Research ArticleANTIVIRAL IMMUNITY

PARP12 suppresses Zika virus infection through PARP-dependent degradation of NS1 and NS3 viral proteins

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Science Signaling  19 Jun 2018:
Vol. 11, Issue 535, eaas9332
DOI: 10.1126/scisignal.aas9332
  • Fig. 1 ISG PARP12 suppresses ZIKV replication in vitro.

    (A) Individual ISGs were knocked out in A549 cells that were subsequently infected with ZIKV. Cell culture supernatants were collected at 24 hours after infection, and ZIKV titers were measured by plaque assay. (B to D) PARP12-overexpressing or control vector–transfected A549 cells were infected with ZIKV. Viral accumulation after 24 hours in the culture supernatants (B and C) and cell lysates (D) was measured by plaque assay (B) or qRT-PCR (C and D). PFU, plaque-forming units. (E to G) PARP12−/− and WT A549 cells were infected with ZIKV. Viral accumulation after 24 hours in the culture supernatants (E and F) and cell lysates (G) was measured by plaque assay (E) or qRT-PCR (F and G). (H) PARP12-overexpressing MEF cells were infected with ZIKV. Viral accumulation after 24 hours in the culture supernatants was measured by plaque assay. (I) PARP12−/− MEF cells were infected with ZIKV. Viral accumulation after 24 hours in the culture supernatants was measured by plaque assay. Viral titers and RT-PCR data (A to I) are means ± SEM from three independent experiments. **P < 0.01 and ***P < 0.001 by Student’s t test (B to I).

  • Fig. 2 PARP12 suppresses ZIKV by reducing NS1 and NS3 protein abundance.

    (A) Control, PARP12-overexpressing, and PARP12−/− A549 cells were incubated with ZIKV at 4°C for 1 hour and then washed extensively with phosphate-buffered saline (PBS). Cell surface binding was assessed by determining the viral copy number in the cell lysates by qRT-PCR. (B) Control, PARP12-overexpressing, and PARP12−/− A549 cells were incubated with ZIKV at 4°C for 1 hour and then allowed to internalize bound ZIKV by incubation at 37°C for another 10 min. Viral entry into cells was assessed by determining the viral copy number in the cell lysates by qRT-PCR. (C) BHK-21 cells were cotransfected with either the PARP12 expression plasmid or empty vector and with RNA from a ZIKV replicon encoding an RLuc reporter. At 24 hours after transfection, luciferase activity in cell lysates was determined. NITD008 was used as a positive control. DMSO, dimethyl sulfoxide. (D) Western blot analysis of lysates from HEK293T cells cotransfected with plasmids encoding one of the ZIKV nonstructural proteins NS1/2B/3/4A/4B/5 and hemagglutinin (HA)–PARP12 or vector control. (E) Densitometry analysis of data in (D). (F) Western blot analysis of lysates from HEK293T cells cotransfected with plasmids encoding one of the ZIKV His-NS1/3/4A and increasing amounts of HA-PARP12 plasmids (0, 100, 300, and 500 ng). (G) Densitometry analysis of data in (F). (H) Western blot analysis of lysates from WT and PARP12−/− HEK293T cells cotransfected with plasmids encoding ZIKV NS1 (top) or NS3 (bottom) and HA-PARP12 or vector control, as indicated. (I) Densitometry analysis of data in (H). (J) Western blot analysis of lysates from WT and PARP12−/− A549 cells infected with ZIKV for 24 hours. (K) Densitometry analysis of data in (J). RT-PCR and luciferase data (A to C) are means ± SEM from three independent experiments. Western blot results (D, F, H, and J) are representative of three independent experiments. Data (E, G, I, and K) are means ± SEM pooled from three independent experiments. **P < 0.01 and ***P < 0.001 by Student’s t test.

  • Fig. 3 The PARP domain of PARP12 is required to reduce NS1 and NS3 protein abundance.

    (A) Map of the functional regions of full-lenght (FL) PARP12 and deletion constructs. (B) Western blot analysis of lysates from PARP12−/− HEK293T cells cotransfected as indicated with His-tagged NS1 or NS3 and the PARP12-deletion constructs. (C) Densitometry analysis of data in (B). (D) qRT-PCR analysis of NS1 and NS3 mRNA expression in HEK293T cells at 24 hours after cotransfection with either His-NS1 or His-NS3 and with PARP12 or control plasmids. (E) Viral replication was assessed by plaque assay of cell culture supernatants taken 24 hours after HeLa cells transfected with PARP12 truncated mutants or control plasmids were infected by ZIKV. (F and G) Viral replication was assessed by plaque assay of cell culture supernatants taken 49 hours after A549 cells were infected with ZIKV for 1 hour and then treated with INO-1001 [3-aminobenzamide (3AB)] (F) or AG-014699 (AG; rucaparib) (G) at the indicated concentrations. NITD008 was used as a positive control. (H and I) Cell viability was assessed by MTT assay of A549 cells pretreated with indicated concentration of INO-1001 (3-aminobenzamide) (H) and AG-014699 (rucaparib) (I) for 48 hours. Data were normalized to DMSO control. Western blot results (B) are representative of three independent experiments. Data (C to I) are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.

  • Fig. 4 PARP12 reduces NS1 and NS3 protein abundance through proteasomal degradation.

    (A) Western blot analysis of lysates immunoprecipitated (IP) for PARP12 from HEK293T cells cotransfected with HA-PARP12 and His-NS1 or His-NS3 plasmids. (B) Confocal microscopy analysis of Vero cells that were cotransfected with EGFP-PARP12 and DsRed-NS1 or DsRed-NS3 plasmids. Scale bars, 5 μm. (C) Western blot analysis of lysates from HEK293T cells cotransfected with HA-PARP12 and His-NS1 or His-NS3, as indicated. After 18 hours, cells were treated with 10 μM MG132 for 10 hours, where indicated. (D) Densitometry analysis of data in (C). (E) Western blot analysis of lysates immunoprecipitated for His-tagged proteins from HEK293T cells cotransfected with HA-ubiquitin, EGFP-PARP12, and His-NS1 or His-NS3 plasmids and treated with MG132, as indicated. (F) Densitometry analysis of data in (E). Western blot (A, C, and E) and confocal (B) results are representative of three independent experiments. Data (D and F) are means ± SEM pooled from three independent experiments. *P < 0.05 and **P < 0.01 by Student’s t test.

  • Fig. 5 Degradation of NS1 and NS3 protein is dependent on PARP12 ADP-ribosylation activity.

    (A) Western blot analysis of lysates immunoprecipitated for His-tagged proteins from HEK293T cells transfected with EGFP-PARP12 and His-NS1 or His-NS3 plasmids and treated with MG132, as indicated. (B) Densitometry analysis of data in (A). (C) Map of the functional regions of WT and G565A PARP12 constructs. A PARP12 mutant with a disruption in the catalytic site (G565A) was generated by site-directed PCR mutagenesis. (D) Western blot analysis of lysates immunoprecipitated for His-tagged proteins from PARP12−/− HEK293T cells transfected with His-NS1 or His-NS3 and with EGFP-PARP12 WT, EGFP-PARP12 G565A, or vector control plasmids. (E) Densitometry analysis of data in (D). (F) Western blot analysis of lysates immunoprecipitated for His-tagged proteins from PARP12−/− HEK392T cells cotransfected with HA-ubiquitin and His-NS1 or NS3 plasmids, as well as EGFP-PARP12 WT, EGFP-PARP12 G565A, or vector control plasmid constructs. (G) Densitometry analysis of data in (F). (H) Western blot analysis of lysates from PARP12−/− HEK392T cells cotransfected with His-NS1 or His-NS3 and with EGFP-PARP12 WT, EGFP-PARP12 G565A, or vector control plasmids. (I) Densitometry analysis of data in (H). (J) Viral replication was assessed by plaque assay of cell culture supernatants taken 24 hours after HeLa cells transfected with EGFP-PARP12 WT, EGFP-PARP12 G565A, or vector control plasmids were infected with ZIKV. Western blots (A, D, F, and H) are representative of three independent experiments. Data (B, E, G, and I) and viral titers (J) are means ± SEM pooled from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.

  • Fig. 6 After PARP12-mediated PAR, ZIKV NS1 and NS3 proteins are ubiquitinated on K48 and degraded.

    (A) Western blot analysis of lysates immunoprecipitated for His-tagged proteins from HEK293T cells were cotransfected with EGFP-PARP12, His-NS1 or His-NS3, and HA-K48 ubiquitin mutants. MG132 was added as indicated. (B) Densitometry analysis of the data in (A). (C) Western blot analysis of lysates immunoprecipitated for His-tagged proteins from HEK293T cells were cotransfected with EGFP-PARP12, His-NS1 or His-NS3, and HA-K63 ubiquitin mutants. MG132 was added as indicated. (D) Densitometry analysis of the data in (C). (E) PARP12 mediates PAR of NS1 and NS3, which are subsequently ubiquitinated by an E3 ligase that is recruited by the PAR modification. Ubiquitinated NS1 and NS3 are then recognized and targeted by the proteasome for degradation. Western blot results (A and C) are representative of three independent experiments. Data (B and D) are means ± SEM pooled from three independent experiments. *P < 0.05 and **P < 0.01 by Student’s t test. Ub, ubiquitin.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/535/eaas9332/DC1

    Fig. S1. A CRISPR/Cas9 screen identifies ISGs that restrict ZIKV infection.

    Fig. S2. The expression of PARP12 can be induced by IFN and ZIKV infection.

    Fig. S3. Construction of PARP12 knockout and PARP12-overexpressing A549 cell lines.

    Fig. S4. The expression and expected size of PARP12 domain deletion mutants.

    Fig. S5. Alignment of ZnF domains from PARP12 and PARP13.

    Table S1. sgRNA sequences used for ISG knockouts.

  • Supplementary Materials for:

    PARP12 suppresses Zika virus infection through PARP-dependent degradation of NS1 and NS3 viral proteins

    Lili Li, Hui Zhao, Ping Liu, Chunfeng Li, Natalie Quanquin, Xue Ji, Nina Sun, Peishuang Du, Cheng-Feng Qin, Ning Lu,* Genhong Cheng*

    *Corresponding author. Email: ninglu{at}ibp.ac.cn (N.L.); gcheng{at}mednet.ucla.edu (G.C.)

    This PDF file includes:

    • Fig. S1. A CRISPR/Cas9 screen identifies ISGs that restrict ZIKV infection.
    • Fig. S2. The expression of PARP12 can be induced by IFN and ZIKV infection.
    • Fig. S3. Construction of PARP12 knockout and PARP12-overexpressing A549 cell lines.
    • Fig. S4. The expression and expected size of PARP12 domain deletion mutants.
    • Fig. S5. Alignment of ZnF domains from PARP12 and PARP13.
    • Table S1. sgRNA sequences used for ISG knockouts.

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    © 2018 American Association for the Advancement of Science

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