DNA mutagenic activity and capacity for HIV-1 restriction of the cytidine deaminase APOBEC3G depend on whether DNA or RNA binds to tyrosine 315

Abstract

APOBEC3G (A3G) belongs to the AID/APOBEC protein family of cytidine deaminases (CDA) that bind to nucleic acids. A3G mutates the HIV genome by deamination of dC to dU, leading to accumulation of virus-inactivating mutations. Binding to cellular RNAs inhibits A3G binding to substrate single-stranded (ss) DNA and CDA activity. Bulk RNA and substrate ssDNA bind to the same three A3G tryptic peptides (amino acids 181-194, 314-320, and 345-374) that form parts of a continuously exposed protein surface extending from the catalytic domain in the C terminus of A3G to its N terminus. We show here that the A3G tyrosines 181 and 315 directly cross-linked ssDNA. Binding experiments showed that a Y315A mutation alone significantly reduced A3G binding to both ssDNA and RNA, whereas Y181A and Y182A mutations only moderately affected A3G nucleic acid binding. Consistent with these findings, the Y315A mutant exhibited little to no deaminase activity in an DNA mutator reporter, whereas Y181A and Y182A mutants retained ∼50% of wild-type A3G activity. The Y315A mutant also showed a markedly reduced ability to assemble into viral particles and had reduced antiviral activity. In uninfected cells, the impaired RNA-binding capacity of Y315A was evident by a shift of A3G from high-molecular-mass ribonucleoprotein complexes to low-molecular-mass complexes. We conclude that Tyr-315 is essential for coordinating ssDNA interaction with or entry to the deaminase domain and hypothesize that RNA bound to Tyr-315 may be sufficient to competitively inhibit ssDNA deaminase-dependent antiviral activity.

Modeling the Embrace of a Mutator: APOBEC Selection of Nucleic Acid Ligands

“…The addition of RNA to an oligomeric complex of A3G assembled on ssDNA induced dissociation of A3G as a homodimer free of nucleic acid [88] . The data suggested a model wherein the mechanism for RNA inhibition of A3G ssDNA binding and catalytic deamination involves competitive RNA binding to Y315 within CD2 [90] .…”

“…The A3F CD2 positively charged surface patch is not conserved in other A3 members, but CD1 of A3G is nearly entirely positively charged and there is a growing body of evidence showing ssDNA binding by A3G CD1 is essential for the catalytic activity of CD2. First, A3G has been shown through mass spectroscopy (MS) of DNA crosslinked A3G to directly bind to DNA through at least three residues (Y181 and Y182 in CD1 domain and Y315 in CD2) [90] . Alanine substitutions at Y181 or Y182 reduced deaminase activity to half that of wild type (WT) A3G and A3G with an alanine substitution at Y315 had little or no activity [90] .…”

“…First, A3G has been shown through mass spectroscopy (MS) of DNA crosslinked A3G to directly bind to DNA through at least three residues (Y181 and Y182 in CD1 domain and Y315 in CD2) [90] . Alanine substitutions at Y181 or Y182 reduced deaminase activity to half that of wild type (WT) A3G and A3G with an alanine substitution at Y315 had little or no activity [90] . Second, atomic force microscopy (AFM) experiments showed A3G may bind ssDNA in different modes depending on DNA length and each involved binding to both CD1 and CD2 [91] .…”

“…MS analysis of tryptic RNA- or DNA-crosslinked A3G peptides revealed that Y315 of A3G CD2 bound to ssRNA as well as ssDNA [90] . As discussed above, A3G Y315A mutants had little or no ability to bind RNA or DNA and were inefficient in assembling ribonucleoprotein (RNP) particles or hypermutating ssDNA [90] . RNA binding by A3G directly inhibited its ability to bind DNA [98] .…”

Structural Insights into APOBEC3-Mediated Lentiviral Restriction

“…A 1.9 Å crystal structure of soluble A3B NTD mutant showed two positively-charged amino acid patches around the NTD domain that likely bind nucleic acid through electrostatic interactions and facilitate cytidine deamination [ 117 ]. In addition, the positively charged CD1 of A3G has been shown to crosslink ssDNA via mass spectrometry and mutations Y181A/Y182A have reduced deaminase activity [ 209 ]. The 2.0 Å co-crystal structure of rhesus A3G-CD1 bound to poly-dT ssDNA shows strong ssDNA-binding affinity, which is likely due to the positively-charged surface of rhesus A3G [ 113 ].…”