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. 2018 Jul 31;92(16):e00539-18.
doi: 10.1128/JVI.00539-18. Print 2018 Aug 15.

Lnc-ISG20 Inhibits Influenza A Virus Replication by Enhancing ISG20 Expression

Wenjia Chai #  1   2 , Jing Li #  1 , Qilin Shangguan  1   2 , Qi Liu  1   2 , Xinda Li  1   2 , Dandan Qi  1 , Xiaomei Tong  1 , Wenjun Liu  1   3 , Xin Ye  4   3
Affiliations

Lnc-ISG20 Inhibits Influenza A Virus Replication by Enhancing ISG20 Expression

Wenjia Chai et al. J Virol. .

Abstract

Long noncoding RNAs (lncRNAs) are involved in many aspects of cellular processes, including the antiviral immune response. To identify influenza A virus (IAV)-related lncRNAs, we performed RNA deep sequencing to compare the profiles of lncRNAs in A549 and HEK293T cells with or without IAV infection. We identified an IAV-upregulated lncRNA named lnc-ISG20 because it shares most of its sequence with ISG20. We found that lnc-ISG20 is an interferon-stimulated gene similar to ISG20. Overexpression of lnc-ISG20 inhibited IAV replication, while lnc-ISG20 knockdown favored viral replication, suggesting that lnc-ISG20 is inhibitory to IAV replication. Further study indicated that overexpression of lnc-ISG20 enhances ISG20 protein levels, while knockdown of lnc-ISG20 reduces ISG20 protein levels in A549 cells induced with poly(I·C) and Sendai virus. We demonstrated that lnc-ISG20 inhibits IAV replication in an ISG20-dependent manner. As lnc-ISG20 did not affect the mRNA level of ISG20, we postulated that lnc-ISG20 may function as endogenous RNA competing with ISG20 to enhance its translation. Indeed, we identified that microRNA 326 (miR-326) is a mutual microRNA for both ISG20 and lnc-ISG20 that targets the 3' untranslated region of ISG20 mRNA to inhibit its translation. We confirmed that lnc-ISG20 can bind miR-326, which in turn decreased the amount of miR-326 bound to ISG20 mRNA. In conclusion, we identified that the IAV-upregulated lnc-ISG20 is a novel interferon-stimulated gene that elicits its inhibitory effect on IAV replication by enhancing ISG20 expression. We demonstrated that lnc-ISG20 functions as a competitive endogenous RNA to bind miR-326 to reduce its inhibition of ISG20 translation. Our results revealed the mechanism by which lnc-ISG20 inhibits IAV replication.IMPORTANCE The replication of influenza A virus is regulated by host factors. However, the mechanisms by which lncRNAs regulate IAV infection are not well understood. We identified that lnc-ISG20 is upregulated during IAV infection and is also an interferon-stimulated gene. We demonstrated that lnc-ISG20 can enhance ISG20 expression, which in turn inhibits IAV replication. Our studies indicate that lnc-ISG20 functions as a competing endogenous RNA that binds miR-326 and reduces its inhibitory effect on ISG20. Taken together, our findings reveal the mechanistic details of lnc-ISG20 negatively regulating IAV replication. These findings indicate that lnc-ISG20 plays an important role during the host antiviral immune response.

Keywords: ISG20; antiviral immunity; influenza A virus; lnc-ISG20; lnc-RNA; miR-326.

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Figures

FIG 1
FIG 1
Identification and characterization of IAV infection-induced lnc-ISG20. (A) A549 or HEK293T cells were infected with IAV (A/WSN/33, A/CA04, or A/PR8) at an MOI of 1 for 8 h. Total RNA was extracted and subjected to RNA-seq analysis. The expression values are shown in shades of red and blue, indicating expression above and below the median expression value across all the samples (log scale 2, from −2 to +2), respectively. (B) Schematic diagram of ISG20 and lnc-ISG20. (C) A549 and HEK293T cells were infected with A/WSN/33, A/CA04, or A/PR/8 at an MOI of 1 for 8 h. Total RNA was extracted and subjected to RT-PCR for lnc-ISG20. (D) A549 and HEK293T cells were infected with A/WSN/33 at the indicated MOI for 0, 4, 8, 12, and 16 h. The amount of lnc-ISG20 was quantified by RT-qPCR. (E) A549 cells were infected with A/WSN/33 for 16 h. Total RNA was prepared for 5′ and 3′ RACE for lnc-ISG20. Images of the RACE PCR products of lnc-ISG20 (left) and sequencing data (right) are presented. (F) A549 cells were infected with A/WSN/33 at an MOI of 0.1 for 16 h. Total RNA was extracted and subjected to Northern blot analysis. The probe used in the assay whose results are presented in the left panel was specific for lnc-ISG20, and the probe used in the assay whose results are presented in the right panel recognizes both the lnc-ISG20 and ISG20 mRNA isoforms. The experiments were repeated twice, and representative data are presented. (G) Full-length lnc-ISG20 was cloned into pcDNA3.1 with an N-terminal start codon and a C-terminal FLAG tag in all three reading frames. The T in lnc-ISG20-T-FLAG and TT in lnc-ISG20-TT-FLAG represent thymidine nucleotide(s). The plasmids were transfected into HEK293T cells for 48 h. Cell lysates were harvested and subjected to immunoblotting with anti-ISG20 or anti-β-actin. The empty vector and FLAG-ISG20 served as the negative and positive controls, respectively. (H) A549 cells were fractionated into cytoplasmic (C) and nuclear (N) fractions. The RNA was extracted from each fraction and subjected to quantitative RT-PCR for lnc-ISG20, and MALAT1, U6, β-actin, and RPS18 mRNAs were used as controls. The fractionated lysates were immunoblotted with lamin B and tubulin. The experiments whose results are presented in panels C, D, and H were repeated twice, and the results were consistent. The data are expressed as the mean for triplicate samples from one independent experiment.
FIG 2
FIG 2
Lnc-ISG20 is inducible as the IFN-β signaling pathway is activated. (A, B, C) A549 cells were infected with Sendai virus at the indicated MOI for 8 h (A) and transfected with poly(I·C) (0.5 μg/ml) (B) or treated with IFN-β (10 ng/ml) (C) for the indicated times. Total RNA was extracted and subjected to RT-qPCR for lnc-ISG20 and ISG20. Samples were normalized to β-actin. Data are shown as the mean ± SD (n = 3).
FIG 3
FIG 3
Lnc-ISG20 inhibits IAV replication. (A) A549 cells were infected with lentivirus carrying lnc-ISG20 or control virus. The GFP-positive cells were sorted by fluorescence-activated cell sorting, expanded, and named A549-lnc-ISG20 and A549-ctrl. The expression level of lnc-ISG20 was determined by RT-qPCR, and the data represent the means for triplicate samples from one independent experiment. The experiments were repeated twice. (B, C) A549-lnc-ISG20 and A549-ctrl cells were A549 cells ectopically expressing lnc-ISG20 and A549 control cells, respectively, infected with the A/WSN/33 virus at an MOI of 0.01 for the indicated times. (B) The cell lysates were harvested for immunoprecipitation and immunoblotting assays. (C) The supernatants of infected cells were collected for plaque assays to measure virus titers. (D, E, F) A549 cells were transfected with lnc-ISG20-specific siRNA (si-lnc-ISG20#1 and -#2) or control siRNA (si-Ctrl) and then infected with the A/WSN/33 virus at an MOI of 0.01 for the indicated times. (D) The expression levels of lnc-ISG20 (left) and ISG20 (right) mRNA were measured by RT-qPCR, and the data represent the means for triplicate samples from one independent experiment. The experiments were repeated three times. (E) The cell lysates were harvested for immunoblotting with the indicated antibodies. (F) The supernatants were collected for plaque assays to measure the virus titers. All experiments for the plaque assays were repeated three times, and the results were consistent. The presented data are expressed as the means for triplicate samples from one experiment. **, P < 0.01; ***, P < 0.001 (Student's t test).
FIG 4
FIG 4
Overexpression of lnc-ISG20 upregulates ISG20 protein levels but does not affect the level of ISG20 mRNA. (A, B) A549-lnc-ISG20 and A549-ctrl cells were transfected with poly(I·C) (0.5 μg/ml) (A) or infected with Sendai virus at the indicated MOI for 8 h (B). (C, D, E) A549 cells were transfected with si-lnc-ISG20 or control siRNA (si-Ctrl) and then transfected with poly(I·C) (C), infected with Sendai virus (D), or infected with A/WSN/33 virus at an MOI of 0.01 (E). Cell lysates were harvested and subjected to immunoblotting with anti-ISG20 or anti-β-actin antibodies. The above-described experiments were repeated 3 times, and representative data are presented. (F) A549-lnc-ISG20 and A549-ctrl cells were infected with Sendai virus for 8 h. Total RNA was extracted for RT-qPCR to quantify ISG20 mRNA. The experiments were independently performed twice, and consistent results were observed. Data were calculated as the means for triplicate samples. ns, not significant.
FIG 5
FIG 5
Lnc-ISG20 inhibits IAV replication in an ISG20-dependent manner. (A, B, C) A549 cells were transduced with lentivirus expressing lnc-ISG20 or control lentivirus and then transfected with si-ISG20 or control siRNA, as indicated (A). Then, the cells were infected with A/WSN/33 virus (MOI = 0.01, 14 h). (B) Cell lysates were harvested for immunoblotting with the indicated antibodies. (C) The supernatants from infected cells were collected for plaque assays to measure the virus titer. The data in panels A and C are expressed as the means for triplicate samples from one independent experiment. The experiments were repeated three times. **, P < 0.01; ***, P < 0.001 (Student's t test).
FIG 6
FIG 6
miR-326 can target lnc-ISG20 and the ISG20 3′ UTR. (A) HEK293T cells were transfected with the pGLO-ISG20 3′ UTR and the indicated miRNA mimics or scrambled miRNA as a negative control. Cell lysates were harvested for dual-luciferase assays. The relative luciferase activity was quantified. The presented data are expressed as the means from one experiment. The experiments were repeated three times. *, P < 0.05 (Student's t test). (B) The pGLO-ISG20 3′ UTR-S (ISG20 3′ UTR) and pGLO-ISG20 3′ UTR-S mutant (ISG20 3′ UTR-mut) were generated by making mutations in the predicted miR-326-binding region. The mutated nucleotides are shown in red. (C) 293T cells were transfected with the miR-326 mimic and reporter plasmids, as indicated. Cell lysates were harvested for dual-luciferase assays. The relative luciferase activity was quantified, and the data represent the means for triplicate samples from one independent experiment. The experiments were repeated twice. **, P < 0.01 (Student's t test). (D) A549 cell lines ectopically expressing S1 aptamer-tagged lnc-ISG20 or lnc-ISG20-mut were generated. Cell lysates were harvested and incubated with streptavidin-coupled beads at 4°C for 3 h. The amount of miR-326 bound to the beads was assessed by RT-qPCR, and the data represent the means for triplicate samples. The experiments were repeated twice. ***, P < 0.01 (Student's t test). (E, F, G) A549 cells were transfected with a control mimic (Ctrl), miR-326 mimic (miR-326), control inhibitor (Ctrl-inhibitor), or miR-326 inhibitor and then transfected with poly(I·C) (0.5 μg/ml, 14 h) (E), infected with SeV at an MOI of 0.2 for 8 h (F), or infected with the A/WSN/33 virus at an MOI of 0.01 for 12 h (G). Cell lysates were harvested and subjected to immunoblotting with anti-ISG20 or anti-β-actin as an internal control. (H) A549 cells were infected with the A/WSN/33 virus at an MOI of 0.01 for the indicated times. The relative amount of miR-326 was measured by RT-qPCR, and the data represent the means for triplicate samples from one independent experiment. The experiments were repeated twice. *, P < 0.05 (Student's t test).
FIG 7
FIG 7
lnc-ISG20 functions as a ceRNA to reduce miR-326 binding to ISG20 mRNA and enhance the expression of ISG20 protein. (A) A549 cells were transduced with lentivirus expressing lnc-ISG20, lnc-ISG20-mut, or control lentivirus. The cell lysates were harvested for immunoprecipitation (IP) with anti-AGO2 antibody by RT-qPCR to quantify AGO2-bound ISG mRNA. The experiment was repeated twice, and the results were consistent. The data are the means for triplicate samples from one experiment. **, P < 0.01 (Student's t test). (B) A549 cells were transduced with lentivirus expressing lnc-ISG20, lnc-ISG20-mut, or control lentivirus, as well as with different concentrations of miR-326 mimics (50, 100, or 200 pmol), followed by transfection with poly(I·C) (0.5 μg/ml). Cell lysates were harvested for immunoblotting with anti-ISG20 antibody and anti-β-actin antibody as an internal control.
FIG 8
FIG 8
Schematic map of lnc-ISG20 regulating IAV replication. IAV infection triggers the transcription of type I interferons, such as IFN-β. IFN-β stimulates the transcription of downstream genes (ISG20 and lnc-ISG20). Lnc-ISG20 acts as a ceRNA by reducing miR-326 binding to target ISG20 mRNA and enhances the translation of ISG20, which in turn inhibits IAV replication. IFN-βR, IFN-β receptor.
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