Biochemical Characterization of APOBEC3H Variants: Implications for Their HIV-1 Restriction Activity and mC Modification

- We purified the proteins of all four SVs and seven haplotypes (Hap I-VII) of A3H in large quantity from E. coli expression system.
- We showed that three of the four SVs and three haplotypes (hap II, V, VII) all have very high deamination activity on C, with hap I showing ~ 1/10 of the activities of other three haplotypes.
- We revealed that for A3H hap II, the deamination activity on mC is almost as efficient as on C for the DNA substrates containing -mCpG- or -CpG- motif.
- We showed that the deaminase activity of different A3H variants correlates well with their reported anti-HIV activity, indicating the importance of deaminase activity for HIV restriction.

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.

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