ABSTRACT
APOBEC3G (APO3G), a cytidine deaminase with two zinc finger domains, inhibits
human immunodeficiency virus type 1 replication in the absence of Vif.
Here, we provide a comprehensive molecular analysis of the deaminase
and nucleic acid binding activities of human APO3G using a pure system
containing only one protein component, i.e., highly purified,
catalytically active enzyme expressed in a baculovirus system. We
demonstrate that APO3G deaminates cytosines in single-stranded DNA
(ssDNA) only, whereas it binds efficiently to ssDNA and ssRNA, about
half as well to a DNA/RNA hybrid, and poorly to double-stranded DNA and
RNA. In addition, the base specificities for deamination and binding of
ssDNA are not correlated. The minimum length required for detection of
APO3G binding to an ssDNA oligonucleotide in an electrophoretic
mobility shift assay is 16 nucleotides. Interestingly, if nucleocapsid
protein and APO3G are present in the same reaction, we find that they
do not interfere with each other's binding to RNA and a complex
containing the RNA and both proteins is formed. Finally, we also
identify the functional activities of each zinc finger domain. Thus,
although both zinc finger domains have the ability to bind nucleic
acids, the first zinc finger contributes more to binding and APO3G
encapsidation into virions than finger two. In contrast, deamination is
associated exclusively with the second zinc finger. Moreover, zinc
finger two is more important than finger one for the antiviral effect,
demonstrating a correlation between deaminase and antiviral
activities.
Proteomimetic zinc finger domains with modified metal‐binding
β‐turns
