Mutational comparison of the single-domained APOBEC3C and double-domained APOBEC3F/G anti-retroviral cytidine deaminases provides insight into their DNA target site specificities

Abstract

Human APOBEC3F and APOBEC3G are double-domained deaminases that can catalyze dC→dU deamination in HIV-1 and MLV retroviral DNA replication intermediates, targeting T–C or C–C dinucleotides, respectively. HIV-1 antagonizes their action through its vif gene product, which has been shown (at least in the case of APOBEC3G) to interact with the N-terminal domain of the deaminase, triggering its degradation. Here, we compare APOBEC3F and APOBEC3G to APOBEC3C, a single-domained deaminase that can also act on both HIV-1 and MLV. We find that whereas APOBEC3C contains all the information necessary for both Vif-binding and cytidine deaminase activity in a single domain, it is the C-terminal domain of APOBEC3F and APOBEC3G that confer their target site specificity for cytidine deamination. We have exploited the fact that APOBEC3C, whilst highly homologous to the C-terminal domain of APOBEC3F, exhibits a distinct target site specificity (preferring Y–C dinucleotides) in order to identify residues in APOBEC3F that might affect its target site specificity. We find that this specificity can be altered by single amino acid substitutions at several distinct positions, suggesting that the strong dependence of APOBEC3-mediated deoxycytidine deamination on the 5′-flanking nucleotide is sensitive to relatively subtle changes in the APOBEC3 structure. The approach has allowed the isolation of APOBEC3 DNA mutators that exhibit novel target site preferences.

Mice Deficient in APOBEC2 and APOBEC3

“…Transfection experiments then revealed that APOBEC3G was packaged into HIV-1 particles and acted to deaminate cytosine to uracil in the first-strand DNA of the HIV replication intermediates (13,18,22,23,49). Similar antilentiviral activities have subsequently been noted for APOBEC3F and APOBEC3C (3,17,20,45,48,51). That APOBEC3F and APOBEC3G can indeed act on HIV-1 during infection of humans is indicated by analysis of the sequences of hypermutated HIV-1 genomes isolated from infected individuals (2, 3, 4, 12, 20, 44).…”

Comparative analysis of the gene-inactivating potential of retroviral restriction factors APOBEC3F and APOBEC3G

“…Flag-tagged A2, A3F and A3G expression plasmids were constructed previously (Langlois et al, 2005). The single-cycle HIV[p8.9] pseudovirus expressing EGFP and the replicative Moloney murine leukaemia virus (M-MLV) expressing EGFP as an Env-EGFP fusion have been previously described (Bélanger et al, 2013;Langlois et al, 2009).…”

“…The single-cycle HIV[p8.9] pseudovirus expressing EGFP and the replicative Moloney murine leukaemia virus (M-MLV) expressing EGFP as an Env-EGFP fusion have been previously described (Bélanger et al, 2013;Langlois et al, 2009). HIV[p8.9] (referred to as HIV-1 DVif pseudovirus in the text) expresses EGFP from an internal spleen focus-forming virus promoter (Langlois et al, 2005). M-MLV expresses EGFP from the viral LTR promoter as a nested fusion protein with Env where it is inserted in the proline-rich region of the protein (Sliva et al, 2004).…”

“…The bases immediately upstream to the deaminated C (underlined) influence this selection. A3G has a strong preference to deaminate a C preceded by a C (59-CC), while all other human A3 proteins, including A3F, prefer to deaminate C preceded by a thymine (59-TC) (Bogerd et al, 2007;Chelico et al, 2006;Hultquist et al, 2011;Langlois et al, 2005;Love et al, 2012;Rausch et al, 2009). Considering that A3-induced deamination events occur predominantly on the minus-strand viral DNA, deamination in a 59-CC context opposite to a tryptophan (Trp) codon (TGG) during reverse transcription produces a TAG termination codon, and also possibly TGA and TAA non-sense codons if the TGG codon is followed by a G (i.e.…”

Flow-cytometric visualization of C>U mRNA editing reveals the dynamics of the process in live cells

“…Transient transfections were performed using Lipofectamine LTX (Invitrogen -Catalog #15338100) and GeneJuice (Novagen -Catalog #70967-3) according to manufacturer's instructions. Lentiviral infections of Caco-2 cells were performed as detailed in Langlois et al 72 All transfections with multiple plasmids were performed using an equal amount of overall plasmid DNA. Differences in the amount of DNA from experimental constructs were filled with an empty construct.…”