DNA-PKcs restricts Zika virus spreading and is required for effective antiviral response

Abstract

Zika virus (ZIKV) is a single-strand RNA mosquito-borne flavivirus with significant public health impact. ZIKV infection induces double-strand DNA breaks (DSBs) in human neural progenitor cells that may contribute to severe neuronal manifestations in newborns. The DNA-PK complex plays a critical role in repairing DSBs and in the innate immune response to infection. It is unknown, however, whether DNA-PK regulates ZIKV infection. Here we investigated the role of DNA-PKcs, the catalytic subunit of DNA-PK, during ZIKV infection. We demonstrate that DNA-PKcs restricts the spread of ZIKV infection in human epithelial cells. Increased ZIKV replication and spread in DNA-PKcs deficient cells is related to a notable decrease in transcription of type I and III interferons as well as IFIT1, IFIT2, and IL6. This was shown to be independent of IRF1, IRF3, or p65, canonical transcription factors necessary for activation of both type I and III interferon promoters. The mechanism of DNA-PKcs to restrict ZIKV infection is independent of DSB. Thus, these data suggest a non-canonical role for DNA-PK during Zika virus infection, acting downstream of IFNs transcription factors for an efficient antiviral immune response.

Keywords: DNA-PKcs; Zika virus; double-strand DNA breaks; infection; interferon.

Figure 1

DNA-PKcs is critical for control of ZIKV infection. Virus replication measured by plaque assay, expressed as plaque-forming units per mL (PFU/mL), on (A) A549^WT and A549 ^PRKDC-/- or (B) RPE^WT and RPE ^PRKDC-/- cells infected with ZIKV at indicated m.o.i. and time. RT-qPCR analysis to measure ZIKV RNA in (C) A549^WT and A549 ^PRKDC-/- or (D) RPE^WT and RPE ^PRKDC-/- cells infected at indicated m.o.i. and time. (E) A549^WT and A549 ^PRKDC-/- or (F) RPE^WT and RPE ^PRKDC-/- cells infected with 50 PFU of ZIKV at 48 hours in semi-solid medium, then ZIKV-E protein (green) was stained for immunofluorescence analysis, and the relative area of infection percentage was measured using the ImageJ software. The cell nuclei were stained with DAPI (blue). (G) Percentage of ZIKV-infected A549^WT and A549 ^PRKDC-/- cells at indicated m.o.i. and time, analyzed by flow cytometry. (H) Viability analysis by MTT assay of ZIKV-infected A549^WT and A549 ^PRKDC-/- cells relative to uninfected cells (mock) at indicated m.o.i. and time. (I) A549^WT and A549 ^PRKDC-/- were pretreated with NU7441 (0.5 and 1 µM) at 24 hours followed by ZIKV infection (m.o.i. 1) at 24 hours. We used two-way ANOVA with Sidak’s correction in (A–D, G, H), and unpaired two-tailed Student’s t-test was used in (E, F). * p<0.05, n = 3, error bars ± SEM.

Figure 2

ZIKV infection does not induce DSB in A549 cells. A549^WT, A549 ^PRKDC-/- and 1 µM NU7441 pre-treated A549^WT infected with ZIKV (m.o.i. 1) at 24 hours. Stimulation with 3 µM etoposide for 12 hours was used as a DSB positive control. (A) Immunofluorescence to analyze γH2AX (green) in the ZIKV-infected cells (red, ZIKV-E protein). The cell nuclei were stained with DAPI (blue). (B) Percentage of γH2AX foci per cell showed in (A). *Compared with WT cells; #Compared with mock. We used two-way ANOVA with Sidak’s correction. * or # p<0.05, n = 3, error bars ± SEM.

Figure 3

DNA-PKcs regulates interferon-related genes during ZIKV infection. (A) RT-qPCR to measure the expression of mRNA for indicated genes on A549^WT and A549^RIGI-/- cells infected with ZIKV m.o.i. 1 for 24 hours. (B) RT-qPCR to measure the expression of mRNA for indicated genes on A549^WT and A549 ^PRKDC-/- cells infected with ZIKV m.o.i. 0.1 at the indicated time or (C) m.o.i. 1 at 24 hours. (D) Expression of mRNA for NFKBIA and IL6, determined by RT-qPCR on A549^WT and A549 ^PRKDC-/- cells infected with ZIKV m.o.i. 1 at 24 hours. We used unpaired two-tailed Student’s t-test in (A, C, D), and two-way ANOVA with Sidak’s correction in (B). ND: Non-detected. * p<0.05, n = 3, error bars ± SEM.

Figure 4

ZIKV induces IRF1, IRF3, and p65 nuclei accumulation independent of DNA-PKcs. Immunofluorescence analysis for localization of endogenous (A) IRF3 (red) and (B) IRF1 (red) on A549^WT and A549^RIGI-/- cells infected with ZIKV m.o.i. 1 (green, ZIKV-E protein) at 36 hours (left panel), and quantified by scoring cells with nuclear staining (right panel, n = 3, counts of at least 30 nuclei per slide). (C–E) Immunofluorescence analysis for localization of endogenous (C) IRF3 (red), (D) IRF1 (red), and (E) p65 (red) on A549^WT and A549 ^PRKDC-/- cells infected with ZIKV m.o.i. 1 (green, ZIKV-E protein) at 24 hours (left panel), and quantified by scoring cells with nuclear staining (right panel, n = 3, counts of at least 30 nuclei per slide). TNF was used as a positive control in (E). Cell nuclei were stained with DAPI (grey). We used unpaired two-tailed Student’s t-test. N = 3, error bars ± SEM. ND: non-detected.