Zinc finger protein ZFP36L1 inhibits influenza A virus through translational repression by targeting HA, M and NS RNA transcripts

Abstract ZFP36L1, a CCCH-type zinc finger protein, is an RNA-binding protein that participates in controlling cellular mRNA abundance and turnover by posttranscriptional regulation. Here, we demonstrated that ZFP36L1 has an important role in host defense against influenza A virus (IAV) infection. Overexpression of ZFP36L1 reduced IAV replication via translational repression of HA, M and NS RNA segment transcripts. IAV infection upregulated cellular ZFP36L1 expression, and endogenous ZFP36L1 knockdown significantly enhanced IAV replication. ZFP36L1 directly binds to IAV NS1 mRNA in the cytoplasm and blocks the expression and function of NS1 protein. Mutation of CCCH-type zinc finger domains of ZFP36L1 lost its antiviral potential and NS1 mRNA binding. Thus, ZFP36L1 can act as a host innate defense by targeting HA, M and NS mRNA transcripts to suppress viral protein translation.


INTRODUCTION
Control of posttranscriptional RNA regulation via cellular mRNA decay and translation inhibition mechanisms plays an important role in the host defense against RNA virus infection. Recently, growing evidence indicates that RNA binding protein-mediated mRNA decay and trans-lational repression machinery by CCCH-type zinc finger (ZF) proteins can function as a host innate defense against virus infection. Several of these CCCH-type ZF proteins identified as RNA-binding proteins involved in host antiviral defense by diverse antiviral mechanisms are tristetraprolin (TTP) (1), monocyte chemotactic protein-induced protein-1 (MCPIP1) (2)(3)(4), zinc-finger antiviral protein (ZAP) (5), target of Egr1 (TOE1) (6), tetrachlorodibenzop-dioxin (TCDD)-inducible poly (ADP-ribose) polymerase (TIPARP/PARP7) (7) and long isoform of PARP12 poly(ADP-ribose) polymerase 12 (PARP12/ZC3H1) (8). TTP itself inhibits HIV-1 production by directly binding to genomic RNA and enhancing HIV RNA transcript splicing. MCPIP1 has broad-spectrum antiviral effects against Japanese encephalitis virus (JEV) (2), dengue virus (DENV) (2), hepatitis C virus (HCV) (3) and HIV (4) via viral RNA binding and degradation by its own RNase activity. CCCHtype ZAP exhibits antiviral activity by preventing the accumulation of viral mRNA via directly binding to viral RNA and recruiting 3 -5 exoribonuclease exosome complex for further degradation (9,10) as well as translational repression of viral mRNA by interrupting the interactions of translational initiation factors eIF4A and eIF4G (11). TOE1, a downstream target of the immediate early gene Egr1, functions as an inhibitor of HIV-1 replication by specifically binding to a HIV-1 transactivator response element and inhibiting its activity (6). TIPARP, a ZAP-like protein, binds to sindbis virus (SINV) RNA via its ZF domain and recruits an exosome to promote the degradation of viral RNA (7). PARP12L, an interferon-stimulated gene, functions as a host defense against infection with Venezuelan equine encephalitis virus (VEE) by downregulating cellular translation (8).
Influenza A virus (IAV) is a segmented, negative-sense single-stranded RNA virus in the family Orthomyxoviridae. The IAV genome possesses eight RNA segments that are encapsidated in the form of ribonucleoprotein (RNP) complexes and encode at least 16 viral proteins (29). There are nine structural proteins: the two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), are the receptor-binding protein and glycoside hydrolase enzyme, respectively, which mainly mediate virus entry and maturevirus particle release, respectively. M2, a proton-selective channel protein represents a minor amount on the virion surface. The major structural protein matrix protein (M1) lies beneath the viral envelope. The polymerase basic proteins 1 and 2 (PB1 and PB2), polymerase acidic protein (PA) and nucleoprotein (NP) form a viral ribonucleoprotein (vRNP) complex for viral transcription and replication (30,31). Nuclear export protein/nonstructural protein 2 (NEP/NS2) and M1 are involved in vRNPs nuclear ex-port during the viral life cycle (32,33). Nonstructural proteins such as nonstructural protein 1 (NS1) or PB1-F2 play important roles in influencing host immune responses by interferon (IFN)-antagonist activities to expedite efficient viral replication (34,35).
Here we examined whether human ZFP36L1 had potent antiviral activity against IAV by blocking the translational process of IAV mRNA, mainly the NS, HA and M RNA segments.
Nucleic Acids Research, 2020, Vol. 48, No. 13 7373 Immunofluorescence assay A549 cells were transduced with the lentiviral vector expressing HA-tagged ZFP36L1 protein for 72 h, then infected with IAV for 24 h. Cells were fixed with 4% formaldehyde and permeabilized in phosphate buffered saline (PBS) with 0.5% Triton X-100. IAV NP protein expression was detected with a rabbit anti-IAV NP antibody and Alexa Fluor 488 goat anti-rabbit secondary antibody (Molecular Probes). The expression of HA-tagged ZFP36L1 was detected with a mouse anti-HA antibody and Alexa Fluor 568 goat anti-mouse secondary antibody (Molecular Probes). Nuclei were stained with 4 ,6 -diamidino-2-phenylindole (DAPI; Molecular Probes).

Western blot analysis
The preparation of cell lysates, SDS-PAGE and western blot analysis were as described previously (2). Data were quantified by using ImageJ.

Lentivirus generation and establishment of shRNAexpressing stable cell lines
The lentivirus preparation and establishment of shRNAexpressing stable cell lines were as described previously (2).

Polysome profiling
The A549 cells were transduced with the lentiviral vector expressing the HA-tagged ZFP36L1 or control LacZ proteins for 72 h, and then infected with IAV (MOI = 1) for 6 h. Cells were treated with 100 g/ml cycloheximide (Sigma) for 5 min at 37 • C, and then washed twice with ice-cold PBS containing 100 g/ml cycloheximide. Cells were then lysed using 0.5 ml polysome lysis buffer (10 mM Tris-HCl [pH7.4], 3 mM MgCl 2 , 150 mM NaCl, 35 g/ml digitonin and 100 g/ml cycloheximide) supplemented with 100 U RNasin Plus inhibitor (Promega) and protease inhibitor (Thermo) on ice for 30 min. Cell debris was removed by centrifugation at 16 000 g for 7 min, and then cell extracts were loaded onto 10 ml 15-40% sucrose gradients (comprising 10 mM Tris-HCl [pH7.4], 3 mM MgCl 2 , 150 mM NaCl) and centrifuged at 39 000 rpm for 90 min in a Beckman SW41-Ti rotor at 4 • C. Gradients were fractionated and the optical density at 254 nm was continuously recorded using an Isco sucrose gradient fractionation system (Teledyne Isco, Inc). Proteins from the each fraction were precipitated with trichloroacetic acid (TCA) and analyzed by western blotting using mouse monoclonal anti-40S ribosomal protein S6 (RSP6) (Invitrogen) and rabbit polyclonal anti-60S ribosomal protein L11 (RPL11) antibodies (Invitrogen). Total RNA was extracted from each fraction, and then 18S and 28S rRNA was resolved on a 1% agarose gel electrophoresis and stained with ethidium bromide.

Isolation of nuclear and cytoplasmic fractions
IAV-infected A549 cells were fractionated into nuclear and cytoplasmic proteins by using the Nuclear/Cytosol fractionation kit (BioVision) following the manufacturer's instruction. The samples were analyzed by immunoblotting with the indicated antibodies.

RNA immunoprecipitation (RIP) assay
A549 cells were transduced with lentiviral vectors expressing the HA-ZFP36L1 wild-type or mutant protein for 72 h, then infected with IAV (multiplicity of infection [MOI] = 0.1) for 16 h. Cell extracts were mixed with prewashed HA beads (Sigma) and incubated at 4 • C overnight. Viral RNA from the complex was extracted by use of the RNeasy Total RNA kit (Qiagen). RT-PCR involved the primer pair sequences for IAV NS1 (5 -AGCAAAAGCAGGGTGAC AAA-3 and 5 -AGTAGAAACAAGGGTGTTTTTTAT TATTA-3 ). The PCR product from IAV NS1 was analyzed by 2% agarose gel electrophoresis and detected by SYBR Green I staining.

Human ZFP36L1 inhibits IAV infection
ZFP36L1 contains two CCCH-type ZF domains characterized by three Cys residues and one His residue ( Figure 1A) that can directly bind to the AREs of certain mRNAs to promote mRNA deadenylation and decay (13,14,16,25). To assess the antiviral potential of human ZFP36L1 against IAV infection, we established A549 cells overexpressing Data are mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; NS: not significant. ZFP36L1 by a lentivirus expression system. Cells overexpressing ZFP36L1 or control EGFP were infected with IAV at low multiplicity of infection (MOI = 0.1). Viral load and protein expression levels were examined at 24 h postinfection (hpi). As compared with the EGFP control, overexpression of human ZFP36L1 reduced the expression of IAV proteins (PB1, PB2 and PA) as measured by western blotting in infected cells ( Figure 1B). Furthermore, infectious IAV production and the expression of IAV protein NP were also significantly decreased by measurement of infectious virus titer and immunofluorescence assay, respectively ( Figure 1C and D). Thus, human ZFP36L1 exhibited potent antiviral activity against IAV replication in human cells.

ZFP36L1 inhibits the expression of IAV proteins HA, M1, M2, NS1 and NS2
To identify the specific targets of IAV proteins by ZFP36L1, we transfected T-REx-293 cells overexpressing ZFP36L1 induced by doxycycline (Dox) treatment with a plasmid expressing individual viral proteins. Dox-induced overexpression of ZFP36L1 greatly inhibited the protein expression of HA, M1, and NS1 and to a lesser extent PA, PB2, NP and NA (Figure 2). To further verify the ability of ZFP36L1 to downregulate the expression of these viral proteins in infected cells, ZFP36L1-or control EGFP-overexpressing A549 cells were infected with IAV (MOI = 1) at 6 hpi, and the expression of 10 viral proteins was measured by western blot analysis. Similar results were obtained: ZFP36L1overexpressing cells mainly reduced the protein expression of HA, M1, M2, NS1 and NS2 as compared with the control EGFP (Supplementary Figure S1). These results suggest that ZFP36L1 inhibits IAV replication by blocking the expression of HA, M1, M2, NS1 and NS2 proteins derived from HA, M and NS RNA segments.

ZFP36L1 inhibits the expression of IAV proteins HA, M1 and NS1 via translational repression
ZFP36L1-mediated mRNA decay is connected to the CCR4-NOT deadenylase complex as well as sequential degradation of the target mRNA via 5 -3 exonucleolytic decay by exonuclease XRN1 and 3 -5 exonucleolytic decay by the RNA exosome complex (13,14,25). We next examined whether the viral mRNA levels of HA, M1 and NS1 were decreased in cells with ZFP36L1 overexpression. Overexpression of ZFP36L1 in IAV-infected A549 cells blocked the expression of HA, M1, and NS1 proteins, with no change in viral mRNA expression ( Figure 3A and B). Likewise, T-REx-293 cells with ZFP36L1 overexpression were transfected with a plasmid expressing HA, M1, NS1 or NP. The protein but not mRNA expression of viral HA, M1 and NS1 was greatly decreased in cells overexpressing ZFP36L1 (Supplementary Figure S2A and B). We next used polysomal profile analysis to assess ZFP36L1 role in the translation regulation of the IAV HA, M and NS mRNAs. The distribution of the IAV HA, M and NS mRNAs from polysome fractions in ZFP36L1-overexpressing cells was a tendency towards the 40S and 60/80S polysome fractions ( Figure 3C), indicating that ZFP36L1 contributes to translation repression of the IAV proteins.
To further evaluate whether cellular deadenylation and RNA decay machineries participated in the anti-IAV activity of ZFP36L1, we used a lentivirus expressing shRNAtargeted CNOT6 (a component of the CCR4-NOT deadenylase complex), XRN1 or EXOSC5 (a component of the RNA exosome complex) in ZFP36L1 and EGFPoverexpressing cells. As compared with knockdown control shLacZ, knock down of CNOT6 (shCNOT6) (Supplementary Figure S3), XRN1 (shXRN1) (Supplementary Figure S4A) or EXOSC5 (shEXOSC5) (Supplementary Figure  S4B) did not lose the anti-IAV effect of ZFP36L1, which suggests that deadenylation and RNA decay machineries are not involved in ZFP36L1-mediated antiviral activity against IAV.
Furthermore, to verify whether proteasomal degradation is involved in the ZFP36L1-reduced IAV protein levels, T-REx-293 cells induced to express ZFP36L1 were treated with the proteasomal inhibitor MG132 or solvent control (DMSO), then transfected with a plasmid expressing M1 or NS1. With MG132 treatment, the expression levels in M1 and NS1 protein was not rescued in cells with overexpression of ZFP36L1 as compared with DMSO treatment (Supplementary Figure S5). In contrast, MG132 treatment could stabilize and enhance the expression level of ZFP36L1, resulting in a higher reduction on the protein levels of NS1 and M1 (Supplementary Figure S5). Thus, ZFP36L1 exhibits antiviral activity against IAV via translational repression of IAV proteins.

ZFP36L1 blocks the nuclear export of viral ribonucleoproteins (vRNPs) by blocking the expression of IAV NS2 and M1 proteins
Next, we examined whether ZFP36L1 inhibits the protein levels of both NS2 and M1 to lead to retention of vRNPs in the nucleus. Cells were transduced with lentiviruses expressing ZFP3L1 or control EGFP, then infected with IAV (MOI = 1) for 24 h. As expected, the protein levels of IAV NS2 and M1 but not NP were nearly inhibited in ZFP36L1-overexpressing cells ( Figure  4A). The nuclear export of vRNPs was monitored by detecting the expression of NP in the cytoplasmic fraction by nuclear/cytosolic fractionation coupled with immunoblotting analysis and the cellular localization of NP by immunofluorescence assay. By nuclear/cytosolic fractionation, immunoblotting revealed lower protein level of NP in the cytoplasm of ZFP36L1-than control EGFPoverexpressing cells ( Figure 4B). Similarly, immunofluorescence assay revealed vRNPs predominantly localized in the cytoplasm in control EGFP-overexpressing cells but in the nucleus in ZFP36L1-overexpressing cells ( Figure  4C). Thus, ZFP36L1 can block the expression of NS2 and M1 proteins, thereby blocking the nuclear export of vRNPs.

CCCH-type ZF domains of ZFP36L1 are critical for its antiviral activity
To evaluate the importance of the ZF domains in ZFP36L1 in antiviral activity against IAV, we constructed a   C135R/C173R mutant with a single point mutation at the conserved CCCH residues (ZF1:C135 and ZF2:C173) of the ZF domains ( Figure 5A). As compared with ZFP36L1overexpressing cells, cells overexpressing the ZFP36L1-C135R/C173R mutant lost nearly all inhibitory activity as determined by the protein expression of M1, NS1 and HA ( Figure 5B) and viral production ( Figure 5C), as did control EGFP, which suggests that the ZF domains of ZFP36L1 are required for its antiviral activity against IAV.

ZFP36L1 interacts with NS1 mRNA via its ZF domains, which inhibits the expression and function of NS1.
ZFP36L1 contains two highly conserved CCCH-type ZF domains with RNA-binding potential that directly bind to certain mRNA via ARE-dependent mechanisms (13,14,16). To verify whether ZFP36L1 is involved in viral mRNA binding and whether the CCCH-type ZF domains are essential for viral mRNA binding activity, cells were overexpressed with the HA-tagged ZFP36L1 or ZFP36L1- C135R/C173R mutant, then infected with IAV. The RNAbinding capacity of ZFP36L1 was analyzed by immunoprecipitation with an antibody against the HA tag, followed by RT-PCR for mRNA levels of HA, M1 or NS1. The mRNA of NS1 was pulled down by ZFP36L1 in IAV-infected cells ( Figure 6A). ZFP36L1-C135R/C173R mutant lost its viral RNA-binding activity ( Figure 6A). We further examined whether ZFP36L1 binds NS1 mRNA in the cytoplasm or nucleus. Co-localization of IAV NS1 mRNA and ZFP36L1 protein was clearly seen in the cytoplasm of IAV-infected cells (Supplementary Figure S6).
To further verify whether the expression and function of NS1 were blocked in cells with ZFP36L1 overexpression, we constructed a lentiviral vector carrying a silent mutation in the splice acceptor site of NS1 and established a stable cell line expressing NS1 by lentivirus transduction in A549 cells. ZFP36L1-but not LacZ-overexpressing or ZFP36L1-C135R/C173R mutant-overexpressing cells showed abolished NS1 expression ( Figure 6B). NS1 functions as a double-stranded RNA (dsRNA) binding protein that blocks the activation of IFN regulatory factor 3 (IRF3), nuclear translocation of IRF3 and downstream induction of the type I IFN genes (38). Cells stably expressing NS1 blocked the IRF3 nuclear translocation mediated by poly IC (Supplementary Figure S7). As compared with control LacZ, ZFP36L1 overexpression blocked the nuclear translocation of IRF3, whereas overexpression of the ZFP36L1-C135R/C173R mutant lost the inhibitory effect on nuclear translocation of IRF3 ( Figure 6C). These data indicate that ZFP36L1 can bind to NS1 mRNA in the cytoplasm via its ZF domains and block the expression and function of IAV NS1.

Antiviral potential of endogenous ZFP36L1 against IAV infection
To assess the role of endogenous ZFP36L1 in IAV infection, A549 cells were infected with IAV (MOI = 1) at various times and the protein expression of endogenous ZFP36L1 was measured. IAV-infected cells showed upregulated protein level of ZFP36L1, especially at 9, 16 and 24 hpi but to a lesser extent at 6 hpi ( Figure 7A), as well as the expression level of endogenous ZFP36L1 was obviously induced by TNF-␣ treatment ( Supplementary Figure S8). Lentivirus knockdown of endogenous ZFP36L1 (shZFP36L1) in A549 cells enhanced the protein level of NS1 at low MOI (MOI = 0.1) ( Figure 7B) and high MOI (MOI = 1) (Supplementary Figure S9). Infected cells with knockdown ZFP36L1 expression showed increased NS1 protein level and IAV viral production at 24 hpi ( Figure  7C). Furthermore, the rescue of the ZFP36L1 knockdown by transduction with a lentiviral vector overexpressing ZFP36L1 significantly decreased the expression of IAV protein (Supplementary Figure S10A) and infectious IAV production (Supplementary Figure S10B), respectively. These results indicate that IAV infection can induce endogenous ZFP36L1 level and knockdown of ZFP36L1 enhances viral replication, so ZFP36L1 may play a role in the host antiviral defense against IAV.

DISCUSSION
ZFP36L1 is a family member of the CCCH-type of ZF proteins and was identified as an RNA-binding protein, which has important roles in various cellular and biological functions such as inflammation, apoptosis, proliferation, differentiation and angiogenesis (13). However, its role in the host defense response against viral infection has not been addressed. In this study, we used gene overexpression to explore the antiviral potential of human ZFP36L1 protein: ZFP36L1 overexpression could exhibit potent antiviral activity against IAV infection. Moreover, the protein level of endogenous ZFP36L1 was upregulated in response to IAV infection, and ZFP36L1 knockdown enhanced the viral replication of IAV. These findings indicate that human ZFP36L1 can function as a cellular antiviral factor to restrict viral replication in IAV-infected cells and extend the biological function of ZFP36L1 in host antiviral defense.
Post-transcriptional and -translational regulation by CCCH ZF proteins such as MCPIP1 and ZAP restrict IAV replication with distinct functions and diverse mechanisms. MCPIP1 ribonuclease, which exhibits antiviral effects via direct binding and degradation of viral RNA by itself, functionally inhibits IAV replication (2,39). The short form of ZAP (ZAPS), which lacks the C-terminal PARP domain, posttranscriptionally inhibits IAV replication at an early stage of infection and blocks the expression of PA, PB2 and NA, then reduces the encoding viral mRNA levels such as PB2 (40). The long form of ZAPL (isoforms ZAPL), with a PARP domain, has distinct antiviral activity against IAV, directly binding the IAV proteins PA and PB2 for subsequent viral protein degradation by the proteasome-dependent pathway (41). Different from the antiviral functions of MCPIP1, ZAPS and ZAPL against IAV, our data demonstrated that ZFP36L1 inhibited IAV replication by blocking protein translation without affecting viral mRNA levels (Figure 3), so the antiviral effect of ZFP36L1 participates in repressing viral mRNA translation but not viral mRNA decay. Furthermore, knockdown of CNOT6 (shCNOT6), XRN1 (shXRN1) and EXOSC5 (shEXOSC5), which are the components of cellular mRNA decay machinery recruited by ZFP36L1, did not support the antiviral effect of ZFP36L1 against IAV (Supplementary Figures S3 and S4). These findings support that the RNA decay pathway does not participate in the antiviral activity of ZFP36L1.
Recently, the ARE-binding protein ZFP36L2 was found to mediate the translational repression of pre-formed mRNA in memory T cells (42). We found that ZFP36L1 inhibited IAV replication, which mainly blocked the protein expression of HA, M1, M2, NS1 and NS2 (Supplementary Figure S1 and Figure 3) without affecting their viral mRNA expression (Figure 3). In addition, the protein but not mRNA expression of viral HA, M1, and NS1 was greatly reduced in ZFP36L1-overexpressing cells (Supplementary Figure S2). As well, the presence of MG132, a proteasome inhibitor, did not prohibit the ZFP36L1-mediated translational repression of NS1 (Supplementary Figure S6); hence, the proteasomal degradation pathway should not be involved in ZFP36L1 reducing the expression of IAV proteins. Together, our results provide strong evidence that ZFP36L1 exerts its antiviral activity against IAV via translational repression of viral mRNA transcripts.
Besides regulating mRNA stability, TTP, a ZFP36 family member, has been found to have an ARE-mRNA translational repression function (43). ZAP blocks translation of viral mRNA target by disrupting the interaction between the translational initiation factors eIF4A and eIF4G (11). Moreover, the translation of influenza virus mRNAs has been found required for the two components of the eIF4F translation initiation factor, eIF4A and eIF4G proteins (44). Thus, whether ZFP36L1 represses the translation of influenza virus mRNAs by disrupting the translational initiation factors eIF4A and eIF4G remains to be clarified.
ZFP36L1, an RNA-binding protein, contains two highly conserved CCCH-type ZF domains that are responsible for binding to AREs in the 3 UTR of the mRNA, which leads to the instability and degradation of mRNA (14,16). The preferential binding region of ZFP36 family proteins to the consensus RNA sequence is UUAUUUAU (45)(46)(47)(48). Mutation of the CCCH-type ZF domains of ZFP36L1 lost its antiviral activity against IAV ( Figure 5B and C), so the CCCH-type ZF domains of human ZFP36L1 are essential for antiviral activity against IAV infection. Analysis of the mRNA sequence of IAV HA, M1 and NS1 revealed that these mRNAs contained a core sequence of AUUUA, a short ARE-containing region, which might be sufficient for binding ZFP36L1. Therefore, the mRNA transcripts of IAV might bind to ZFP36L1. Indeed, we found that NS1 mRNA bound to ZFP36L1 in IAV-infected cells, and mutation of Nucleic Acids Research, 2020, Vol. 48, No. 13 7383 the CCCH-type ZF domains of ZFP36L1 lost its RNAbinding activity and translation inhibition for NS1 mRNA ( Figure 6A and B).
NS1 mRNA contains two ARE-containing regions in its 3 UTR, ARE1 and ARE2 (Supplementary Figure S11A). To determine whether ARE regions of NS1 mRNA are critical for translation inhibition by ZFP36L1, we constructed the NS1-ARE1, -ARE2 and -ARE1/2 mutant with AUUUA mutated to AGGGA as well as 3 UTRtruncated mutant for RNA transcripts. However, the effect of ZFP36L1 on translational repression remained unchanged (Supplementary Figure S11B), so the ARE regions of NS1 mRNA are not critical for ZFP36L1 to inhibit translation.
Besides involvement in a classical ARE-dependent pathway, TTP has been reported to downregulate the expression of mRNAs via a non-ARE-dependent pathway (49). Thus, ZFP36L1 might downregulate the translation of NS1 mRNA by binding to a non-ARE-containing target sequence. Also, ZFP36L1 might trigger a distinct mechanism to block the translation of IAV mRNA, which contributes to restrict IAV replication. More work is needed to delineate the underlying mechanism for the potent antiviral effect of ZFP36L1 on IAV.