  Citation: Tsukimoto, S.; Hakata, Y.; Tsuji-Kawahara, S.; Enya, T.; Tsukamoto, T.; Mizuno, S.; Takahashi, S.; Nakao, S.; Miyazawa, M. Distinctive High Expression of Antiretroviral APOBEC3 Protein in Mouse Germinal Center B Cells. Viruses 2022, 14, 832. https:// doi.org/10.3390/v14040832 Academic Editor: Vinay Pathak Received: 7 March 2022 Accepted: 13 April 2022 Published: 17 April 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). viruses Article Distinctive High Expression of Antiretroviral APOBEC3 Protein in Mouse Germinal Center B Cells Shota Tsukimoto 1,2 , Yoshiyuki Hakata 1 , Sachiyo Tsuji-Kawahara 1 , Takuji Enya 1,3 , Tetsuo Tsukamoto 1,† , Seiya Mizuno 4 , Satoru Takahashi 5 , Shinichi Nakao 2 and Masaaki Miyazawa 1,6, * 1 Department of Immunology, Faculty of Medicine, Kindai University, 377-2 Ohno-Higashi, Osaka-Sayama 589-8511, Osaka, Japan; s-tsukimoto@med.kindai.ac.jp (S.T.); hakata@med.kindai.ac.jp (Y.H.); skawa@med.kindai.ac.jp (S.T.-K.); enyatakuji@yahoo.co.jp (T.E.); ttsukamoto@med.kindai.ac.jp or tetsuo-tsukamoto@nuhw.ac.jp (T.T.) 2 Department of Anesthesiology, Faculty of Medicine, Kindai University, 377-2 Ohno-Higashi, Osaka-Sayama 589-8511, Osaka, Japan; nakaos@med.kindai.ac.jp 3 Department of Pediatrics, Faculty of Medicine, Kindai University, 377-2 Ohno-Higashi, Osaka-Sayama 589-8511, Osaka, Japan 4 Laboratory Animal Resource Center in Transborder Medical Research Center, Department of Laboratory Animal Science, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Ibaraki, Japan; konezumi@md.tsukuba.ac.jp 5 Laboratory Animal Resource Center in Transborder Medical Research Center, Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Ibaraki, Japan; satoruta@md.tsukuba.ac.jp 6 Anti-Aging Center, Kindai University, 3-4-1 Kowakae, Higashiosaka 577-8502, Osaka, Japan * Correspondence: masaaki@med.kindai.ac.jp † Present address: Department of Health Informatics, Niigata University of Health and Welfare, 1398 Shimami-cho, Kita-ku, Niigata-shi 950-3198, Niigata, Japan. Abstract: Tissue and subcellular localization and its changes upon cell activation of virus-restricting APOBEC3 at protein levels are important to understanding physiological functions of this cyti- dine deaminase, but have not been thoroughly analyzed in vivo. To precisely follow the possible activation-induced changes in expression levels of APOBEC3 protein in different mouse tissues and cell populations, genome editing was utilized to establish knock-in mice that express APOBEC3 protein with an in-frame FLAG tag. Flow cytometry and immunohistochemical analyses were per- formed prior to and after an immunological stimulation. Cultured B cells expressed higher levels of APOBEC3 protein than T cells. All differentiation and activation stages of freshly prepared B cells expressed significant levels of APOBEC3 protein, but germinal center cells possessed the highest levels of APOBEC3 protein localized in their cytoplasm. Upon immunological stimulation with sheep red blood cells in vivo, germinal center cells with high levels of APOBEC3 protein expression increased in their number, but FLAG-specific fluorescence intensity in each cell did not change. T cells, even those in germinal centers, did not express significant levels of APOBEC3 protein. Thus, mouse APOBEC3 protein is expressed at distinctively high levels in germinal center B cells. Antigenic stimulation did not affect expression levels of cellular APOBEC3 protein despite increased numbers of germinal center cells. Keywords: cytidine deaminase; virus restriction; APOBEC3; protein expression; genome editing; tissue distribution; germinal center 1. Introduction Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3) proteins are polynucleotide cytidine deaminases that belong to the activation-induced cyti- dine deaminase (AID)/APOBEC family proteins and have emerged in placental mammals through gene duplication and diversification which most likely occurred under a conflict Viruses 2022, 14, 832. https://doi.org/10.3390/v14040832 https://www.mdpi.com/journal/viruses