Biochemical activities of highly purified, catalytically active human APOBEC3G: correlation with antiviral effect.
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.
Iwatani
Yasumasa
Y
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, MD 20892-2780, USA.
Takeuchi
Hiroaki
H
Strebel
Klaus
K
Levin
Judith G
JG
D000071480:APOBEC-3G Deaminase; D000998:Antiviral Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics; D002384:Catalysis; D003001:Cloning, Molecular; D003564:Cytidine Deaminase; D004277:DNA, Single-Stranded / Q000378:metabolism; D006801:Humans; D009700:Nucleoside Deaminases / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D009841:Oligonucleotides / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D014771:Virion / Q000378:metabolism; D016335:Zinc Fingers
D000998:Antiviral Agents; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D012097:Repressor Proteins; D012313:RNA; D009700:Nucleoside Deaminases; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase