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Stress Granules in Infected Cells Regulation of PKR Phosphorylation 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 ol.1302813 Downloaded from Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 28, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 kinase (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 gene 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
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