Title Two Genetic Determinants Acquired Late in Mus Evolution Regulate the Inclusion of Exon 5 , which Alters Mouse APOBEC 3 Translation Efficiency

Mouse apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like editing complex 3 (mA3), an intracellular antiviral factor, has 2 allelic variations that are linked with different susceptibilities to betaand gammaretrovirus infections among various mouse strains. In virus-resistant C57BL/6 (B6) mice, mA3 transcripts are more abundant than those in susceptible BALB/c mice both in the spleen and bone marrow. These strains of mice also express mA3 transcripts with different splicing patterns: B6 mice preferentially express exon 5-deficient (D5) mA3 mRNA, while BALB/c mice produce exon 5-containing full-length mA3 mRNA as the major transcript. Although the protein product of the D5 mRNA exerts stronger antiretroviral activities than the full-length protein, how exon 5 affects mA3 antiviral activity, as well as the genetic mechanisms regulating exon 5 inclusion into the mA3 transcripts, remains largely uncharacterized. Here we show that mA3 exon 5 is indeed a functional element that influences protein synthesis at a post-transcriptional level. We further employed in vitro splicing assays using genomic DNA clones to identify two critical polymorphisms affecting the inclusion of exon 5 into mA3 transcripts: the number of TCCT repeats upstream of exon 5 and the single nucleotide polymorphism within exon 5 located 12 bases upstream of the exon 5/intron 5 boundary. Distribution of the above polymorphisms among different Mus species indicates that the inclusion of exon 5 into mA3 mRNA is a relatively recent event in the evolution of mice. The widespread geographic distribution of this exon 5-including genetic variant suggests that in some Mus populations the cost of maintaining an effective but mutagenic enzyme may outweigh its antiviral function. Citation: Li J, Hakata Y, Takeda E, Liu Q, Iwatani Y, et al. (2012) Two Genetic Determinants Acquired Late in Mus Evolution Regulate the Inclusion of Exon 5, which Alters Mouse APOBEC3 Translation Efficiency. PLoS Pathog 8(1): e1002478. doi:10.1371/journal.ppat. 1002478 Editor: Sara Sawyer, University of Texas at Austin, United States of America Received July 5, 2011; Accepted November 26, 2011; Published January 19, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, including the High-Tech Research Center and Anti-Aging Center projects, those from the Japan Society for Promotion of Sciences, KAKENHI (B), and those from the Ministry of Health, Labor and Welfare of Japan for Research on HIV/AIDS (all given to MM), and by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Sciences, KAKENHI 21790450, given to YH. This work was also supported by the Intramural Research Program of the NIH, NIAID, given to CAK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hakata@med.kindai. ac.jp (YH); masaaki@med.kindai. ac.jp (MM) ¤ Current address: Department of Emerging Infectious Diseases, Nagasaki University Institute of Tropical Medicine, Nagasaki, Japan