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.

Figure 1

APOBEC3C has an anti-retroviral effect on MLV. (A) APOBEC3 proteins but not AID or U2AF are packaged in MLV virus-like particles. Virions were produced by co-transfection of 293T cells with M5p, Gag-Pol, VSV-G and Flag-tagged APOBEC expression vectors. Three days after transfection, viruses were isolated from the supernatant by ultracentrifugation and producer cells were collected. Viral pellet and producer cells were then lysed and submitted to western blot analysis using anti-FLAG HRP-conjugated monoclonal antibody. Sample loading was controlled using an anti-p30gag antibody. (B) Upper panel: inhibition of MLV infection by various AID or APOBEC3 proteins. 293T cells were co-transfected with MLV producer plasmids in combinations with either of the FLAG-tagged APOBEC3 or of the AID expression plasmids. Expression of eGFP was measured 48 h later by FACS analysis. Histograms represent the mean of at least three independent experiments and error bars represent the corresponding standard error. Lower panel: titration effect of various FLAG-tag APOBEC3 proteins on MLV infectivity. Increasing APOBEC3/AID:M5P ratios were used to co-transfect 293T cell. Only the amount of APOBEC3/AID plasmid was modified and the total amount of DNA was adjusted with empty pCDNA3.1 vector. EGFP expression was monitored by FACS 48 h post-infection. Each point represents the average of two independent experiments. (C) Upper panel: mutation analysis of the MLV-encoded eGFP gene that successfully integrated into the genome of 293T target cells. Viruses were produced in the presence of APOBEC3C, APOBEC3F or APOBEC3G. Numbers correspond to the frequency in percentage at which the adjacent bases are represented in the mutated sequences. ‘n’ represents the number of independent G→A mutations. Lower panel: sequences of the eGFP gene of MLV amplified from 293T target cells. Vertical lines depict the relative locations of G→A mutations. (D) APOBEC3C, APOBEC3F and APOBEC3G inhibit viral genome integration in target 293T cells. DNA was extracted from 293T cells infected with MLV virus-like particles, digested and a Southern blot analysis was performed using an eGFP cDNA probe to detect viral DNA sequences and a genomic APOBEC2 probe for sample normalization. The ethidium bromide-stained gel before DNA transfer is also shown.

Figure 2

APOBEC3C inhibits HIV infection and is sensitive to Vif. (A) Left panel: inhibition of HIV-1 and HIV-1 (Δvif) by various AID or APOBEC3 proteins. 293T cells were co-transfected with HIV-1 or HIV-1 (Δvif) producer plasmids in combinations with either of the FLAG-tagged APOBEC3 or of the AID expression plasmids. Histograms represent the mean of at least three independent experiments and error bars represent the corresponding standard error. Right panel: titration effect of various FLAG-tagged APOBEC3 proteins on HIV-1 (Δvif) infectivity. Increasing APOBEC3/AID:CSGW ratios were used to co-transfect 293T cell. Each point represents the average of two independent experiments. (B) 293T cells were co-transfected with peGFP-C3, eGFP-APOBEC3C or eGFP-APOBEC3G and plasmids expressing either Vif or ΔVif (Ctrl). Fluorescence was monitored for 5 days by FACS analysis. Each point represents the average and standard error of three independent measurements. ^*Significantly different by Student's t-test (P = 0.007). (C) 293T cells were co-transfected with pVif alone (Ctrl) or pVif and peGFP-C3, eGFP-APOBEC3C or eGFP-APOBEC3G, and cell lysates were prepared 48 h later. The bulk of the lysate was submitted to immunoprecipitation with an anti-eGFP monoclonal antibody. Immunoprecipitates and cell lysates were then submitted to SDS–PAGE and the western blots were probed with anti-Vif monoclonal antibodies. (D) Expression of APOBEC3C, APOBEC3F and APOBEC3G in CEM and CEM-SS cells. An RT–PCR was performed on RNA isolated from either CEM (upper panel) or CEM-SS (lower panel). Results reveal that the CEM-SS clone has lost the expression of APOBEC3C, -3F and -3G.

Figure 3

The second domain of APOBEC3F and APOBEC3G enclose the elements governing deamination spectrum specificity. (A) Western blot analysis of the different chimeric APOBEC3 proteins. An HRP-conjugated anti-eGFP antibody was used to reveal the bands. (B) F1C and F1G2 chimeric APOBEC3 proteins show a strong antiviral effect on MLV virus-like particles. Histograms represent the mean of three independent experiments and error bars represent the standard error. (C and D) Schematic representation of the different chimeric APOBEC3F proteins and their respective deamination spectrum at positions −2 and −1 in relation to the deaminated dC is depicted in pie charts. ‘n’ represents the number of independent G→A mutations.

Figure 4

The deamination spectrum of APOBEC3F is altered by single amino acid substitutions. (A) Sequence alignment of APOBEC3C and the second domain of APOBEC3F. Numbers indicate residues that were changed to generate the 12 mutant APOBEC3F proteins. (B) Histograms depicting the proportion of each nucleotide at positions −2 and −1 in relation to the deaminated dC.