The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA. CEM15, also known as APOBEC3G, is an endogenous inhibitor of HIV-1 replication and functions as a DNA mutator. It induces G to A hypermutation in newly synthesized viral DNA, which can be counteracted by the HIV-1 virion infectivity factor (Vif). This hypermutation may lead to lethal mutations or instability in incoming viral reverse transcripts, contributing to the Vif-defective phenotype. The accumulation of CEM15-mediated non-lethal hypermutation in the viral genome can significantly contribute to the genetic variation of primate lentiviruses. HIV-1 Vif is essential for viral replication in vivo and in some non-permissive cells. Vif-defective viruses from non-permissive cells cannot complete reverse transcription or the newly synthesized DNA cannot persist in target cells. CEM15 is an endogenous inhibitor of HIV-1 present only in non-permissive cells and is counteracted by Vif. CEM15 shares homology with other cytidine deaminases that edit RNA and can edit HIV-1 genomic or spliced RNA. Sequencing of HIV-1 RNA from Δvif virions showed some mutations, but no mutations were found in spliced RNA. CEM15, along with AID and APOBEC1, can act as a DNA mutator in E. coli. Both CEM15 and Vif can be packaged into HIV-1 virions. The study investigated whether newly synthesized HIV-1 DNA is a substrate for CEM15. The presence of Vif reduced the G to A substitutions in Δvif viruses. The mutation frequency in various regions was similar. The pattern of G to A hypermutation in Δvif viruses from H9 cells was consistent with that in wild-type viruses in cell culture and in vivo. The hypermutation occurs preferentially in GpA or GpG dinucleotide contexts or in groups of G nucleotides. Other substitutions were found but with lower frequency. CEM15 was shown to have cytidine deaminase activity, which can be inhibited by tetrahydrouridine (THU). CEM15 contains two zinc finger domains, which are important for its deaminase activity. Mutations in these domains significantly reduced deaminase activity. CEM15 was found to induce G to A hypermutation in newly synthesized viral DNA through its deaminase activity. The study also showed that CEM15 can induce non-lethal hypermutation in the viral DNA of wild-type viruses in long-term culture. CEM15 does not significantly affect dNTP pools in SupT1 cells. The study suggests that CEM15 may directly deaminate dC in newly synthesized viral DNA, converting it to dU, whichThe cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA. CEM15, also known as APOBEC3G, is an endogenous inhibitor of HIV-1 replication and functions as a DNA mutator. It induces G to A hypermutation in newly synthesized viral DNA, which can be counteracted by the HIV-1 virion infectivity factor (Vif). This hypermutation may lead to lethal mutations or instability in incoming viral reverse transcripts, contributing to the Vif-defective phenotype. The accumulation of CEM15-mediated non-lethal hypermutation in the viral genome can significantly contribute to the genetic variation of primate lentiviruses. HIV-1 Vif is essential for viral replication in vivo and in some non-permissive cells. Vif-defective viruses from non-permissive cells cannot complete reverse transcription or the newly synthesized DNA cannot persist in target cells. CEM15 is an endogenous inhibitor of HIV-1 present only in non-permissive cells and is counteracted by Vif. CEM15 shares homology with other cytidine deaminases that edit RNA and can edit HIV-1 genomic or spliced RNA. Sequencing of HIV-1 RNA from Δvif virions showed some mutations, but no mutations were found in spliced RNA. CEM15, along with AID and APOBEC1, can act as a DNA mutator in E. coli. Both CEM15 and Vif can be packaged into HIV-1 virions. The study investigated whether newly synthesized HIV-1 DNA is a substrate for CEM15. The presence of Vif reduced the G to A substitutions in Δvif viruses. The mutation frequency in various regions was similar. The pattern of G to A hypermutation in Δvif viruses from H9 cells was consistent with that in wild-type viruses in cell culture and in vivo. The hypermutation occurs preferentially in GpA or GpG dinucleotide contexts or in groups of G nucleotides. Other substitutions were found but with lower frequency. CEM15 was shown to have cytidine deaminase activity, which can be inhibited by tetrahydrouridine (THU). CEM15 contains two zinc finger domains, which are important for its deaminase activity. Mutations in these domains significantly reduced deaminase activity. CEM15 was found to induce G to A hypermutation in newly synthesized viral DNA through its deaminase activity. The study also showed that CEM15 can induce non-lethal hypermutation in the viral DNA of wild-type viruses in long-term culture. CEM15 does not significantly affect dNTP pools in SupT1 cells. The study suggests that CEM15 may directly deaminate dC in newly synthesized viral DNA, converting it to dU, which