Jiang Gu1, Qihan Chen1, Xiao Xiao2, Fumiaki Ito1, Aaron Wolfe2, Xiaojiang S Chen3. 1. Molecular and Computational Biology Program, Departments of Biological Sciences and Chemistry, University of Southern California, Los Angeles, CA 90089, USA. 2. Molecular and Computational Biology Program, Departments of Biological Sciences and Chemistry, University of Southern California, Los Angeles, CA 90089, USA; Genetic, Molecular and Cellular Biology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA. 3. Molecular and Computational Biology Program, Departments of Biological Sciences and Chemistry, University of Southern California, Los Angeles, CA 90089, USA; Center of Excellence in NanoBiophysics, University of Southern California, Los Angeles, CA 90089, USA; Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90089, USA; Genetic, Molecular and Cellular Biology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA. Electronic address: xiaojiac@usc.edu.
Abstract
APOBEC3H (A3H) is the most polymorphic member of the APOBEC3 family. Seven haplotypes (hap I-VII) and four mRNA splicing variants (SV) of A3H have been identified. The various haplotypes differ in anti-HIV activity, which is attributed to differences in protein stability, subcellular distribution, and/or RNA binding and virion packaging. Here, we report the first comparative biochemical studies of all the A3H variants using highly purified proteins. We show that all haplotypes were stably expressed and could be purified to homogeneity by Escherichia coli expression. Surprisingly, four out of the seven haplotypes showed high cytosine (C) deaminase activity, with hap V displaying extremely high activity that was comparable to the highly active A3A. Furthermore, all four haplotypes that were active in C deamination were also highly active on methylated C (mC), with hap II displaying almost equal deamination efficiency on both. The deamination activity of these A3H variants correlates well with their reported anti-HIV activity for the different haplotypes, suggesting that deaminase activity may be an important factor in determining their respective anti-HIV activities. Moreover, mC deamination of A3H displayed a strong preference for the sequence motif of T-mCpG-C/G, which may suggest a potential role in genomic mC modification at the characteristic "CpG" island motif.
APOBEC3H (n class="Gene">A3H) is the most polymorphic member of the APOBEC3 family. Seven haplotypes (hap I-VII) and four mRNA splicing variants (SV) of A3H have been identified. The various haplotypes differ in anti-HIV activity, which is attributed to differences in protein stability, subcellular distribution, and/or RNA binding and virion packaging. Here, we report the first comparative biochemical studies of all the A3H variants using highly purified proteins. We show that all haplotypes were stably expressed and could be purified to homogeneity by Escherichia coli expression. Surprisingly, four out of the seven haplotypes showed high cytosine (C) deaminase activity, with hap V displaying extremely high activity that was comparable to the highly active A3A. Furthermore, all four haplotypes that were active in C deamination were also highly active on methylated C (mC), with hap II displaying almost equal deamination efficiency on both. The deamination activity of these A3H variants correlates well with their reported anti-HIV activity for the different haplotypes, suggesting that deaminase activity may be an important factor in determining their respective anti-HIV activities. Moreover, mC deamination of A3H displayed a strong preference for the sequence motif of T-mCpG-C/G, which may suggest a potential role in genomic mC modification at the characteristic "CpG" island motif.
Keywords:
comparative in vitro functional studies; expression/purification of all APOBEC3H variants; mC deamination activity for mCpG; mC selectivity factor
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