NF90 Exerts Antiviral Activity through Regulation of PKR Phosphorylation and Stress Granules in Infected Cells

This information is current as Xi Wen, Xiaofeng Huang, Bobo Wing-Yee Mok, Yixin of September 28, 2021. Chen, Min Zheng, Siu-Ying Lau, Pui Wang, Wenjun Song, Dong-Yan Jin, Kwok-Yung Yuen and Honglin Chen J Immunol published online 12 March 2014 http://www.jimmunol.org/content/early/2014/03/12/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 12, 2014, doi:10.4049/jimmunol.1302813 The Journal of Immunology

NF90 Exerts Antiviral Activity through Regulation of PKR Phosphorylation and Stress Granules in Infected Cells

Xi Wen,*,† Xiaofeng Huang,*,† Bobo Wing-Yee Mok,*,† Yixin Chen,‡ Min Zheng,*,† Siu-Ying Lau,*,† Pui Wang,*,† Wenjun Song,*,† Dong-Yan Jin,x Kwok-Yung Yuen,*,† and Honglin Chen*,†

NF90 was shown to exhibit broad antiviral activity against several viruses, but detailed mechanisms remain unclear. In this study, we examined the molecular basis for the inhibitory effect of NF90 on virus replication mediated through protein (PKR)- associated translational regulation. We first verified the interaction between NF90 and PKR in mammalian cells and showed that NF90 interacts with PKR through its C-terminal and that the interaction is independent of NF90 RNA-binding properties. We further showed that knockdown of NF90 resulted in significantly lower levels of PKR phosphorylation in response to dsRNA in- duction and influenza virus infection. We also showed that high concentrations of NF90 exhibit negative regulatory effects on PKR Downloaded from phosphorylation, presumably through competition for dsRNAvia the C-terminal RNA-binding domain. PKR activation is essential for the formation of stress granules in response to dsRNA induction. Our results showed that NF90 is a component of stress granules. In NF90-knockdown cells, dsRNA treatment induced significantly lower levels of stress granules than in control cells. Further ev- idence for an NF90–PKR antiviral pathway was obtained using an NS1 mutated influenza A virus specifically attenuated in its ability to inhibit PKR activation. This mutant virus replicated indistinguishably from wild-type virus in NF90-knockdown cells, but not in scrambled control cells or Vero cells, indicating that NF90’s antiviral function occurs through interaction with PKR. http://www.jimmunol.org/ Taken together, these results reveal a yet-to-be defined host antiviral mechanism in which NF90 upregulation of PKR phosphor- ylation restricts virus infection. The Journal of Immunology, 2014, 192: 000–000.

F90 (also known as NFAR1 or DRBP76) was first iden- NF90 was found to be involved in host antiviral mechanisms tified as an IL-2 promoter-binding protein in activated targeting various viruses. It suppresses the function of Ebola virus N T cells and was found to regulate IL-2 expression via polymerases through interaction with VP35 (14), inhibits HIV stabilization of IL-2 mRNA (1–5). A variety of independent studies replication through interaction with HIV-1 TAR RNA (15, 16),

subsequently found that NF90 has dsRNA-binding properties. NF90 represses internal ribosome entry sites in rhinoviruses (17, 18) and by guest on September 28, 2021 is predominantly localized within the nucleus (6, 7), although it also negatively regulates influenza virus replication through interaction has been found in the cytoplasm (8). It interacts with PKR and is a with viral nucleoprotein (NP) (19). However, other studies (20–23) substrate for phosphorylation by PKR (7–11). Further studies (12, found that NF90 is required for the replication of some positive- 13) revealed that phosphorylation of NF90 by PKR is necessary stranded RNA viruses and is important for expression of E6 protein for association of the NF90/NF45 complex, shuttling of NF90 be- in human papillomavirus–infected cells. It is postulated that, al- tween the nucleus and cytoplasm, and NF90 function in transla- though members of the NF90 family generally serve as compo- tional regulation and host antiviral defense. nents of host antiviral responses, some viruses may have adapted a mechanism to hijack NF90, retasking it for viral replication and *State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, weakening host defenses (20). A study in which NF90 (NFAR) was The University of Hong Kong, Hong Kong Special Administrative Region, People’s depleted in mouse embryonic fibroblast (MEF) cells suggested that Republic of China; †Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong Special Administrative Region, People’s Republic of NF90 exerts its antiviral activity through modulation of translation China; ‡National Institute of Diagnostics and Vaccine Development for Infectious in host cells in a PKR-dependent manner (12, 13). Diseases, School of Life Sciences, Xiamen University, Xiamen 361005, China; and x In addition to its involvement in the regulation of the IL-2 Department of Biochemistry, The University of Hong Kong, Hong Kong Special Administrative Region, People’s Republic of China promoter in T cells, dsRNA-dependent protein kinase (PKR) is Received for publication October 18, 2013. Accepted for publication February 18, the most well-defined host factor interacting with NF90. PKR is 2014. an IFN-inducible gene that plays a critical role in host antiviral This work was supported in part by the Research Grants Council of the Hong Kong responses (24, 25). It is natural to hypothesize that the antiviral Special Administrative Region (7620/10M and 7629/13M) and the Areas of Excel- function exerted by NF90 may be signaled through PKR-related lence Scheme of the University Grants Committee (Grant AoE/M-12/06). pathways. The primary role of PKR in the antiviral response is its Address correspondence and reprint requests to Dr. Honglin Chen, State Key Labo- ratory for Emerging Infectious Diseases, Department of Microbiology, The University inhibition of translation of viral mRNAs through phosphorylation of Hong Kong, 21 Sassoon Road, Laboratory Block, Pokfulam, Hong Kong Special of eukaryotic initiation factor 2 a (eIF2a) (26). PKR is also rec- Administrative Region, People’s Republic of China. E-mail address: [email protected] ognized for its role in regulating cellular inflammatory signals (27, Abbreviations used in this article: co-IP, coimmunoprecipitation; eIF2a, eukaryotic 28). A previous study (29) found that NF90 is able to inhibit yeast initiating factor 2 a; G3BP, Ras GAP SH3-domain–binding protein; MEF, mouse embryonic fibroblast; MOI, multiplicity of infection; NP, nucleoprotein; NS1, non- growth when coexpressed with PKR, but not on its own, implying structural protein 1; PKR, protein kinase; poly(I:C), polyinosinic-polycytidylic acid; that NF90 may activate PKR to cause translational inhibition. RBD, RNA-binding domain; RBDm, RNA-binding domain mutant; siRNA, small However, this same study (29) found no evidence of NF90 activa- interfering RNA; WT, wild-type. tion of PKR in an in vitro assay. Conversely, it was found that NF90 Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 inhibited PKR phosphorylation, presumably through competition for

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302813 2 NF90 REGULATES ACTIVATION OF PKR dsRNA binding, because the C-terminal of NF90 possesses RNA- reverse primers. NF90-T188/T315A mutant was made using primers 59-G- binding ability (6). TATTAGCTGGAGAAGCGCTATCAGTCAACG-39 and 59-CAACGGGA- 9 Activation of PKR leads to the phosphorylation of eIF2a and AGATATCGCACAGAGTGCGCAGC-3 and their complementary reverse primers. NF90 (1–369 aa) and NF90 (370 aa-c) truncation mutants were results in the stalling of mRNA translation in cells (30). Activation constructed by inserting NF90 1–369 aa or 370 aa-c fragments into pCDNA3. of eIF2a triggers formation of stress granules that are composed 1-Flag vectors. PKR (GenBank accession number NM_002759) was cloned of multiple RNA-binding proteins, including TIA-1 and Ras GAP into pCDNA3.1-V5. Truncated mutants of PKR, including PKR (1–257) and SH3-domain–binding protein (G3BP)-1 (31, 32). Interaction be- (258–551), also were constructed in the same vector. tween influenza A virus and host stress granules was demonstrated Small interfering RNA knockdown of NF90 recently (33, 34). Activation of PKR to induce the formation of stress granules is regarded as a hallmark of the cellular response Transient knockdown of NF90 was achieved by transfection of NF90-specific small interfering RNA (siRNA) (NM_004516_stealth_1155 59-AAAGA- to virus infection (34–36). This study explored the possibility of AGCCACUGAUGCUAUUGGGC-39 and its reverse complementary se- NF90 involvement in PKR activation and the formation of stress quence) or 39-untranslated region siRNA (59-AATAGGACCAAGTTCA- granules in response to dsRNA treatment or virus infection of AAGGC-39 and its reverse complementary sequence), which were obtained cells. We found that NF90 interacts with PKR via its C-terminal from Life Technologies and Sigma, respectively. HEK 293T cells were domain and further demonstrated that NF90 is a previously un- transfected with 50 nM siRNA using Lipofectamine RNAiMAX transfec- tion reagent (Invitrogen). After 24 h of incubation, NF90-silenced cells characterized component of cellular stress granules formed when were transfected with 100 ng the different NF90 constructs and incubated cells are treated with stress-inducing agents, such as polyinosinic- for an additional 24 h. Cells were subsequently treated with 150 ng poly(I:C) polycytidylic acid [poly(I:C)] or arsenite. NF90 may inhibit virus for 6 h. The effect of the various NF90 constructs on stress granule formation replication by regulating levels of PKR phosphorylation. Com- was visualized using an LSM700 confocal microscope in the Core facility at Downloaded from Li Ka Shing Faculty of Medicine, University of Hong Kong. pared with control cells, stable NF90-knockdown 293T cells exhibit lower levels of activated PKR and form significantly fewer stress Transfection and coimmunoprecipitation granules following treatment with poly(I:C). Using an NS1 mutant For transfection experiments, subconfluent (70%) monolayer cells were influenza A virus, which is specifically attenuated in its ability to transiently transfected or cotransfected with plasmids using TransIT-LT1 antagonize PKR activation but unaltered with respect to other transfection reagent (Mirus). A total of 10–500 ng plasmid was added to functions, this study confirms that NF90 is required for PKR ac- each well in a 24-well plate, in accordance with the user manual. For dsRNA http://www.jimmunol.org/ tivation in response to virus infection. NF90 may serve as a reg- treatment, 100 ng poly(I:C) (Invitrogen) and 400 ng pUC19 carrier DNA ulator for PKR activation in response to dsRNA in cells. were combined to make a mix containing 500 ng DNA, which was then transfected into cells (34). Coimmunoprecipitation (co-IP) was performed using Dyna Beads Materials and Methods (Invitrogen). Specific Abs were added to the beads and incubated at room $ Cells and viruses temperature for 20 min. Cell lysates were obtained by adding lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, and protease HEK 293T cells, MEF cells, HeLa cells, and Vero cells were maintained in inhibitor mixture; Roche) to the cells, shaking for 15 min on ice, and then Dulbecco’s minimal essential medium, whereas MDCK cells were cul- centrifuging. Cell lysate supernatants were added to the Ab-coupled beads, tured in Eagle’s MEM. Culture media were supplemented with 10% FBS, and the mixture was incubated at room temperature for 2 h or overnight at 100 IU penicillin G/ml, and 100 ml streptomycin sulfate/ml, and cells were 4˚C. Finally, beads were washed three times with lysis buffer, and Western by guest on September 28, 2021 incubated at 37˚C in a 5% CO2 atmosphere. NF90-knockdown or scram- blotting was performed. bled control 293T cells were kindly provided by Dr. Christopher Basler Immunoprecipitation products or diluted cell lysate supernatants were (Mount Sinai School of Medicine, New York, NY) (14). PKR-knockout mixed with SDS loading buffer and heated at 95˚C for 10 min. Samples were (PKR2/2)andwild-type(WT)(PKR+/+) primary MEF cells were a generous fractionated by 12% SDS-PAGE and then blotted onto nitrocellulose mem- gift from Drs. John C. Bell (Ottawa Health Research Institute, Ottawa, ON, branes (Bio-Rad). After blocking with 3% skim milk, membranes were Canada) and Craig McCormick (Dalhousie University, Halifax, NS, Canada) incubated with the primary Abs anti–Flag M2 (Sigma) at 1:2000 dilution, (34). anti-V5 (Invitrogen) at 1:2000 dilution, anti-PKR (Santa Cruz) at 1:1000 WT and NS1-mutated influenza viruses (A/WSN/1933 [H1N1]) were dilution, anti–p-PKR (T446; Abcam) at 1:1000 dilution, anti-NP (19) at propagated in MDCK cells cultured in MEM containing antibiotics and 1 1:2000 dilution, anti-DRBP76 (NF90; BD) at 1:500 dilution, anti-tubulin mg TPCK-trypsin/ml at 37˚C for 48 h. Supernatants were harvested at (Sigma) at 1:5000 dilution, or anti-NS1 (33) at 1:2000 dilution. IRDye designated time points, and viruses were titrated in MDCK cells by plaque 680– or IRDye 780–labeled donkey anti-mouse or anti-rabbit secondary assay. Briefly, virus culture supernatant samples were serially diluted in Abs (Li-Cor Biosciences) were used at a dilution of 1:5000. Membrane PBS and adsorbed onto confluent MDCK cells for 1 h at 37˚C. The in- blots were scanned to visualize Ab–protein bands using the Odyssey im- oculum was removed, and the cells were washed with PBS and covered aging system (Li-Cor Biosciences). Relative levels of protein present in with 2 ml an agar medium (1% agarose, 1 mg TPCK-trypsin/ml in MEM). bands were analyzed by estimation of band density using ImageJ software. After 2 d of incubation, plaques were counted, and the virus titer (PFU/ml) was calculated. Immunofluorescence assay and microscopy Plasmid construction Cells were grown on Millicell EZ slides (Millipore) and transiently transfected with plasmids or infected with virus. For indirect immunoflu- The NP, PB1, PB2, and PA , derived from the H5N1 strain A/VNM/ orescence assay, cells were fixed for 15 min using 4% paraformaldehyde in 1194/2004 and the H1N1 strain A/WSN/1933, were cloned into pCDNA3.1- PBS, followed by permeabilization with 0.2% Triton X-100 in PBS for Flag/-V5 and pCMV-Flag vectors, respectively. The NS1 gene was cloned 5 min. The cells were washed with PBS and blocked with 5% normal into a Flag-tagged vector (33). For construction of recombinant virus, the donkey serum in PBS at 37˚C for 30 min, followed by incubation for 1 h at viral genomes were cloned into the pHW2000 vector (37). The QuikChange 37˚C with primary Abs diluted in PBS as follows: NP 1:500, DRBP76 II Site-Directed Mutagenesis Kit (Stratagene) was used to introduce R38A (NF90) 1:50, G3BP 1:200, PABP1 1:200, V5 1:200, and Flag 1:200. The and R35A (19, 33) and I123A/M124A/K126A/N127A (33, 38) point mu- slides were washed with PBS and incubated with anti-mouse, anti-rabbit, tations into NS1, as well as to construct an NS1-deletion mutant (delNS1) or anti-chicken conjugates (FITC, rhodamine, or aminomethylcoumarin with stop codons introduced into residues C13 and L15. Plasmids pcDNA3.1- acetate, respectively), as appropriate, for 30 min at room temperature. For NF90-V5 (NF90-V5) and pcDNA3.1-NF90-Flag were generated by cloning costaining procedures in which primary Abs raised from the same species the NF90 gene (GenBank accession number NM_004516; https://www.ncbi. were used, the primary Abs were coupled with Zenon mouse IgG labeling nlm.nih.gov/genbank/) into pcDNA3.1-V5 and pcDNA3.1-Flag vectors, reagents (Invitrogen) prior to application onto the cells, and the use of respectively. NF90-385-388A mutant was generated using the primer, 59- secondary Abs was omitted (33). After staining, the cells were washed GAGGAGAAGTCGCCCAGCGCAGCGGCGGCGAAGATTCAGAAGA- several times and mounted in mounting buffer, with or without DAPI AAGAG-39, and a complementary reverse primer. NF90-432/555A mutant (VECTASHIELD). Signals were examined using an LSM 700 confocal was made using primers 59-ACGACAAGCGCGCCGTCATGGAGGT-39 microscope (Zeiss). Images were taken using a 633 or 403 oil-immersion and 59-ATGCCCCCATCGCTACCATGTCTGT-39 and their complementary lens, and captured images were processed using ZEN software (Carl Zeiss). The Journal of Immunology 3

Quantitative real-time PCR NF90 and V5-tagged full-length and C-terminal– and N-terminal– NF90-knockdown or scrambled 293T cells were infected with WSN WT truncated versions of PKR (Fig. 1A). NF90 and PKR plasmids were or mutant viruses at a multiplicity of infection (MOI) of 0.1. Total coexpressed in 293T cells, and co-IP with anti-V5 Ab showed that were extracted at 24 h postinfection using RNAiso (Takara). cDNAs were both N- and C-terminals of PKR interact with NF90 (Fig. 1B). synthesized by reverse transcription using SuperScript II Reverse Tran- NF90 contains a DZF motif in the N-terminal and an RNA-binding scriptase (Invitrogen) and random primers. A SYBR Green–based real-time domain (RBD) in the C-terminal (Fig. 1A). We then investigated the PCR method (Roche) was used to detect target mRNAs with the Light- Cycler system (Roche). Primers for detecting IFN-b were 59-GCCGCA- PKR-interacting domain on the NF90 molecule by constructing 1– TTGACCATCT-39 and 59-CACAGTGACTGTACTCCT-39. Actin mRNA 369 N-terminal and 370–702 C-terminal truncates and RBD mutant was quantified, and the information was used to normalize the total RNA (RBDm) of NF90. These Flag-tagged truncated or mutated forms concentration between different samples, as described previously (19). A of NF90 were coexpressed with V5-tagged full-length PKR in reaction mix of 20 ml was composed of 10 pmol each gene-specific primer, 10 ml SYBR Green Master Mix, and 2 ml cDNA. The amplification pro- 293T cells and coprecipitated with anti-Flag. Our result showed that gram was as follows: 95˚C for 10 min, followed by 40 cycles of 95˚C for only the C-terminal of NF90 interacts with PKR and that the in- 10 s, 60˚C for 10 s, and 72˚C for 15 s. The specificity of the assay was teraction is independent of NF90 RNA-binding properties (Fig. 1C). confirmed by melting-curve analysis at the end of the amplification program. NF90 enhances phosphorylation of PKR upon induction by Results dsRNA NF90 interacts with PKR Previous reports (6, 7, 9) demonstrated that NF90 interacts with NF90 was shown previously to interact with PKR using yeast two- PKR and is one of the substrates of PKR. The phosphorylated form hybrid and GST pull-down assays (6, 29). To verify their interaction of NF90, MPP4, also was identified as a phosphoprotein detected Downloaded from in mammalian cells and map the interacting domains on both pro- during the M phase of the cell cycle (9, 39). Other studies indicated teins, we first tested interactions between Flag-tagged full-length that NF90 may activate PKR and increase eIF2a phosphorylation http://www.jimmunol.org/

FIGURE 1. Interaction between NF90 and by guest on September 28, 2021 PKR. (A) Illustration of NF90 domain required for interaction with PKR. (B) Co-IP analysis of PKR and NF90. V5-tagged full length (FL) and N-terminal (N) and C-terminal (C) truncates of PKR were coexpressed with Flag-tagged NF90 by transfection into 293T cells. At 48 h post- transfection, cell lysates were prepared and precipitated with anti-V5 Ab and then blotted with anti-Flag Ab. (C) Reciprocal co-IP analysis of NF90 and PKR. Flag-tagged full-length (FL), N-terminal (N), and C-terminal (C) truncates and RBD mutated NF90 were coexpressed with V5-tagged PKR by transfection in 293T cells for 48 h. Cell lysates were subjected to co-IP with anti-Flag and blotted with anti-V5 Abs. Right panels in (B)and(C) show co-IP input material: 1/20 diluted lysates. The strong low m.w. bands seen in (B)and(C)areM2-V5(M2 protein of influenza A virus (A/Vietnam/1194/04) and GFP-Flag (full-length GFP), respectively, which were included as negative controls. 4 NF90 REGULATES ACTIVATION OF PKR in yeast, but these positive effects on PKR phosphorylation could NF90 into NF90-knockdown cells to reveal the effect of NF90 on not be seen when purified proteins were used in vitro (29, 40), PKR phosphorylation in response to dsRNA. We found that PKR suggesting that additional factors may be missing in the in vitro phosphorylation was enhanced in response to low amounts of assay. However, in the in vitro study, it was demonstrated that high transfected NF90, but the positive effect disappeared when higher concentrations of NF90 inhibited PKR activation, possibly through concentrations (500 ng) of NF90 were introduced into the NF90- competitive binding to dsRNA (40). These observations from yeast knockdown cells (Fig. 2C). To test whether PKR and NF90 may and in vitro studies prompted us to investigate the effect of NF90 on be competing for dsRNA binding in poly(I:C)-treated cells, we PKR phosphorylation in mammalian cells. In this study, we ex- used NF90 truncated at either the N-terminal or RBD (C-terminal) amined the levels of the phosphorylated form of PKR in both NF90- or containing mutations abolishing RNA-binding ability (RBDm) knockdown and scrambled control 293T cells following PKR ac- (Fig. 1A) (41). We first confirmed that the full-length and N-terminal tivation induced by transfection of either dsRNA [poly(I:C)] or viral forms of NF90 bind poly(I:C) and that NF90 RBDm does not bind RNA. We found that, although expression of PKR protein is not dsRNA by performing poly(I:C) bead pull-down assays (data not changed, the level of the phosphorylated form of PKR is signifi- shown). Interestingly, it was found that, at the NF90 concentration cantly lower (∼3–4–fold) in NF90-knockdown cells compared with that exhibited inhibitory effects (500 ng), WT (full-length) NF90 that in scrambled siRNA-treated cells (Fig. 2A). The observation and the mutant form containing only the C-terminal significantly that NF90 is required for PKR phosphorylation in response to inhibited PKR activation following poly(I:C) treatment of 293T dsRNA treatment was confirmed in a pair of 293T cell lines: one in cells, whereas N-terminal and RBDm NF90 exerted little or no which NF90 was stably knocked down with specific short hairpin inhibitory effect, respectively (Fig. 2D) (41). The different p-PKR

RNA and the other a control created using a scrambled short hairpin expression levels associated with the NF90 mutants also may be Downloaded from RNA (14) (Fig. 2B). To delineate the mechanism for the regulation due to the positive or negative effect of these clones on PKR acti- of PKR activation by NF90, we transfected increasing amounts of vation. It appears that NF90 has dual and conflicting functions, in http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. NF90 is required for phosphorylation of PKR in response to dsRNA activation. (A) Levels of PKR phosphorylation were determined by Western blotting of lysates of 293T cells in which knockdown of NF90 with siRNA for 56 h was followed by treatment with poly(I:C) (100 ng) or viral RNA for 6 h. Different cell treatments are indicated in the figure. Efficiency of NF90 knockdown (right panel). A scrambled siRNA (sc) was used as a control. (B)LevelsofPKR phosphorylationinNF90stablyknocked-downcells(kd)and scrambled control cells (sc) were determined as in (A). Levels of NF90 in both NF90-knockdown and scrambled cells also were examined (right panel). b-tubulin was detected to normalize cell numbers in (A)and(B). (C) Dose-dependent NF90 regulation of PKR phosphorylation in cells treated with poly(I:C) dsRNA. NF90-knockdown cells were transfected with different amounts of NF90-encoding plasmid 18 h prior to dsRNA treatment. Cells were treated with poly(I:C), collected, and lysed for Western blot analysis. The amounts of NF90 transfected are indicated; empty vector wasusedtobringthetotalDNAineachwellto500ng.(D) 293T cells were transfected with 500 ng of NF90 WT, truncation mutants 1–369 aa (N) or 370 aa-c (C), site-mutated mutant 432/555A (RBDm) (Fig. 1A), or control pCDNA3.1 vector (vec) before treatment with poly(I:C). Sample preparation and Western blotting were carried out as described above. Levels of NF90 produced from transfected plasmids were analyzed using anti-Flag Ab. Relative levels of protein present in bands were analyzed by estimation of band density using ImageJ software in Western blots. The Journal of Immunology 5 that it can support PKR phosphorylation (Fig. 2A) while inhibiting translation through induction of stress granules in virus-infected the level of PKR phosphorylation through sequestration of dsRNA cells (25, 34, 35). Taken together with the observed interaction and consequent prevention of PKR activation via the C-terminal between NF90 and PKR, a mechanism for regulation of protein RBD (Fig. 2C, 2D). A study (13) found that PKR phosphorylated synthesis involving these two proteins seems likely. This study T188 and T315 of NF90 and regulated NF90 cellular shuttling. tested the possibility that NF90 is an undefined component of The T188A/T315A (attenuated) and the T188D/T315D (constitutively PKR-induced stress granules. We examined stress granules induced active) form of NF90 did not exhibit any altered activity with effect by treatment with arsenite and poly(I:C) for the presence of NF90. on PKR phosphorylation in response to dsRNA treatment compared NF90 was found to colocalize with the stress granule markers with WT NF90 (data not shown). These results establish a mech- PABP1 and G3BP (Fig. 3A), as well as TIA-1 (data not shown), in anism in which NF90 is involved in a complex process of host both arsenite- and poly(I:C)-induced granules. In untreated control regulation of exerted through the PKR pathway. cells, NF90 predominantly localized in the nucleus and did not colocalize with PABP1 in the cytoplasm. To investigate whether NF90 is a component of stress granules NF90 is involved in the formation of stress granules, we used the NF90 was shown to inhibit replication of a broad range of viruses pair of NF90 stable knockdown 293T cell lines described above and to form complexes with NF45 and shuttle between the nucleus (14) (Fig. 2B). It is notable that significantly more stress granules and cytoplasm (13–19). However, a detailed mechanism describing were observed in the scrambled control cells compared with the how NF90 negatively regulates virus replication remains unclear. NF90-knockdown cells following induction with poly(I:C) (Fig. One of the well-defined pathways set in motion following PKR 3B, p , 0.05). Interestingly, there was no significant difference in activation was found to associate with the regulation of mRNA granule formation between NF90-knockdown and scrambled cells Downloaded from

FIGURE 3. NF90 colocalizes with http://www.jimmunol.org/ markers of stress granules. (A) 293T cells were transfected with poly(I:C) for 6 h, treated with sodium arsenite for 30 min, or left untreated, and im- munofluorescence assays were per- formed as described previously (33). Stress granules were examined using AbsspecificforG3BPorPABP1and

NF90 (anti-DRBP76) and visualized by guest on September 28, 2021 using a confocal microscope. DAPI isshowninblueinmergedimages (original magnification 363). (B) Scrambled or NF90 stable knockdown (kd) cells were treated with poly(I:C) or arsenite and examined for the formation of stress granules (SG) by staining with anti-G3BP and DAPI. Stress granules (G3BP) pres- ent in the cytoplasm are shown in green. (C) 293T cells were treated with poly(I:C) or mock treated for 4 h; treatment medium was removed and replaced with medium containing 2-AP (with final working concentra- tion of 10 mM) in vehicle (acetic acid) or vehicle only. For statistical analysis of cells containing stress granules (SG), ∼100–200 cells were examined for the presence of SG in each of three independent experi- ments, and the average percentage of SG+ cells was calculated. Error bars represent SD. (D) Enlarged images of stress granules in scrambled and NF90-knockdown (kd) cells after treatment with poly(I:C) (original magnification 340). *p , 0.05, **p , 0.01. 6 NF90 REGULATES ACTIVATION OF PKR treated with arsenite (Fig. 3B, lower panels). Because poly(I:C), was not able to localize to stress granules (Fig. 4A). PKR was but not arsenite, activates PKR (42), these results suggest that reported to phosphorylate T188 and T315 of NF90, thereby regu- NF90’s role in the formation of stress granules requires PKR. To lating NF90 cellular shuttling (13). We found that the phosphoryla- further verify that NF90’s facilitation of stress granule induction tion-deficient T188A/T315A mutant (NF90-A) (Fig. 1A) exhibited in cells in response to poly(I:C) treatment is mediated by PKR, we a deficiency in localization to stress granules in response to dsRNA tested whether treatment with the PKR inhibitor, 2-AP (43), af- treatment (Fig. 4A). The control, 293T cells transfected with vector, fected stress granule induction. Our results showed that 2-AP, but showed no stress granules (data not shown). By using siRNA not the vehicle-only control, reduced stress granule formation in targeting the 39-untranslated region of NF90, we were able to knock NF90 WT cells treated with poly(I:C) (Fig. 3C, p , 0.01). These down NF90 expression to a minimal level in 293T cells (Fig. 4B). results clearly demonstrate that NF90-regulated stress granule We then examined different NF90 mutants for the ability to restore formation in response to dsRNA induction is mediated by PKR. formation of stress granules in NF90-knockdown cells. Although In contrast to the scrambled 293T cells, in which distinct stress both RBDm (NF90-RBDm) and phosphorylation mutant (NF90-A) granules were observed, PABP1 exhibited a diffuse pattern of ex- possess low-level stress granule formation activity, the ability of pression in NF90-knockdown cells (Fig. 3D), suggesting that NF90 these mutants to restore stress granule formation in poly(I:C)- may be a core nucleating component for the formation of stress treated NF90 knockdown cells was considerably weaker than that granules in response to dsRNA induction. Just as we established of WT NF90 (Fig. 4C, 4D). Taken together, these results suggest that the C-terminal of NF90 is required for interaction with PKR that RNA binding and phosphorylation of NF90 are required for (Fig. 1C), it was important to identify which domain of NF90 induction of stress granules via dsRNA activation of PKR. is associated with stress granules. Because NF90 predominantly Downloaded from localizes to the nucleus in normal conditions, we created a mutant Mutant of influenza A virus associated with PKR activation NF90 protein that does not have the ability to localize to the nucleus NF90 was shown to be involved in the host antiviral response and to better understand NF90’s association with stress granules. When to limit influenza virus replication (12, 19). We attempted to de- four substitutions were introduced into the putative nuclear- termine whether there is an NF90–PKR antiviral pathway that localization signal of NF90 (residues 385–388) (Fig. 1A), this mu- enhances activation of PKR, using influenza virus as a model. The

tated NF90 (NF90-NLSm) was found exclusively in the cytoplasm nonstructural protein 1 (NS1) of influenza A virus is able to inhibit http://www.jimmunol.org/ of poly(I:C)-treated cells, where it localized to stress granules (Fig. PKR activation and stress granule formation induced by PKR 4A). This seems to suggest that the nuclear-localization ability of activation, thereby supporting virus replication (33, 34). We pre- NF90 is not associated with its interaction with stress granules. In viously described a panel of NS1 mutant viruses that is not able to contrast, an RBDm (NF90-RBDm) that lacked RNA-binding ability inhibit formation of stress granules (33) (Fig. 5A). We confirm in

FIGURE 4. NF90 RNA binding and phos- by guest on September 28, 2021 phorylation by PKR are required for localiza- tion to stress granules in response to poly(I:C) treatment. (A) Plasmids containing V5-tagged NF90 (NF90-WT) and NF90 nuclear localiza- tion (NF90-NLSm) and RNA binding (NF90- RBDm) mutants, as well as Flag-tagged phos- phorylation mutant (NF90-A), as shown in Fig. 1A, were transfected into 293T cells. Cells were treated with poly(I:C) for 6 h, fixed, and stained with anti-V5 or Flag, along with anti-PABP1 or anti-TIA Abs, to assess colocalization of NF90 and stress granules (original magnification 363). (B) Western blot analysis of NF90 expression in 293T cells treated with NF90-specific siRNA or scrambled siRNA or mock treated. b-tubulin was used as a loading control. (C) NF90 was knocked down with siRNA, as described above, and cells were transfected with WT NF90 or RNA-binding (NF90-RBDm) or phosphorylation (NF90-A) mutants. Formation of stress granules (SG) in response to poly(I:C) treatment was estimated in cells (left panel). Values represent the means of 10 randomly selected microscopic field of views for each sample (∼100 cells/field of view); error bars show SD. Expression of NF90 constructs was confirmed by Western blotting (lower panel). *p , 0.05, **p , 0.005, ****p , 0.0001. (D)Repre- sentative images of stress granules (red) in cells expressing WT or different mutants of NF90 (original magnification 340). The Journal of Immunology 7 this study that the NS1 mutants that cannot inhibit stress granule of PB1, PB2, PA, and NP) were coexpressed with NF90, only NP formation also have lost the ability to inhibit PKR activation in- was targeted to stress granules (Fig. 6A, lower panel). Using NP duced by poly(I:C) or virus infection (Fig. 5B, 5C). Consistent with as a marker for influenza A virus–induced stress granules, we found previous observations, the R38A mutant form of NS1, which is that, although WT virus suppresses formation of stress granules, NP attenuated in its ability to suppress host IFN expression, retains was clearly detectable in stress granules in 293T, MEF, and HeLa the ability to inhibit PKR phosphorylation similarly to WT NS1 cells infected with 123-127A mutant influenza virus at 24 h postin- (33, 44). The R35A mutant, which has lost both RNA-binding and fection. In addition, the nonfunctional R35A NS1 and del-NS1 dimerization ability (45), was used as a nonfunctional NS1 control mutants also failed to inhibit formation of NP-associated stress and showed no activity in inhibiting PKR activation. Although granules in infected cells (Fig. 6B–D). Quantitation of stress RNA-binding activity is not affected, as described in a previous granule–forming 293T cells infected with WT or mutant viruses study (33), NS1 mutants that combine I123A/M124A/K126A/N127A further confirmed that inhibition of PKR blocks induction of stress (123-127A) quadruple mutations exhibited a reduced ability to granules upon virus infection (Fig. 6E). The characteristics of inhibit PKR activation induced by poly(I:C) or virus infection these NS1 mutants of influenza A virus indicated their suitability (Fig. 5B, 5C). Using this PKR-specific NS1 mutant, we further for further use in studying the PKR-related antiviral pathway. demonstrated that knockdown of NF90 produced a negative effect on PKR phosphorylation upon virus infection (Fig. 5D). NF90 antiviral activity is not associated with expression of b We then examined whether the 123-127A quadruple mutant had IFN- lost the ability to suppress formation of stress granules, in addition We next performed a series of infections, using WT and mutant to being unable to inhibit PKR activation. We first examined influenza A viruses, to demonstrate the role of NF90 in the PKR- Downloaded from whether any influenza A virus components were targeted to stress induced antiviral pathway and to confirm that it is independent of granules. We showed previously that NP interacts with RAP55 and IFN expression. We previously showed that there is a significant colocalizes to P-bodies and stress granules (33). Moreover, NP also increase in the replication of PR8 (H1N1) and 1194 (H5N1) in- interacts with NF90 (19). Because NF90 is associated with stress fluenza viruses in cells in which NF90 has been transiently knocked granules, as shown above, it seems possible that it negatively down using siRNA targeting NF90 compared with cells treated

regulates influenza A virus replication by targeting viral NP to with control siRNA (19). Using stable NF90-knockdown cells, we http://www.jimmunol.org/ stress granules. To test this idea, individual PB1, PB2, PA, and NP observed a similar significant negative effect (p = 0.0001) of NF90 influenza virus proteins were expressed in cells, and their coloc- on virus replication in cells infected with another H1N1 virus alization with stress granules was examined following treatment strain, A/WSN/1933 (Fig. 7A). To verify that the altered virus- with poly(I:C). Only NP colocalized with the stress granule marker replication efficiency is associated with the NF90–PKR pathway, G3BP following poly(I:C) induction (Fig. 6A, upper panel). A we compared the growth kinetics of WT WSN virus with those of colocalization assay with another stress granule marker, PABP1, mutant WSN viruses containing different NS1 mutations, which showed a similar result (data not shown). When RNP (composed were attenuated in inhibition of PKR activation (123-127A) or by guest on September 28, 2021

FIGURE 5. Characterization of an NS1 mutant of influenza A virus that is attenuated in the ability to inhibit PKR phosphorylation in response to dsRNA activation. (A) Illustration of the NS1 protein of influenza A virus (A/WSN1933), showing locations of mutations used in this study. (B) 293T cells were transfected with pCMV expression vectors containing WT or R35A, R38A, or 123-127A quadruple NS1 mutants or with vector expressing GFP, or they were left untransfected prior to treatment with poly(I:C). Cells were harvested and lysed for Western blot analysis of NS1 and PKR expression and PKR phosphorylation. Expression of b-tubulin was analyzed for normalization of protein loading. (C) 293T cells were infected with WT or R38A or 123-127A NS1 mutant viruses for 24 h, and PKR activation levels were examined. Virus infection is shown by viral NP expression. (D) 293T cells were treated with siRNA targeting NF90 or scrambled siRNA for 56 h and then infected with WT or 123-127A NS1 mutant virus or were mock infected. Cell lysates were examined for levels of PKR phosphorylation at 24 h postinfection. Levels of total PKR also were estimated as a control. Relative levels of protein present in bands were analyzed by estimation of band density using ImageJ software in Western blots, as described above. 8 NF90 REGULATES ACTIVATION OF PKR

FIGURE 6. The influenza A virus mu- tant with attenuated ability to inhibit PKR phosphorylation also lost the ability to suppress formation of stress granules upon virus infection. (A) Plasmids expressing NP and individual subunits of the RNP complex derived from the A/Vietnam/ 1194/2004 (H5N1) strain of influenza A virus were transfected into 293T cells for 16 h. Cells were then treated with poly(I:C) for 6 h before fixation. Coloc- alization of NP, PB1, PB2, and PA with G3BP was examined as described above. V5-tagged NP and Flag-tagged PB1, PB2, and PA were coexpressed in 293T cells (upper panel, original magnification 363), and cells were examined for colocalization with G3BP (lower panel, original mag- nification 363). Cells were stained with

Abs specific for G3BP, V5 (NP), and Flag Downloaded from (PB1, PB2, and PA). NP-associated stress granules were examined in 293T (B), MEF (C), and HeLa (D) cells infected with WT or 123-127A or R35A mutant viruses by examining cells for colocali- zation of G3BP and NP in the cytoplasm

(original magnification 363). (E)Forma- http://www.jimmunol.org/ tion of stress granules (SG) in 293T cells infected with WSN WT or 123-127A, R35A, or R38A NS1 mutant viruses. Stress granules were examined by staining cells with G3BP-specific Ab at 24 h post- infection. Virus-infected cells were visu- alized by staining for the presence of NP protein. For statistical analysis, 60–300 cells were examined, and the percentage of infected cells containing stress gran- by guest on September 28, 2021 ules was determined. The average values from two independent experiments were calculated and graphed; error bars rep- resent SD.

IFN expression (R38A) (as described in Fig. 5A), in both NF90 with mutant viruses that are unable to inhibit PKR activation (NS1 stable-knockdown and scrambled 293T cells. In the scrambled R35A or 123-127A) induces stress granules in PKR WT (PKR+/+) 293T cells, antiviral mechanisms involving expression of IFN-b cells, but not in PKR knockout (PKR2/2) cells with normal NF90 and activation of PKR are intact, with both 123-127 and R38A (Fig. 7F, 7G), and expression of PKR by transfection restores mutant viruses replicating to a significantly lower level (p = 0.0001) stress granule–forming ability in PKR2/2 cells (Fig. 7G, lower compared with the WT virus (Fig. 7B). In the NF90-knockdown panel). PKR is an IFN-inducible gene, and it is not known whether cells, it is notable that 123-127A quadruple mutant virus showed the differences in stress granule induction may be due to variations no sign of attenuation and replicated to a level similar to the WT in inhibition of IFN expression by WT and mutant NS1 proteins. virus, whereas replication of R38A mutant virus was severely However, examination of inhibition of IFN-b expression in WT attenuated, presumably due to the antiviral effects of IFN (Fig. 7C). and mutant virus-infected cells revealed that both the WT and 123- The R38A mutant is well recognized for its reduced ability to inhibit 127A quadruple mutant viruses are able to inhibit IFN-b expres- the host IFN antiviral response (46). To differentiate the effects sion, but R38A virus cannot (Fig. 7E). Taken together, these re- caused by the IFN antiviral response from those associated with sults support the idea of an underlying mechanism whereby NF90 the NF90–PKR pathway, we compared virus-replication efficiency is associated with PKR activation in the host antiviral response, in Vero cells, which are deficient in IFN expression. In contrast to and the related antiviral signaling is independent of induction of the result obtained in NF90-knockdown cells, R38A mutant virus IFN-b expression (Fig. 8). is able to replicate to a level close to that of WT virus, whereas the 123-127A quadruple mutant is significantly attenuated (p = 0.001) Discussion (Fig. 7D). PKR-knockout (PKR2/2) and WT (PKR+/+) primary NF90 was found to play an important role in host innate immunity MEF cells were used to further verify the linkage of NF90 and PKR. against various virus infections (12–15). We (19) previously found WesternblotshowedNF90expressioninbothPKR-knockout that NF90 negatively regulates influenza A virus replication through (PKR2/2)andWT(PKR+/+) primary MEF cells. Using this influ- interaction with viral NP. Although the significance of NF90 in enza A virus–infection model, we further demonstrated that PKR is restricting virus replication is evident from the magnitude of virus a downstream effector for NF90 antiviral activity, because infection replication increase in NF90-knockdown cells (12, 19), the molecular The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 FIGURE 7. NF90 inhibition of influenza A virus is mediated via the PKR pathway. (A) Growth kinetics in NF90 stable-knockdown and scrambled 293T cells infected with the influenza A virus A/WSN/1933 at an MOI of 0.005. Growth kinetics of influenza viruses containing specific mutations that attenuate their ability to inhibit PKR activation were determined in different cell lines. Scrambled (B) and NF90-knockdown (C) 293T cells and Vero cells (D) were infected with WT A/WSN/1933 (H1N1) or mutant viruses containing NS1 R38A or 123–127A quadruple mutations at an MOI of 0.05. Supernatants were collected at 8, 24, and 48 h postinfection (h.p.i.) for titration by plaque assay. (E) NF90 inhibition of influenza A virus is not caused by expression of IFN-b. NF90-knockdown (kd) cells and scrambled control cells were infected with WT or NS1 R38A or 123-127A mutant viruses at an MOI of 0.1 or were mock infected (PBS). At 24 h postinfection, cellular RNAs were extracted, and quantitative real-time PCR was performed. IFN-b mRNA levels were determined after normalization. (F) Western blot showing NF90 and PKR expression in both PKR-knockout (PKR2/2) and WT (PKR+/+) primary MEF cells. Expression of b-tubulin was assessed as a loading control. (G) PKR-knockout (PKR2/2) and WT (PKR+/+) primary MEF cells were infected with R35A or 123–127A virus for 24 h before being subjected to immunofluorescence assay for visualization of stress granules (upper panel, original magnification 363). G3BP, NP, and DAPI are shown in green, red, and blue, respectively. PKR expression in PKR-knockout MEF cells was restored by transfection of a V5-tagged PKR construct (lower panel, original magnification 363). MEF cells with restored PKR expression were infected with 123–127A mutated influenza A virus. Formation of antiviral stress granules was examined by immunostaining using anti-V5 (PKR), anti-NP, and anti- G3BP Abs. Error bars represent SD. *p , 0.05, **p , 0.01, ***p , 0.001, t test. details of the NF90-associated antiviral mechanism remain largely that binding of either dsRNA or PACT to PKR may interrupt unknown. NF90 is one of the evolutionarily conserved members of intramolecular interaction between the kinase and dsRNA-binding the dsRNA-binding and is expressed abundantly in domains, leading to an open conformation of the PKR molecule various human cells. Previous studies (6, 7, 9, 10, 13, 40) identified and allowing autophosphorylation to occur (49). NF90 is phos- and confirmed PKR as the key interacting partner for NF90 in cells, phorylated by PKR in its RBD (6). Previous studies (7, 9, 29) in addition to its function in regulating IL-2 gene expression in showed that NF90 interacts with PKR in the yeast two-hybrid and T cells (4). PKR plays an important role in mediating innate im- GST-pull down assays. We confirmed interaction between NF90 munity to viral infection and is required for NF90 antiviral activity, and PKR in 293T cells using a co-IP assay and showed that it is as demonstrated in a vesicular stomatitis virus infection experiment independent of RNA binding of NF90, because the RBDm could using PKR-knockout (PKR2/2) and control (PKR+/+) MEFs (13, interact with PKR (Fig. 1C). On testing the effect of NF90 on PKR 25). Thus, it is natural to reason that the antiviral activity exerted activation, we found that low concentrations of NF90 restore some by NF90 may lie within the PKR-signaling pathway. PKR activity in response to poly(I:C) induction in NF90 stably Phosphorylation of PKR is essential for activation of its function, knocked-down cells. However, when high concentrations of NF90 and it can be regulated by host RNA-binding proteins other than were introduced by transfection, PKR phosphorylation was in- dsRNA (47–49). Two models currently describe PKR activation: hibited in these cells. Furthermore, the results showed that the in- the autoinhibition and dimerization models (50). It was suggested hibitory effect is exerted by the dsRNA-binding domain, presumably 10 NF90 REGULATES ACTIVATION OF PKR Downloaded from

FIGURE 8. Working model of PKR-mediated NF90 antiviral pathway. In normal conditions, NF90 migrates to the cytoplasm to facilitate PKR phos- http://www.jimmunol.org/ phorylation in response to virus infection or the presence of dsRNA during virus replication. Activation of PKR leads to the phosphorylation of eIF2a and subsequently triggers formation of stress granules that involve NF90, PKR, and other stress granule components. However, viruses adapt different mechanisms to antagonize NF90–PKR antiviral activity; for instance, influenza A viruses express NS1 to inhibit phosphorylation of PKR, which also may interact with NF90, to enhance virus replication. PKR also may be activated by IFN to exert antiviral activity in an NF90-independent manner. through competition with PKR for dsRNA ligand in an RNA- We found that NF90 is regularly associated with stress granules binding–dependent manner, as was suggested previously (6). This formed in response to treatments with oxidative substances (ar- hypothesis was confirmed in this study using an NF90 mutant senite) or dsRNA mimics [poly(I:C)]. It was further demonstrated containing mutations in the RBD, which shows no inhibition of that NF90 may be required for the formation of stress granules in by guest on September 28, 2021 PKR phosphorylation when transfected into 293T cells at high the dsRNA-induced host response, because there is a significant concentrations (Fig. 2D). NF90 predominantly localizes to the nu- reduction in the levels of stress granules formed in response to cleus but may be exported to the cytoplasm to perform its functions poly(I:C) treatment in NF90 stably knocked-down cells. However, (5, 8). NF90 is also phosphorylated by PKR, and activated NF90 there was no significant difference in granule formation between does not inhibit PKR activation (6). It was proposed that shuttling NF90 knockdown and scrambled cells treated with arsenite, which of NF90 between nucleus and cytoplasm is regulated through phos- does not involve PKR activation (42) (Fig. 3B), whereas treatment phorylation of NF90 by PKR (13). However, these studies did not with a PKR phosphorylation inhibitor, 2-AP (43), inhibited stress investigate whether NF90 exerted any effect on PKR. Our results granule induction (Fig. 3C). It seems likely that NF90’s associa- suggest that a physiological concentration of NF90 is required for tion with stress granules is coupled with its ability to interact with optimal activation of PKR in response to stimuli, such as cellular PKR and regulate its activation. Stable knockdown of NF90 ren- dsRNA or intermediate dsRNA products generated during virus ders 293T cells ineffective in induction of phosphorylation of PKR replication. Because PKR phosphorylation can activate down- in response to poly(I:C) treatment. stream antiviral cascades, including NF-kB and inflammasomes To further verify the linkage between NF90 and PKR signaling (28, 51), it is postulated that NF90 may serve as a regulator for in the host antiviral response, we generated an influenza A virus PKR activation in response to cellular dsRNA stimuli or virus specifically mutated in the NS1 gene that is attenuated in its ability infection. Our findings suggest a mechanism in which NF90 may to inhibit PKR activation but otherwise functions normally. We act to regulate PKR activity in cells through balancing PKR phos- found that NP of this mutant is targeted to stress granules, presumably phorylation in response to dsRNA via its N- and C-terminal functions. through interaction with NF90 and/or other NP-interacting proteins. The PKR-activation function of NF90 lies in the N-terminal region. Using this PKR-specific virus model, this study clearly demonstrated In contrast, interaction between NF90 and PKR also may modulate that this mutant virus is able to replicate to a similar level as WT virus the activation of PKR through competition with dsRNA via its in NF90-knockdown cells but not in scrambled control cells or in Vero C-terminal RBD; this also was reported in an in vitro study (6). cells, which are deficient in expression of IFN-b. Our experiments also Hosts use multiple antiviral mechanisms to restrict virus infec- showed that this specific mutant virus is able to inhibit IFN-b ex- tion; formation of stress granules is an important antiviral response pression just as WT virus does but that the R38A mutant, which has that limits virus replication by stalling viral protein synthesis (52, lost RNA-binding ability, is attenuated in terms of suppression 53). Activation of PKR is required for the formation of stress gran- of IFN. These lines of evidence strongly support the hypothesis ules in response to virus infection (34–36, 54). This study attempts to that NF90 antiviral function is signaled through the PKR pathway. delineate the mechanism of NF90 antiviral activity through exami- Although further studies are needed to reveal the details of how nation of NF90 involvement in PKR activation and formation of NF90 enhances PKR phosphorylation and how NF90 is recycled antiviral stress granules using the influenza A virus–infection model. back to the nucleus after engaging in PKR activation in the The Journal of Immunology 11 cytoplasm, it is tempting to postulate that, in normal conditions, NF90 12. Pfeifer, I., R. Elsby, M. Fernandez, P. A. Faria, D. R. Nussenzveig, I. S. Lossos, B. M. Fontoura, W. D. Martin, and G. N. Barber. 2008. NFAR-1 and -2 modulate migrates to the cytoplasm to facilitate PKR phosphorylation in re- translation and are required for efficient host defense. Proc. Natl. Acad. Sci. USA sponse to virus infection or the presence of dsRNA during virus 105: 4173–4178. replication. Activation of PKR leads to the phosphorylation of eIF2a 13. Harashima, A., T. Guettouche, and G. N. Barber. 2010. Phosphorylation of the NFAR proteins by the dsRNA-dependent protein kinase PKR constitutes a novel and subsequently triggers formation of stress granules that involve mechanism of translational regulation and cellular defense. Genes Dev. 24: both NF90 and PKR (Fig. 8). We also found that phosphorylation of 2640–2653. NF90 by PKR is required for NF90–PKR signaling to induce an- 14. Shabman, R. S., D. W. Leung, J. Johnson, N. Glennon, E. E. Gulcicek, K. L. Stone, L. Leung, L. Hensley, G. K. Amarasinghe, and C. F. Basler. 2011. tiviral stress granules (Fig. 4). Based on a previous study (13) that DRBP76 associates with Ebola virus VP35 and suppresses viral polymerase reported that NF90 is phosphorylated by PKR and retained in the function. J. Infect. Dis. 204(Suppl. 3): S911–S918. cytoplasm during the interaction, it seems reasonable to suggest that 15. Agbottah, E. T., C. Traviss, J. McArdle, S. Karki, G. C. St Laurent, III, and A. Kumar. 2007. Nuclear Factor 90(NF90) targeted to TAR RNA inhibits tran- NF90 and PKR are mutually regulated. It may be speculated that an scriptional activation of HIV-1. Retrovirology 4: 41. accumulation of NF90 in the cytoplasm may subsequently compete 16. Hoque, M., R. A. Shamanna, D. Guan, T. Pe’ery, and M. B. Mathews. 2011. with PKR for dsRNA binding and, thereby, form a feedback loop to HIV-1 replication and latency are regulated by translational control of cyclin T1. J. Mol. Biol. 410: 917–932. restore PKR back to its latent form; similarly, NF90 would be 17. Merrill, M. K., E. Y. Dobrikova, and M. Gromeier. 2006. Cell-type-specific re- recycled back to the nucleus after the stimuli dissipates. Further pression of internal ribosome entry site activity by double-stranded RNA-binding protein 76. J. Virol. 80: 3147–3156. studies are needed to unveil the regulatory loop of the NF90–PKR 18. Merrill, M. K., and M. Gromeier. 2006. The double-stranded RNA binding pathway. Although we showed that IFN-b is not involved in NF90– protein 76:NF45 heterodimer inhibits translation initiation at the rhinovirus type PKR activation, it would be interesting to study how NF90 and IFN 2 internal ribosome entry site. J. Virol. 80: 6936–6942. 19. Wang, P., W. Song, B. W. Mok, P. Zhao, K. Qin, A. Lai, G. J. Smith, J. Zhang, coordinately activate PKR in the antiviral state induced during virus T. Lin, Y. Guan, and H. Chen. 2009. Nuclear factor 90 negatively regulates in- Downloaded from infection. Host innate immunity relies on pattern recognition fluenza virus replication by interacting with viral nucleoprotein. J. Virol. 83: receptors, which reside primarily in the cytosol or cellular mem- 7850–7861. 20. Isken, O., C. W. Grassmann, R. T. Sarisky, M. Kann, S. Zhang, F. Grosse, branes, to recognize pathogen-associated molecule patterns that P. N. Kao, and S. E. Behrens. 2003. Members of the NF90/NFAR protein group are usually intermediate products generated from the viral- are involved in the life cycle of a positive-strand RNA virus. EMBO J. 22: 5655– replication process. Because NF90 predominantly resides in the 5665. 21. Isken, O., M. Baroth, C. W. Grassmann, S. Weinlich, D. H. Ostareck, nucleus, it would be important to examine whether it is a yet-to- A. Ostareck-Lederer, and S. E. Behrens. 2007. Nuclear factors are involved in http://www.jimmunol.org/ be-defined nuclear pattern recognition receptor, like IFI16 (55). RNA replication. RNA 13: 1675–1692. 22. Gomila, R. C., G. W. Martin, and L. Gehrke. 2011. NF90 binds the dengue virus RNA 39 terminus and is a positive regulator of dengue virus replication. PLoS Acknowledgments ONE 6: e16687. We thank Dr. Christopher Basler for the gift of NF90-knockdown and 23. Shamanna, R. A., M. Hoque, T. 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