Fig 2: A synthetic dimer of APOBEC3C has increased antiviral activity.(A) Cartoon schematic showing the sequence of the double-domain APOBEC3C. Restriction of HIV-1 Δvif by APOBEC3G(A3G), APOBEC3C S188 (A3C S188), APOBEC3C I188 (A3C I188) and the double-domain APOBEC3C (S188-S188). 0.3μg APOBEC3 was used in this assay. Error bars represent standard deviation of four independent transfections and infections. A Western blot for expression of the APOBEC3 proteins is shown, and is representative of three experiments. (B) Packaging of APOBEC3C variants. Left side: Intracellular expression. Right side: Proteins in the pelleted virions. The intracellular blot was probed with antibody to the HA tag and with antibody to tubulin. The virion pellet blot was probed with antibody to the HA tag and with an antibody to p24gag. A background band in the virion blot with the HA antibody is marked with an asterisk. The single domain APOBEC3C proteins are marked with a solid arrow, while the synthetic double domain APOBEC3C is marked with an open arrow. Relative quantitation of the amounts of APOBEC3C is shown under the panels. An HIV provirus is transfected in each condition; Lanes 1, no APOBEC3; lanes 2, APOBEC3C I188; lanes 3, APOBEC3C S188; lanes 4; the double-domain APOBEC3C (S188-S188).

Fig 3: APOBEC3C is targeted by Vif.(A) Restriction of HIV-1 Δvif by APOBEC3C S188 (A3C S188), APOBEC3C I188 (A3C I188), and full recovery of infectivity by the presence of HIV-1 (LAI strain) and HIV-2 (ROD strains) vif. 0.4μg of APOBEC3 plasmid was used for each condition, and 0.6μg of each provirus was used. Infectivity of each virus is set to 100% for infection with No APOBEC3 (No A3) present. Error bars represent standard deviation of three independent transfections and infections. Black bars indicate no Vif, orange bars indicate HIV-1 (LAI) Vif, and green bars indicate HIV-2 (ROD) Vif (B) Vif degradation of APOBEC3C S188 and I188 was detected by Western blot analysis. 0.4μg of APOBEC3C and 0.6μg of an HIV-1 provirus (either Δvif, lane labelled as (-)) or containing HIV-1 vif or containing HIV-2 vif, lanes labelled as HIV-1 or HIV-2) were used to transfect 293T cells and lysates were probed for APOBEC3C expression. Tubulin was used as a loading control.

Fig 4: In vitro characterization of APOBEC3C S188 and I188.(Top) The specific activity of APOBEC3C S188 and I188 was determined by incubating the enzyme with a 118 nt ssDNA substrate with an internal fluorescein label (yellow star) and 2 possible sites for cytidine deamination (marked as “C”). Single deaminations of the 5'C and 3'C are detected as the appearance of fluorescently labeled 100 nt and 81 nt fragments, respectively; double deamination of both C residues on the same molecule results in a 63 nt labeled fragment. Substrate usage is quantified for below each lane of the gels. (Bottom) The substrate usage during a 60 min time course was plotted from three independent experiments (bottom left) and used to calculate the specific activity of the enzymes (bottom right).

Fig 5: Isoleucine 188 changes to serine in some hominoids but was maintained or reverted back to isoleucine for some human populations.(A) A phylogram of catarrhines, along with an alignment of the C-terminus of APOBEC3C. All old world monkey sequences contained isoleucine at 188 (15 sequences total). (B) Allele frequency of Ile SNP in global human populations, as well as Homo neanderthalensis. (C) Allele frequency of Ile 188 in chimpanzees, bonobos, gorillas, and orangutans (n = 79). Sequences were derived from the Great Ape Genome Project[39].