Zika virus inhibits type-I interferon production and downstream signaling

Abstract

Zika virus is an emerging mosquito-borne pathogen that is associated with Guillain-Barré syndrome in adults and microcephaly and other neurological defects in newborns. Despite being declared an international emergency by the World Health Organization, comparatively little is known about its biology. Here, we investigate the strategies employed by the virus to suppress the host antiviral response. We observe that once established, Zika virus infection is impervious to interferon treatment suggesting that the virus deploys effective countermeasures to host cell defences. This is confirmed by experiments showing that Zika virus infection impairs the induction of type-I interferon as well as downstream interferon-stimulated genes. Multiple viral proteins affect these processes. Virus-mediated degradation of STAT2 acts to reduce type-I and type-III interferon-mediated signaling. Further, the NS5 of Zika virus binds to STAT2, and its expression is correlated with STAT2 degradation by the proteasome. Together, our findings provide key insights into how Zika virus blocks cellular defense systems. This in turn is important for understanding pathogenesis and may aid in designing antiviral therapies.

Keywords: NS5; STAT2; Zika virus; flavivirus; interferon response.

Figure EV1. The IFN response is delayed during ZIKV infection

A549 cells were infected with ZIKV (MOI = 2) for 18, 24 and 48 h. At each time point, relative levels of Ifnb and Ifit1 mRNA (compared to mock‐infected cells) were determined by qRT–PCR (upper panel). As a positive control for IFN induction, cells were transfected with 2 μg of poly(I:C) for 12 h (lower panel). Levels of Ifnb and Ifit1 mRNA (compared to untreated cells) are shown. Values are expressed as mean ± standard error. N = 3.

Figure 1. ZIKV inhibits induction of the IFN response

1. A549 cells were transfected with 2 μg of poly(I:C) for 6 h and then infected with ZIKV (MOI = 3) for 18 h (pre‐treatment) or infected with ZIKV first for 6 h and then transfected with poly(I:C) for 12 h (post‐treatment). Levels of ZIKV genomic RNA were determined by qRT–PCR. Values are expressed as mean ± standard error. ***P < 0.001 (Student's t‐test), N = 3.

2. A549 cells infected with ZIKV (MOI = 3) for 6 h were transfected with 2 μg poly(I:C) for 12 h after which levels of Ifnb and Ifit1 mRNA were determined by qRT–PCR. Values are expressed as mean ± standard error. ***P < 0.001 (Student's t‐test), N = 3.

3. A549 cells were infected with ZIKV (MOI = 5) for 16 h and then transfected with an IFIT1 promoter‐driven firefly luciferase plasmid (pGL3B/561) and a constitutively active Renilla luciferase construct (pRL‐TK), as well as 1 μg of poly(I:C). Eight hours later, cell lysates were harvested and subjected to luciferase assay. Values are expressed as mean of two independent experiments, N = 2.

4. HEK293T cells were transfected with plasmids encoding each of the 10 ZIKV proteins. Sixteen hours later, they were transfected with an IFIT1 promoter‐driven firefly luciferase plasmid (pGL3B/561) and a constitutively active Renilla luciferase construct (pRL‐TK), as well as 1 μg of poly(I:C). Eight hours later, cell lysates were harvested and subjected to luciferase assay. C, capsid; E, envelope. Values are expressed as mean ± standard error. *P < 0.05, **P < 0.01 (Student's t‐test), N = 4.

5. A549 cells were infected with ZIKV (MOI = 5) for 16 h and then transfected with the indicated promoter‐driven firefly luciferase plasmids and a constitutively active Renilla luciferase construct (pRL‐TK), as well as 1 μg of poly(I:C). Eight hours later, cell lysates were harvested and subjected to luciferase assay. Values are expressed as mean of two independent experiments, N = 2.

6. HEK293T cells were transfected with plasmids encoding individual ZIKV proteins, the indicated promoter‐driven firefly luciferase plasmids and a constitutively active Renilla luciferase construct (pRL‐TK), as well as 0.4 μg of poly(I:C). Twenty‐four hours later, cell lysates were harvested and subjected to luciferase assay. C, capsid; E, envelope. Values are expressed as mean ± standard error. *P < 0.05, **P < 0.01 (Student's t‐test); N = 3.

Figure EV2. ZIKV NS5 reduces NF‐κB reporter activity

HEK293T cells were transfected with plasmids encoding different ZIKV proteins. Sixteen hours later, they were transfected with an NF‐κB promoter‐driven firefly luciferase plasmid (pNF‐κB‐luciferase) and a constitutively active Renilla luciferase construct (pRL‐TK), as well as 1 μg of poly(I:C). Twenty‐four hours later, cell lysates were harvested and subjected to luciferase assay. C, capsid; E, envelope. Values are expressed as mean ± standard error. *P < 0.05, **P < 0.01 (Student's t‐test); N = 3.

Figure 2. ZIKV infection blocks IFN signaling

1. A549 cells were treated with IFN‐α (100 U/ml), IFN‐λ (200 ng/ml), or IFN–γ (10 U/ml) for 6 h after which they were infected with ZIKV (MOI = 3) for 18 h (pre‐treatment). Alternatively, cells were first infected with ZIKV for 6 h followed by treatment with IFN for 12 h (post‐treatment). Total RNA was isolated, and levels of ZIKV genomic RNA were determined by qRT–PCR. Values are expressed as mean ± standard error. *P < 0.05, **P < 0.01 (Student's t‐test); N = 3.

2. A549 cells were infected with ZIKV (MOI = 3) for 6 h prior to addition of IFN‐α (100 U/ml), IFN‐λ (200 ng/ml), or IFN‐γ (10 U/ml) for 12 h. Total RNA was isolated, and levels of Ifit1 RNA were determined by qRT–PCR. Values are expressed as mean ± standard error. *P < 0.05, **P < 0.01 (Student's t‐test); N = 3.

3. A549 cells were infected with ZIKV (MOI = 5) for 6 h and then transfected with ISRE or GAS promoter‐driven firefly luciferase together with a constitutively active Renilla luciferase construct. Sixteen hours later, cells were treated with IFN‐α (100 U/ml) or IFN‐γ (10 U/ml) for 2 h. Cells lysates were harvested, and relative luciferase activity from ISRE and GAS promoters were determined. Values are expressed as mean ± standard error. *P < 0.05 (Student's t‐test); N = 3.

4. HEK293T cells were transfected with ZIKV protein expression constructs together with an ISRE promoter‐driven firefly luciferase plasmid (pGL3B/561) and a constitutively active Renilla luciferase construct (pRL‐TK). After 24 h, they were treated with IFN‐α (100 U/ml) for 10 h. Cell lysates were then harvested and subjected to luciferase assays. C, capsid; E, envelope. Values are expressed as mean ± standard error. *P < 0.05 (Student's t‐test); N = 3.

Figure 3. ZIKV infection causes degradation of human STAT2

1. A549 cells were infected with ZIKV (MOI = 5) for 24–48 h after which cell lysates were processed by SDS–PAGE and immunoblotting. Representative panels are shown. The experiment was repeated three times.

2. A549 cells were infected with ZIKV (MOI = 5) for 24 h and then treated with DMSO, MG132 (20 μM), or epoxomicin (Epox; 400 nM) for 12 h. Cell lysates were processed by SDS–PAGE and immunoblotting. Representative panels are shown. The experiment was repeated three times.

3. A549 cells (MOI = 2) or human primary fetal astrocytes (HFA) (MOI = 5) were infected with ZIKV for 24 and 48 h, respectively, after which IFN‐α (100 U/ml) was added for 2 h. Cells were then fixed and processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40× objective. Dashed line white circles indicated ZIKV‐infected cells. Representative panels are shown. The experiment was repeated two times.

4. Mouse embryonic fibroblasts (MEF) were infected with ZIKV (MOI = 5) for 48 h after which IFN‐α (200 U/ml) was added for 2 h. Samples were processed for immunofluorescence assay as described in panel (C). Dashed line white circles indicated ZIKV‐infected cells. Representative panels are shown. The experiment was repeated two times.

5. STAT2 levels in MEFs were decreased by transfection with siRNAs for 48 h after which the cells were infected with ZIKV (MOI = 5) for another 48 h. The silencing efficiency was determined by immunoblotting. Representative panels are shown. The experiment was repeated three times. The ZIKV replication was measured by qRT–PCR. Values are expressed as mean ± standard error. *P < 0.05, **P < 0.01 (Student's t‐test); N = 3.

Figure EV3. ZIKV infection does not affect STAT1 levels

1. A549 cells or mouse embryonic fibroblasts (MEFs) were infected with ZIKV (MOI = 2) for 24 and 48 h after which IFN‐α (100 U/ml) was added for 2 h. Cells were then fixed and processed for indirect immunofluorescence. Representative panels are shown. The experiment was repeated two times.

2. A549 cells were infected with ZIKV (MOI = 5) for 24 h and then treated with DMSO or bafilomycin‐A1 (Baf‐A1) (400 nM) for 12 h. Cell lysates were processed by SDS–PAGE and immunoblotting. Representative panels are shown. The experiment was repeated three times.

Figure EV4. Expression of ZIKV NS5 induces degradation of STAT2

A549 cells were transfected with control plasmid pcDNA 3.1 or plasmids encoding FLAG‐tagged ZIKV proteins. At 48 h, post‐transfection cells were treated with IFN‐α (100 U/ml) for 2 h and then processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40× objective. NS5‐positive cells are indicated by dashed white circles. Representative panels are shown. The experiment was repeated three times.

Figure 4. NS5 protein interacts with human STAT2 and induces its degradation

1. A549 cells were transfected with control plasmid pcDNA 3.1 or plasmids encoding FLAG‐tagged WT or mutant NS5 proteins. At 48 h, post‐transfection cells were treated with IFN‐α (100 U/ml) for 2 h and then processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40× objective. NS5‐positive cells are indicated by dashed white circles. Representative panels are shown. The experiment was repeated three times.

2. Quantification of nuclear STAT2 was performed using Volocity image analyses software. A minimum of 20 cells were counted for each sample. N.D., not detected. Values are expressed as mean ± standard error. *P < 0.05, **P < 0.01 (Student's t‐test); N = 3.

3. A549 cells were transfected with plasmids encoding FLAG‐tagged ZIKV NS5 or FLAG alone for 24 h and then treated with epoxomicin (400 nM) for 24 h. Cells were harvested and processed for immunoprecipitation (IP) using a mouse anti‐FLAG or anti‐myc antibody followed by SDS–PAGE and immunoblotting. Representative panels are shown. The experiment was repeated three times.

4. A549 cells were transfected with plasmids encoding FLAG‐tagged WT, mutant NS5 proteins, or FLAG alone for 48 h. Cells were harvested and processed for immunoprecipitation (IP) using rabbit anti‐STAT2 followed by SDS–PAGE and immunoblotting. Arrows indicate co‐immunoprecipitated WT NS5 and NS5 mutants that are in a complex with STAT2. Representative panels are shown. The experiment was repeated two times.