GANP Interacts with APOBEC3G and Facilitates Its Encapsidation into the Virions To Reduce HIV-1 Infectivity This information is current as Kazuhiko Maeda, Sarah Ameen Almofty, Shailendra Kumar of September 23, 2021. Singh, Mohammed Mansour Abbas Eid, Mayuko Shimoda, Terumasa Ikeda, Atsushi Koito, Phuong Pham, Myron F. Goodman and Nobuo Sakaguchi J Immunol 2013; 191:6030-6039; Prepublished online 6 November 2013; Downloaded from doi: 10.4049/jimmunol.1302057 http://www.jimmunol.org/content/191/12/6030 Supplementary http://www.jimmunol.org/content/suppl/2013/11/06/jimmunol.130205 http://www.jimmunol.org/ Material 7.DC1 References This article cites 52 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/191/12/6030.full#ref-list-1 Why The JI? Submit online. by guest on September 23, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology GANP Interacts with APOBEC3G and Facilitates Its Encapsidation into the Virions To Reduce HIV-1 Infectivity Kazuhiko Maeda,*,1 Sarah Ameen Almofty,*,1 Shailendra Kumar Singh,* Mohammed Mansour Abbas Eid,* Mayuko Shimoda,* Terumasa Ikeda,†,2 Atsushi Koito,† Phuong Pham,‡ Myron F. Goodman,‡,x and Nobuo Sakaguchi* The ssDNA-dependent deoxycytidine deaminase apolipoprotein B mRNA–editing, enzyme-catalytic, polypeptide-like 3G (A3G) is a potent restrictive factor against HIV-1 virus lacking viral-encoded infectivity factor (Vif) in CD4+ T cells. A3G antiretroviral activity requires its encapsulation into HIV-1 virions. In this study, we show that germinal center–associated nuclear protein (GANP) is induced in activated CD4+ T cells and physically interacts with A3G. Overexpression of GANP augments the A3G encapsidation into the virion-like particles and ΔVif HIV-1 virions. GANP is encapsidated in HIV-1 virion and modulates A3G packaging into the cores together with cellular RNAs, including 7SL RNA, and with unspliced HIV-1 genomic RNA. GANP Downloaded from upregulation leads to a significant increase in A3G-catalyzed G→A hypermutation in the viral genome and suppression of HIV-1 infectivity in a single-round viral infection assay. Conversely, GANP knockdown caused a marked increase in HIV-1 infectivity in a multiple-round infection assay. The data suggest that GANP is a cellular factor that facilitates A3G encapsidation into HIV-1 virions to inhibit viral infectivity. The Journal of Immunology, 2013, 191: 6030–6039. http://www.jimmunol.org/ ctivation-induced cytidine deaminase (AID)/apolipo- troduced genetic materials (1–3). AID has been well characterized protein B mRNA–editing enzyme, catalytic polypeptide- as a critical initiator of Ig somatic hypermutation (SHM) and A like (APOBEC) protein family is composed of 11 cyti- class-switch recombination in germinal center (GC) B cells during dine deaminases that catalyze C→U on ssDNA or RNA to regulate the immune responses to T cell–dependent Ags (4–8). AID ini- Ab diversification during adaptive immune response and to pro- tiates SHM and CRS by catalyzing C→U conversion at actively vide innate resistance against retroviruses and exogenously in- transcribed V region and switch (S) region of Ig genes (4–8). In contrast to AID that acts preferentially at Ig loci in B cell genomic *Department of Immunology, Graduate School of Life Sciences, Kumamoto Univer- DNA, proteins of a subgroup APOBEC3 (A, B, C, DE, F, G, H) sity, Kumamoto 860-8556, Japan; †Department of Retrovirology and Self-Defense, primarily target retroviral cDNA in the cytoplasm to cause lethal by guest on September 23, 2021 Graduate School of Life Sciences, Kumamoto University, Kumamoto 860-8556, hypermutation in viral genomes (9). In particular, APOBEC3G Japan; ‡Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089; and xDepartment of Chemistry, University of Southern Califor- (A3G) possesses a potent antiretroviral activity that restricts HIV-1 nia, Los Angeles, CA 90089 replication in T cells in the absence of viral infectivity factor 1K.M. and S.A.A. contributed equally to this work. (Vif) (10). A3G antiviral activity requires its encapsidation into 2Current address: Department of Biochemistry, Molecular Biology and Biophysics, the HIV-1 virions. Upon entry into the secondary infected target University of Minnesota, Minneapolis, MN. cells, A3G catalyzes deamination of C→U, preferentially at many Received for publication August 1, 2013. Accepted for publication October 8, 2013. 59-YCC (Y = C or T) motifs located on the nascent minus-strand This study was supported in part by a Global Center of Excellence Program (Global cDNA reverse transcribed from HIV-1 genomic RNA (gRNA) (9). Education and Research Center Aiming at the Control of AIDS, Kumamoto University), Subsequently, this creates G→A hypermutation in the HIV-1 ge- Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Sci- entific Research 22021036, a contract research fund from the Ministry of Education, nome that potentially inactivates essential genes required for in- Culture, Sports, Science and Technology for Program of Founding Research Centers for fectivity in the absence of Vif (11). Although Vif has been shown Emerging and Reemerging Infectious Diseases, Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 23390122, 24659224, 22590436, and to inhibit A3G translation and promote A3G degradation through 23590546, a fund from Higo Bank, and in part by National Institutes of Health Grants the core binding factor b-mediated proteosomal degradation (12, GM21422 and ES013192. S.A.A. is supported by the King Abdullah Scholarship Pro- 13), the action of Vif is not absolute, and a few copies of A3G are gram (Ministry of Higher Education in Saudi Arabia). S.A.A and M.M.A.E. are sup- ported by the Advanced Education Program for Integrated Clinical, Basic and Social thought to be encapsidated into virions (14), probably through Medicine (Graduate School of Medical Sciences, Kumamoto University). interaction with HIV-1 gRNA (15, 16) and/or a number of cellular Address correspondence and reprint requests to Prof. Nobuo Sakaguchi, Department of RNAs, including 7SL RNA (17–19). A3G-catalyzed G→A hyper- Immunology, Graduate School of Life Sciences, Kumamoto University, 1-1-1 Honjo, mutated HIV-1 genomes have been isolated from blood cells of Chuo-ku, Kumamoto 860-8556, Japan. E-mail address: [email protected] HIV-1–infected patients at different stages of infection (20, 21). The online version of this article contains supplemental material. A3G encapsidation into HIV-1 virions requires interactions with Abbreviations used in this article: A3G, apolipoprotein B mRNA–editing, enzyme- the nucleocapsid (NC) domain of a viral Gag protein, suggesting catalytic, polypeptide-like 3G; AID, activation-induced cytidine deaminase; APO- BEC, apolipoprotein B mRNA–editing, enzyme-catalytic, polypeptide-like; GANP, that incorporation of A3G into HIV virions occurs during viral germinal center–associated nuclear protein; GC, germinal center; gRNA, genomic assembly (18, 22–24). In T cells, A3G is present in RNase-sen- RNA; HA, hemagglutinin; IP, immunoprecipitation; NC, nucleocapsid; qRT-PCR, quantitative real-time PCR; RNP, ribonucleoprotein; S, switch; SHM, somatic hyper- sitive ribonucleoprotein (RNP) complexes localized in the cyto- mutation; si, small interfering; Vif, viral infectivity factor; virion-IP, virion immuno- plasm (25–27) and enriched at mRNA processing bodies (26–31). precipitation; VLP, virus-like particle; VSV-G, vesicular stomatitis virus G protein; Cellular proteins interacted with the RNP complex might also WB, Western blot; WCL, whole-cell lysate; WT, wild-type. be important for A3G encapsidation. However, there is a little Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 knowledge about the molecular mechanism and host cellular www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302057 The Journal of Immunology 6031 proteins responsible for A3G encapsidation into HIV-1 virions Transfection and cell lysate preparation, immunoprecipitation, (32, 33). and Western blotting GC-associated nuclear protein (GANP) that was discovered as Cells were transfected with FuGENE HD transfection reagent (Roche a protein upregulated in GC B cells during immune responses is Diagnostics). Forty-eight hours after transfection, cells were harvested and physically associated with AID through its C-terminal side region lysed using TNE buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, (34). GANP is a component of transcription and export complex 2 0.5% Nonidet P-40, 0.5% Triton X-100 [pH 7.5]) with a protease inhibitor interacted with RNP complexes and involving mRNA export in mixture (Nacalai Tesque). Immunoprecipitation (IP) and Western blotting (WB) were performed as described previously (38). In some experiments, mammals (35, 36). GANP mutant mice studies have shown a strong cellular and viral RNAs in cell lysates were removed by a treatment with correlation between the levels of GANP expression and SHM at the 25 mg/ml RNase A for 30 min at 37˚C before IP. rearranged IgV loci. B cell–specific GANP-transgenic mice aug- ment the generation of high-affinity Abs against the immunized Ags Viral preparation in vivo (37), suggesting that GANP is an important functional AID Virus-like particles (VLPs) were produced from HIV-1 Gag mutant con- partner in generation of high-affinity Abs in GC B cells.