The zinc finger protein ZFP36L2 inhibits flavivirus infection via the 5'-3' XRN1-mediated RNA decay pathway in the replication complexes

Abstract

Background: The zinc finger protein 36-like (ZFP36L) family is a CCCH-type group consisting of RNA-binding proteins, i.e., ZFP36L1 and ZFP36L2, which regulate cellular mRNA through the RNA decay pathway. ZFP36L1 combats flavivirus infections through the 5'-3' XRN1 and 3'-5' RNA exosome decay pathways. The present study clarified the role of human ZFP36L2 in the defense response of the host against flavivirus infection.

Methods: Cell lines with overexpression or knockdown of ZFP36L2 were established using lentiviral vectors carrying genes for overexpression and short-hairpin RNA targeting specific genes, respectively. A plaque assay was employed to determine the viral titer. Immunofluorescence and real-time quantitative polymerase chain reaction were used to measure the viral RNA levels. The in vitro-transcribed RNA transcript derived from a replication-dead Japanese encephalitis virus (JEV) replicon containing the renilla luciferase reporter gene (J-R2A-NS5mt) was used to assess the stability of the flavivirus RNA. An RNA immunoprecipitation assay was used to detect the protein-RNA binding ability. Confocal microscopic images were captured to analyze protein colocalization.

Results: ZFP36L2 served as an innate host defender against JEV and dengue virus. ZFP36L2 inhibited flavivirus infection solely through the 5'-3' XRN1 RNA decay pathway, whereas ZFP36L1 inhibited JEV infection via the 5'-3' XRN1 and 3'-5' RNA exosome RNA decay pathways. The direct binding between viral RNA and ZFP36L2 via its CCCH-type zinc finger motifs facilitated the degradation of flavivirus RNA mediated by 5'-3' XRN1. Furthermore, ZFP36L2 was localized in processing bodies (PBs), which participate in the 5'-3' XRN1-mediated RNA decay pathway. Nonetheless, the disruption of PBs did not affect the antiviral activity of ZFP36L2, suggesting that its localization is not essential for the function of the protein. Interestingly, the colocalization of ZFP36L2 and XRN1 with viral RNA and NS3 revealed that the antiviral activity of ZFP36L2 occurred within the replication complexes (RCs).

Conclusions: In summary, ZFP36L2 bound to and degraded viral RNA through the XRN1-mediated RNA decay pathway in the RCs, thereby inhibiting flavivirus replication. These findings provide valuable insights into the diverse antiviral mechanisms of the ZFP36-like family of proteins in the innate immune response against flavivirus infection.

Keywords: 5′- 3′ XRN1; Antiviral mechanism; CCCH-type zinc finger protein; Processing bodies; RNA decay pathway; Replication complexes; ZFP36L2.

Fig. 1

Antiviral activity of human ZFP36L2 against JEV and DENV infection. A549 cells were transduced with lentiviruses expressing EGFP, HA-tagged ZFP36L1 (HA-ZFP36L1) or HA-tagged ZFP36L2 (HA-ZFP36L2) (MOI = 2) for 72 h. Subsequently, these cells were infected with JEV A, B and DENV, C, D (MOI = 5). At 24 hpi, both cell lysates and culture supernatants were collected. A, C Cell lysates were used to determine the levels of the viral JEV or DENV NS3 protein, HA-ZFP36L2, and actin by western blot analysis. B, D Culture supernatants were used to measure viral titers by plaque assay. Representative data are presented as the mean ± SD (n = 3), and statistical significance was analyzed by two-tailed Student’s t-test; ** P ≤ 0.01

Fig. 2

Enhancement of both JEV and DENV replication in ZFP36L2-knockdown cells. A, B A549 cells were mock infected or infected with JEV (A) or DENV (B) (MOI = 5) for the indicated periods. A Western blot analysis of the levels of ZFP36L2, viral NS3, and actin (as the loading control) is shown. The ZFP362 protein level normalized to actin was quantified using the ImageJ software. Data are representative of three independent experiments, as shown in the supplementary data (Fig. S2), and are expressed as mean ± SD (n=3). C A549 cells with either shLacZ or shZFP36L2 were infected with JEV or DENV (MOI = 5) for 24 h. Subsequently, the culture supernatants were used to determine the viral titer. Data from three independent experiments are presented as the mean ± SD. Statistical significance was analyzed by two-tailed Student’s t-test. * P ≤ 0.05, ** P ≤ 0.01, *** P < 0.001

Fig. 3

ZFP36L1 and ZFP36L2 reduce JEV viral RNA levels and inhibit JEV replication by destabilizing the viral RNA. A549 cells were transduced with lentiviruses expressing EGFP, HA-ZFP36L1, or HA-ZFP36L2 (MOI = 2) for 72 h, then infected with JEV (MOI = 5) for indirect IFA and RT-qPCR analysis. A At 24 hpi, indirect IFAs of cells stained with viral dsRNA (red), HA-tagged ZFP36L1 (green), HA-tagged ZFP36L1 (green), and DAPI (blue) were photographed using a fluorescence microscope. B RT-qPCR analysis was employed to measure the JEV RNA levels after the indicated time points. The relative JEV RNA levels were normalized to that of GAPDH and presented as the mean ± SD (n = 3). C T-REx-293 cells with inducible expression of HA-ZFP36L1 or HA-ZFP36L2 were cultured in medium without (−) or with (+) Dox (1 μg/mL) for 16 h were co-transfected with 5ʹ-capped RdRP-dead JEV replicon RNA and control firefly luciferase (Fluc) RNA. The relative RNA levels of replicon normalized to that of firefly luciferase were analyzed using RT-qPCR at 4 h and 6 h post-transfection. D T-REx-293 cells overexpressing HA-ZFP36L1 or HA-ZFP36L2 induced without (−) or with (+) Dox (1 μg/mL) for 16 h were co-transfected with 5ʹ-capped RdRP-dead JEV replicon RNA or 5ʹ-capped renilla luciferase (Rluc) RNA and control firefly luciferase (Fluc) RNA. The relative luciferase activity (Rluc/Fluc) was measured by dual-luciferase reporter assay at 24 h post-transfection. Data from three independent experiments are presented as the mean ± SD. Statistical significance was analyzed by two-tailed Student’s t-test. * P ≤ 0.05, ** P ≤ 0.01, *** P < 0.001

Fig. 4

The zinc-finger motifs of ZFP36L2 are critical for its viral RNA-binding and antiviral activities. A Schematic representation of wild-type (WT) ZFP36L2 and the ZF domains of the ZFP36L2 (C174R/C212R) mutant. B 293T/17 cells were infected with JEV (MOI = 5) for 16 h or DENV (MOI = 5) for 36 h, then transfected with the indicated plasmids expressing HA-tagged ZFP36L2 (WT or C174R/C212R mutant) or EGFP for 16 h. The viral RNA bound to HA-ZFP36L2 (WT or C174R/C212R mutant) and EGFP was pulled down with anti-HA beads and amplified by RT-PCR using JEV or DENV 3′-UTR-specific primers (middle panel). Input RT-PCR used for the determination of the RNA levels of the JEV or DENV 3′ UTR in virus-infected cells (bottom panel). A western blot analysis was conducted to estimate the expression of the immunoprecipitated HA-ZFP36L2 (WT and C174R/C212R mutant) (top panel). C A549 cells were transduced with the lentiviral vector (MOI = 2) expressing HA-tagged ZFP36L2 (WT or C174R/C212R mutant) or EGFP for 72 h, then infected with JEV or DENV (MOI = 5) for 24 h. The culture supernatants were collected to determine the viral titers by plaque assay, and the cell lysates were examined by western blot analysis

Fig. 5

Distinct antiviral pathways employed by human ZFP36L1 and ZFP36L2 against JEV infection. A549 cells with shLacZ, shXRN1, shEXOSC5, and shXRN1/shEXOSC5 were transduced with lentiviruses expressing EGFP, HA-ZFP36L1, or HA-ZFP36L2 (MOI = 2) for 72 h. Subsequently, these cells were infected with JEV (MOI = 5). At 24 hpi, cell lysates and culture supernatants were collected. A, C, E Cell lysates were examined by western blot analysis of the indicated proteins. B, D, F Culture supernatants were used to measure the viral titers by plaque assay. Representative data are presented as the mean ± SD (n = 3), and statistical significance was analyzed by two-tailed Student’s t-test. ** P ≤ 0.01, *** P ≤ 0.001, NS: not significant

Fig. 6

Localization of the human ZFP36L2 protein in cytoplasmic PBs and SGs. A, B A549 and COS-7 cells were co-transfected with either control mCherry (red) or mCherry-ZFP36L2 (red) plus EGFP-Dcp1a (a PB marker; green) or EGFP-G3BP1 (an SG marker; green) for 24 h. Cells were fixed and permeabilized, and the subcellular localization was analyzed by confocal microscopy

Fig. 7

Disruption of PBs does not impact the ZFP36L2-mediated antiviral activity against JEV. A A549 cells with either shLacZ or sheIF4E-T were transduced with lentiviruses expressing EGFP-Dcp1a (MOI = 2) for 72 h, then treated with 0.5 mM sodium arsenite (0.5 mM) for 1 h. The formation of PBs by EGFP-Dcp1a (green) was observed using confocal microscopy. Nuclei were stained with DAPI (blue). B, C A549 cells with either shLacZ or sheIF4E-T were transduced with lentiviruses expressing EGFP or HA-ZFP36L2 (MOI = 2) for 72 h. Subsequently, these cells were infected with JEV (MOI = 5). At 24 hpi, cell lysates were harvested for western blotting using the indicated antibodies (B). Culture supernatants were used to measure the viral titers by plaque assay (C). Representative data are presented as the mean ± SD (n = 3), and statistical significance was analyzed by two-tailed Student’s t-test. NS: not significant

Fig. 8

Subcellular localization of ZFP36L2, XRN1, and viral RNA in the RCs. A549 cells were infected with JEV (MOI = 5) for 3 h, then transfected with the indicated plasmids expressing either control mCherry (red) or mCherry-ZFP36L2 (red) plus EGFP-XRN1 (green) (A–C) for 16 h. Subsequently, the cells were fixed and permeabilized for confocal microscopy. A–C Viral RNA was detected using a mouse anti-dsRNA Ab and an Alexa Fluor 647 goat anti-mouse Ab (purple) (A). The JEV NS3 protein was detected using a mouse anti-JEV NS3 Ab and an Alexa Fluor 647 goat anti-mouse Ab (purple) (B). The ER was stained with an Alexa Fluo 647 anti-calreticulin Ab (purple) (C). Nuclei were stained with DAPI (blue). D Pairwise colocalization analysis was performed using Pearson’s correlation coefficient (PCC). Means and SD were calculated from 20 cells for each group. ** P ≤ 0.01, *** P < 0.001