G to A hypermutation in viral DNA of Δvif viruses from non-permissive cells or semi-permissive cells. a, The DNA sequence alignments in the U3-R region. The clones with the most-identical sequences from the total clones (n = 8) are listed. The newly synthesized viral DNA are listed in the top panel. The rows are: (1) wild-type viruses from H9 cells to infect C8166 cells; (2) Δvif viruses from H9 cells to infect C8166 cells; (3) Δvif viruses from H9 cells to process ERT; (4) wild-type viruses from SupT1 cells to infect C8166 cells; (5) Δvif viruses from SupT1 cells to infect C8166 cells. After two passages, the viral DNA in C8166 cells was sequenced (bottom panel). The rows are: (6) wild-type viruses; (7) Δvif viruses; (8) Δvif viruses passaged in SupT1 cells. b, Comparison of mutation frequency of G to A in the newly synthesized DNA of viruses generated from H9 or SupT1 cells (301 nucleotides in U3-R-U5 region, n = 8). The G to A mutation frequency of Δvif viruses from H9 cells to infect C8166 cells is significantly higher than that of others (asterisk, P < 0.001, t-test). WT, wild type. c, Comparison of the G to A mutation frequency in the viral DNA after passage in C8166 cells (n = 8). The G to A mutation frequency of Δvif viruses passaged in C8166 is significantly higher than that of wild-type viruses (asterisk, P < 0.001, t-test).

G to A hypermutation in newly synthesized DNA of viruses generated from cells containing CEM15. a, DNA sequence alignment in HIV-1 U3-R region. The clones with the most-identical sequences from the total clones (n = 8) are listed. Viruses were from 293T (top) cells or passaged in SupT1 (bottom) cells. In the top panel, the rows are: (1) wild-type viruses, with CEM15, to infect C8166 cells; (2) wild-type viruses, with CEM15, to process ERT; (3) wild-type viruses, without CEM15, to infect C8166 cells; (4) Δvif viruses, with CEM15, to infect C8166 cells; (5) Δvif viruses, with CEM15, to process ERT; (6) Δvif viruses, without CEM15, to infect C8166 cells. In the bottom panel, the rows are: (7) wild-type viruses, without CEM15; (8) wild-type viruses, with CEM15. b, Comparison of G to A mutation frequency in newly synthesized DNA (301 nucleotides in U3-R-U5 region, n = 8). The G to A mutation frequency of Δvif viruses from 293T or SupT1 cells containing CEM15 is significantly higher than that of others (asterisk, P < 0.001, t-test). c, Comparison of G to A mutation frequency in the viral DNA after passage (n = 8). The G to A mutation frequency of wild-type viruses passaged in SupT1 cells containing CEM15 is significantly higher than that in SupT1 cells without CEM15 (asterisk, P < 0.001, t-test). d, The wild-type virions, after various passages in SupT1 cells containing CEM15, were purified. After normalization with HIV-1 p24, the virion-associated Vif protein was detected by western blot.

CEM15 has cytidine deaminase activity in vitro. a, GST–CEM15, but not GST alone, converts deoxycytidine to deoxyuridine in vitro in a concentration-dependent manner. This figure represents five independent experiments. b, Mutations at zinc finger domains significantly decrease cytidine deaminase activity (asterisk, P < 0.001, t-test). THU significantly inhibits cytidine deaminase activity (asterisk, P < 0.001, t-test).

CEM15 without cytidine deaminase activity can neither inhibit the infectivity nor induce hypermutation in the newly synthesized DNA of Δvif viruses. pNL4-3 or pNL4-3Δvif were transfected into 293T cells with CEM15 or CEM15 mutants. a, The viruses were used to infect HLCD4-CAT cells. CAT assays were performed. This figure represents three independent experiments. b, The newly synthesized viral DNA in C8166 cells was amplified by PCR and sequenced. The G to A mutation frequency of Δvif viruses from 293T cells containing mutant CEM15 genes is significantly lower than that containing wild-type CEM15 (asterisk, P < 0.01, t-test).