 (OAS2), myxovirus resistance protein A (MxA), myxovirus resistance protein B (MxB), virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible (Viperin), and guanylate binding protein 5 (GBP5). Further investigation showed that the activation of RIG-I was largely responsible for poly(dA:dT)-mediated HSV-2 inhibition and IFN/ISGs induction in the cervical epithelial cells, as RIG-I knockout abolished the poly(dA:dT) actions. These observations demonstrate the importance for design and development of AT-rich dsDNA-based intervention strategies to control HSV-2 mucosal transmission in FRT.

Keywords: herpes simplex virus type 2; human cervical epithelial cells; interferon; interferon-stimulated gene; poly(dA:dT); retinoic acid-inducible gene-I.

Figure 1

Effect of poly(dA:dT) on End1/E6E7 cells. (A) Cell lysates of End1/E6E7 cells, End1/E6E7 V2 control cells and RIG-I knockout End1/E6E7 cells were subjected to Western blot assay for RIG-I expression and GAPDH was used as a protein loading control. (B, C) End1/E6E7 cells were treated with poly(dA:dT) at the indicated concentrations, and cell viability was assessed by MTT assay (B) and annexin V/7-AAD assay (C) 72h post poly(dA:dT) treatment.

Figure 2

Poly(dA:dT) inhibits HSV-2 infection. (A, C, E) End1/E6E7 cells were pretreated with poly(dA:dT) at indicated concentrations for 24h prior to HSV-2 (MOI = 0.001) infection. Forty-eight hours after HSV-2 infection, (A) intracellular DNA, (C) extracellular DNA, and (E) total cellular proteins were collected and subjected to the real-time PCR or Western blot for HSV-2 gene expression. (B, D, F) End1/E6E7 cells were treated with either poly(dA:dT) (0.5μg/ml) for 24h prior to HSV-2 (MOI=0.001) infection (before) or poly(dA:dT) and infected with HSV-2 simultaneously (simul) or infected with HSV-2 for 2h prior to poly(dA:dT) treatment (after). At 48h post HSV-2 infection, (B) intracellular DNA, (D) extracellular DNA, and (F) total cellular proteins were collected and analyzed by the real-time PCR or Western blot for HSV-2 gene expression. Data shown are the mean ± SD of three independent experiments. Asterisks indicate statistically significant differences. (*P < 0.05, **P < 0.01).

Figure 3

Effect of poly(dA:dT) on HSV-2 gene expression. End1/E6E7 cells were transfected with or without poly(dA:dT) (0.5μg/ml) at indicated concentrations for 24h prior to HSV-2 infection (MOI = 0.001). Cellular RNAs were collected from the virus-infected cells at 12 or 24h post infection and subjected to the real-time PCR for HSV-2 immediate early genes (A, B), early genes (C, D) and late genes (E, F) expression. The results were measured as HSV-2 gene levels relative (%) to control (without treatment, which is defined as 100%). Data are shown as mean ± SD of three independent experiments. Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01).

Figure 4

Effect of poly(dA:dT) on IFNs and IRFs expression. End1/E6E7 cells were transfected with or without poly(dA:dT) (0.5μg/ml) for the indicated times. (A, C) Total cellular RNAs were extracted and subjected to the real-time PCR for IFN-β, IFN-λ1, IFN-λ2/3, and IRF3, IRF7 expression. (B) The cell-free supernatant was subjected to ELISA assay to determine IFN-β, IFN-λ1/3, and IFN-λ2 protein levels. (D) Total cellular proteins were collected and subjected to Western blot with the antibodies against IRF3, IRF7, p-IRF3, p-IRF7, and GAPDH. Data are shown as mean ± SD of three independent experiments.

Figure 5

Effect of poly(dA:dT) on JAK/STAT signaling pathway. End1/E6E7 cells were transfected with or without poly(dA:dT) (0.5μg/ml) for the indicated times. (A) Total cellular RNAs were extracted and subjected to the real-time PCR for STAT1, STAT2, and STAT3 expression. (B) Total cellular proteins were collected and subjected to Western blot with the antibodies against STAT1, STAT2, STAT3, p-STAT1, p-STAT2, p-STAT3, ISGF-3γp48, and GAPDH. Data are shown as mean ± SD of three independent experiments.

Figure 6

Effect of poly(dA:dT) on ISGs expression. End1/E6E7 cells were transfected with or without poly(dA:dT) (0.5μg/ml) for the indicated times. (A, B) Total cellular RNAs were isolated and subjected to the real-time PCR for ISG15, ISG56, OAS1, OAS2, MxA, MxB, viperin, and GBP5 expression. (C, D) Total cellular proteins were collected and subjected to Western blot with antibodies against ISG15 and ISG15-conjugates, ISG56, OAS1, OAS2, MxA, MxB, Viperin, GBP5, and GAPDH. Data shown represent the mean ± SD of three independent experiments.

Figure 7

Effect of poly(dA:dT) on the DNA sensors and RIG-I. End1/E6E7 cells were transfected with or without poly(dA:dT) (0.5μg/ml) for the indicated times. (A) Total cellular RNAs were extracted and the messenger RNA (mRNA) levels of DNA sensors (IFI16, cGAS, STING, DAI, AIM2, and DHX29) were measured by the real-time PCR. (B) Total cellular proteins were collected and subjected to Western blot with the antibodies against IFI16, cGAS, p-STING, DAI, AIM2, DHX29, and GAPDH. (C) Total cellular RNAs were extracted and subjected to the real-time PCR for measuring RIG-I mRNA. (D) Total cellular proteins were collected and subjected to Western blot with the antibodies against RIG-I and GAPDH. Representative data are the mean ± SD of three independent experiments.

Figure 8

Effect of RIG-I knockout on poly(dA:dT)-mediated HSV-2 inhibition. End1/E6E7 control cells (V2 control) and RIG-I knockout End1/E6E7 cells (RIG-I^−/−) were transfected with or without poly(dA:dT) (0.5μg/ml) for 24h prior to HSV-2 infection (MOI = 0.001). (A, B) Total DNAs extracted from cells (intracellular) and culture supernatant (Extracellular) were measured by the real-time PCR for HSV-2 gD expression. (C) Total cellular proteins were collected and subjected to Western blot with antibodies against HSV-2 gB, HSV-2 gD, RIG-I, and GAPDH. The results are the mean ± SD of three independent experiments. Asterisks indicate that the differences between the indicated groups are statistically significant (**P < 0.01).

Figure 9

Effect of RIG-I knockout on poly(dA:dT)-mediated inhibtion of HSV-2 gene expression. End1/E6E7 control cells (V2 control) and RIG-I knockout End1/E6E7 cells (RIG-I^−/−) were transfected with or without poly(dA:dT) (0.5μg/ml) for 24h prior to HSV-2 infection (MOI = 0.001). At 24h post HSV-2 infection, cellular RNAs were collected and subjected to the real-time PCR for HSV-2 immediate early genes (A, B), early genes (C, D), and late genes (E, F) expression. The results were measured as HSV-2 gene levels relative (%) to control (without treatment, which is defined as 100%). Data are shown as mean ± SD for three independent experiments. Asterisks indicate that the differences between the indicated groups are statistically significant (*P < 0.05, **P < 0.01).

Figure 10

Effect of RIG-I knockout on poly(dA:dT)-induced expression of IFNs and IRFs. End1/E6E7 control cells (V2 control) and RIG-I knockout End1/E6E7 cells (RIG-I^−/−) were transfected with or without poly(dA:dT) (0.5μg/ml) for 12h. (A, C) Total cellular RNAs were extracted and subjected to the real-time PCR for IRF3, IRF7, IFN-β, IFN-λ1, and IFN-λ2/3 mRNA expression. (B) The cell-free supernatant was collected and subjected to ELISA assay to determine IFN-β, IFN-λ1/3, and IFN-λ2 protein levels. (D) Total cellular proteins were collected and subjected to Western blot with antibodies against IRF3, IRF7, p-IRF3, p-IRF7, and GAPDH. The results are the mean ± SD of three independent experiments. Asterisks indicate that the differences between the indicated groups are statistically significant (ND, not detected, **P < 0.01).

Figure 11

Effect of RIG-I knockout on poly(dA:dT)-induced expression of STATs. End1/E6E7 control cells (V2 control) and RIG-I knockout End1/E6E7 cells (RIG-I^−/−) were transfected with or without poly(dA:dT) (0.5μg/ml) for 12h. (A) Total cellular RNAs were collected and subjected to the real-time PCR for STAT1, STAT2, and STAT3 expression. (B) Total cellular proteins were collected and subjected to Western blot with the antibodies against STAT1, STAT2, STAT3, p-STAT1, p-STAT2, p-STAT3, ISGF-3γp48, and GAPDH. The results are the mean ± SD of three independent experiments. Asterisks indicate that the differences between the indicated groups are statistically significant (**P < 0.01).

Figure 12

Effect of RIG-I knockout on poly(dA:dT)-induced ISGs expression. (A, B) End1/E6E7 control cells (V2 control) and RIG-I knockout End1/E6E7 cells (RIG-I^−/−) were transfected with or without poly(dA:dT) (0.5μg/ml) for 12h. Total cellular RNAs were extracted and subjected to the real-time PCR for ISG15, ISG56, OAS1, OAS2, MxA, MxB, viperin, and GBP5 expression. (C) V2 control and RIG-I^−/− cells were transfected with or without poly(dA:dT) (0.5μg/ml) for 24h. Cellular proteins were collected and subjected to Western blot with the antibodies against ISG15, ISG56, OAS1, OAS2, MxA, MxB, viperin, GBP5, and GAPDH, respectively. The results are the mean ± SD of three independent experiments. Asterisks indicate that the differences between the indicated groups are statistically significant (*P < 0.05, **P < 0.01).

Figure 13

Schematic diagram of mechanisms for poly(dA:dT)-mediated HSV-2 inhibition in human cervical epithelial cells. Poly(dA:dT) can be recognized by RNA polymerase III which then converts DNA into 5’-ppp RNA, the ligand for RIG-I. The activation of RIG-I facilitates the phosphorylation and translocation of IRF3 and IRF7, resulting in the production of IFNs. The released IFNs bind to their corresponding receptors on the cell surface, and trigger JAK/STAT signaling pathway, facilitating STATs phosphorylation and inducing the antiviral ISGs (ISG15, ISG56, OAS1, OAS2, MxA, MxB, viperin, GBP5) which have the ability to block or inhibit HSV-2 at different steps of replication cycle.