Virus and cells
ZIKV strain GZ01/2016 (Genbank accession number KU820898) and VSV virus was used at a multiplicity on infection (MOI) of 0.1 in this study, except where indicated otherwise (33). The IFNAR1-/- HEK293T and A549 cell lines were generated as described (19). A549, BHK-21, Vero, HeLa and HEK293T cells were purchased from America Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (37°C, 5% CO2) supplemented with 10% fetal bovine serum (FBS),100U/mL penicillin and 50 μg/mL streptomycin.
Plaque assay
BHK-21 cells were seeded in a 12-well plate for 12 hr. Cells were washed with PBS once and infected with virus samples for 1 hr. The culture supernatant was aspirated and replaced with DMEM containing 1% low-melting agarose and 2% FBS. Viral plaques were stained and counted 4 days after infection. The titer of ZIKV was quantified by plaque assay and normalized to control.
DNA constructs and stable cell line generation
pMOI-GFP and pMOI-PARP11 (Homo sapiens) expression plasmids were purchased from GeneCopoeia and described previously (34). The viral RNA of the GZ01/2016 strain was isolated and used in reverse transcription PCR experiments to obtain the complementary DNA (cDNA) sequence of ZIKV nonstructural NS1 and NS3 proteins. ZIKV NS1 and NS3 genes were cloned into the pcDNA6/V5-His expression vector (Invitrogen) using standard molecular techniques and verified by sequencing. DsRed-PARP11, EGFP-PARP12, Flag-PARP11, HA-PARP12, EGFP-PARP11 WWE domain, EGFP-PARP11 PARP domain, YFP-PARP11, Flag-PARP13 and HA-PARP11 mutants were cloned using standard molecular cloning and oligonucleotide mutagenesis methods. To create a stable cell line for PARP11 expression, PARP11 was cloned into the pMXsIG-IgkFLAG vector and co-transfected into HEK293T cells with VSV glycoprotein and pCpG helper plasmids. 48 hr after transfection, the culture supernatant was collected and added into WT or PARP12-/- A549 cells for infection. The cells were collected 72 hr after infection, and the PARP11-overexpressing cells were then sorted by fluorescence-activated cell sorting (FACS).
Western blotting
WT, PARP11-/- and PARP12-/- HEK293T cells were cotransfected with the plasmids listed in the main text and where indicated. 28 hr after transfection, cells were treated with lysis buffer [ 50mM tris-HCI (pH 7.5), 150 mM NaCI, 5 mM EDTA, 1% NP-40, 1 mM PMSF, and 1хprotein inhibitor (Roche)]. The cell extracts were immunoblotted with the indicated antibodies to measure the level of the expressed proteins. Mouse anti-β-actin (ZSGB-Bio), rabbit anti-GFP (Abcam), rabbit anti-PARP11 (Thermo Fisher Scientific), mouse anti-poly(ADP-ribose) (GeneTex) and mouse anti-HA, anti-His, and anti-Flag tag antibodies (Sigma-Aldrich) were used for detection at the appreciated dilutions.
Co-immunoprecipitation assay
HEK293T cells were transfected with the indicated plasmids. 30 hr after transfection, protein was extracted using solution A [ 50mM tris-HCI (pH 7.5), 150 mM NaCI, 5 mM EDTA, 1% Triton-X100, 1 mM phenylmethylsulfonylfluoride (PMSF), and 1хprotein inhibitor (Roche)]. An aliquot of the extracts was immunoblotted with the indicated antibodies. The remaining extracts were immunoprecipitated using Sepharose beads bound to anti-Flag or anti-HA antibodies (Sigma-Aldrich) at 4°C overnight. After washing the Sepharose beads four times with solution B [ 50mM tris-HCI (pH 7.5), 150 mM NaCI, 5 mM EDTA, 0.2% Triton-X100, and 1 mM PMSF], proteins were eluted by heating the beads to 98°C in 1хSDS-polycrylamid gel electrophoresis loading buffer [ 50 mM Tris-HCl (pH 6.8), 2% (V/V) SDS, 6% (V/V) glycerol, and 2% (V/V) β-mercaptoethanol]. The eluted was analyzed by Western blotting with the indicated antibodies.
Gene knockout by the CRISPR/Cas9 system
To knockout PARP11 and PARP12 in A549 and HEK293T cell lines, two small guide RNAs (SgRNAs) (~100 bp gap sequence) targeting the PARP11 and PARP12 genes were designed and cloned into sgRNA expression vectors under the control of human U6 promotor. A549 or HEK293T cells were transfected with sgRNAs and Cas9 expression plasmids, followed by puromycin selection, as described previously (35, 36). Sigle clones were isolated by FACS and confirmed by PCR genotyping and sequencing.
RNA isolation, reverse transcription, and PCR
Total RNA from cells or viruses was extracted with the PureLink RNA Extraction kit (Thermo Fisher Scientific). Viral RNA copies were measured by qRT-PCR (37) with the One Step PrimeScript RT-PCR kit (Takara). ZIKV primers and TaqMan probes were described previously (38). Primers used to amplify corresponding genes were obtained from PrimerBank (http://pga.mgh.harvard.edu/primerbank/). SYRB Green qPCR mix (TransGen Biotec) was used to analyze mRNA levels on an ABI 7500 (Applied Biosystems) analyzer.
Immunofluorescence staining and confocal imaging
Vero cells were seeded in a confocal dish (Solarbio) and transfected with EGFP-PARP12 and DsRed-PARP11 plasmids. After 24 hr, cells were fixed with 0.4% paraformaldehyde for 15 min and permeabilized in 0.2% Triton-X100 for 15 min at room temperature. The cells were washed three times with PBS supplemented with 0.05% Tween-20. Nuclei were stained with 4’6-diamidino-2-phenylindole (Thermo Fisher Scientific). Cells were imaged on a LSM700 (Carl Zeiss) confocal microscope, and the images were analyzed with ImageJ software.
Statistical analysis
All data were analyzed using Prism software (Graphpad). Statistical evaluation was performed by two-way Student’s t test. Data are mean±SEM, and P values are indicated by *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. All cellular experiments were repeated at least three times.