The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA

Abstract

High mutation frequency during reverse transcription has a principal role in the genetic variation of primate lentiviral populations. It is the main driving force for the generation of drug resistance and the escape from immune surveillance. G to A hypermutation is one of the characteristics of primate lentiviruses, as well as other retroviruses, during replication in vivo and in cell culture. The molecular mechanisms of this process, however, remain to be clarified. Here, we demonstrate that CEM15 (also known as apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G; APOBEC3G), an endogenous inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, is a cytidine deaminase and is able to induce G to A hypermutation in newly synthesized viral DNA. This effect can be counteracted by the HIV-1 virion infectivity factor (Vif). It seems that this viral DNA mutator is a viral defence mechanism in host cells that may induce either lethal hypermutation or instability of the incoming nascent viral reverse transcripts, which could account for the Vif-defective phenotype. Importantly, the accumulation of CEM15-mediated non-lethal hypermutation in the replicating viral genome could potently contribute to the genetic variation of primate lentiviral populations.

Figure 1

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).

Figure 2

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.

Figure 3

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).

Figure 4

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).