At 53 h post-infection, cells were collected. followed by autoradiography. Real-time PCR 293TRex-ZAP cells were infected with NL4C3-luc. At 5 h post-infection, tetracycline was added to induce ZAP expression, and SB216763 was added to inhibit GSK3. At 53 h post-infection, cells were collected. Ten percent of the cells were lysed to measure luciferase activity. The rest of the cells were used to extract cytoplasmic mRNA, followed by reverse transcription. above the sequence are the numbers used to identify the serines studied in this work. There are eight serine residues in ZAP in the region of amino acids 255C295 (numbered 1C8 from the N terminus in this report (Fig. 1and and and and and and and and and and and and and and and and and and and and and luciferase activity expressed from pRL-TK. -Fold inhibition was calculated as the normalized luciferase activity in the mock-treated cells divided by that in the tetracycline-treated cells. Relative -fold inhibition was calculated as the -fold inhibition with GSK3 divided by that without GSK3 ( 0.05. To test whether endogenous GSK3 modulates ZAP activity, endogenous GSK3 was down-regulated by RNAi, and the effect around the antiviral activity of ZAP against MMLV-luc was evaluated. To confirm the specificity of the shRNA (Gi-5) to target GSK3, a GSK3-expressing plasmid (GSK3M) that cannot be targeted by Gi-5 was constructed (Fig. 4mRNAs by real-time PCR. RNA -fold inhibition was calculated as the mRNA level in the mock-treated cells divided by that in the tetracycline-treated cells (kinase assays. One possible explanation is usually that GSK3 can execute phosphorylation without phosphorylation of the priming site, but when the priming site is usually phosphorylated, GSK3 works more efficiently. Similar observations have also been reported for the phosphorylation of tau and -catenin by GSK3 (18C20). GSK3 plays regulatory roles in various diseases (21), including diabetes (22, 23), Alzheimer Triisopropylsilane disease (24, 25), bipolar mood disorder (26), and cancer (27). GSK3 is also involved in innate and adaptive immune responses (28C30). Lithium has been used as a GSK3 inhibitor in the treatment of bipolar disorder. Other GSK3 inhibitors are being tested for the treatment of Alzheimer disease (31C33), type 2 diabetes (32, 34), and osteoporosis (31). Our results showing that inhibition of GSK3 compromises the antiviral activity of ZAP suggest that precautions should be taken in the clinical use of GSK3 inhibitors. *This work was supported by Ministry of Science and Technology 973 Program Grant 2012CB910203, National Science Foundation Grants 30530020 and 81028011, and Ministry of Health of China Grant 2012ZX10001-006 (to G. G.). 4L. Sun and G. Gao, unpublished data. 3The abbreviations used are: ZAPzinc-finger antiviral proteinMMLVMoloney murine leukemia virusGSK3glycogen synthase kinase 3lucluciferase. REFERENCES 1. Gao G., Guo X., Goff S. P. (2002) Inhibition of retroviral RNA production by ZAP, a CCCH-type zinc-finger protein. Science 297, 1703C1706 [PubMed] [Google Scholar] 2. Zhu Y., Chen G., Triisopropylsilane Lv F., Wang X., Ji X., Xu Y., Sun J., Wu L., Zheng Y. T., Gao G. (2011) Zinc-finger antiviral protein inhibits HIV-1 contamination by selectively targeting multiply spliced viral mRNAs for degradation. Proc. Natl. Acad. Sci. U.S.A. 108, 15834C15839 [PMC free article] [PubMed] [Google Scholar] 3. Mller S., M?ller P., Bick M. J., Wurr S., Becker Triisopropylsilane S., Gnther S., Kmmerer B. M. (2007) Inhibition of filovirus replication by the zinc-finger antiviral protein. J. Virol. 81, 2391C2400 [PMC free article] [PubMed] [Google Scholar] 4. Zhang Y., Efnb2 Burke C. W., Ryman K. D., Klimstra W. B. (2007) Identification and characterization of interferon-induced proteins that inhibit alphavirus replication. J. Virol. 81, 11246C11255 [PMC free article] [PubMed] [Google Scholar] 5. Bick M. J., Carroll J. W., Gao G., Goff S. P., Rice C. M., MacDonald M. R. (2003) Expression of the zinc-finger antiviral protein inhibits alphavirus replication. J. Virol. 77, 11555C11562 [PMC free article] [PubMed] [Google Scholar] 6. Wang N., Dong Q., Li J., Jangra R. K., Fan M., Brasier A. R., Lemon S. M., Pfeffer L. M., Li K. (2010) Viral induction of the zinc-finger antiviral protein is usually IRF3-dependent but NF-B-independent. J. Biol. Chem. 285, 6080C6090 [PMC free article] [PubMed] [Google Scholar] 7. MacDonald M. R., Machlin E. S., Albin O. R., Levy D. E. (2007) The zinc-finger antiviral protein acts synergistically with an interferon-induced factor for maximal activity against alphaviruses. J. Virol. 81, 13509C13518 [PMC free article] [PubMed] [Google Scholar] Triisopropylsilane 8. Chen G., Guo X., Lv F., Xu Y., Gao G. (2008) p72 DEAD box RNA helicase is required for optimal function of the zinc-finger antiviral protein. Proc. Natl. Acad. Sci. U.S.A. 105, 4352C4357.