Background:  HIV-1 vif blocks the antiviral activity of human APOBEC3G, a cellular cytosine deaminase.  APOBEC3G encapsidated into the vif-deleted virions deaminates cytosines of the viral reverse transcripts to uracil on the next round of infection.  HIV-1 vif does not block the antiviral activity of primate and mouse APOBEC3G.  Here, we present a detailed study of the mechanism of deamination and the target site specificity of human, mouse and African green monkey APOBEC3G.

Methods:  Wild type and vif^- HIV-1 produced in the presence or absence of APOBEC3G was used to infect cells. The newly synthesized and integrated viral DNA from the infected cells was amplified.  Nucleotide sequences of LTR, gag, pol, env, and nef were determined.

Results:  APOBEC3G deaminated the HIV-1 reverse transcripts over the entire genome.  Deamination occurred in a graded frequency over the genome in a direction corresponding to the predicted length of time during reverse transcription that each region is not double-stranded DNA.  Mutations were most frequent in env and least frequent in U3.  Although mutations are almost exclusively G>A changes in the plus-strand, C>T changes were found in regions where the plus-strand transiently becomes single stranded during reverse transcription.  The consensus target sites for human APOBEC3G deamination in di-, tri-, and quadruple-nucleotide context were determined.  African green monkey APOBEC3G had a consensus target sequence similar to the human enzyme, whereas mouse APOBEC3G was less stringent.  A small number of integrated proviruses was generated from vif^- virions containing human APOBEC3G. The integrated proviruses were heavily mutated and unable to encode functional proteins. Interestingly, most of the integrated proviruses contained mismatched DNA strands, with fewer mutations in the minus strand than in the plus strand.

Conclusions:  The hypermutation rate is related to the length of time that a region is not in double-stranded DNA.  Human APOBEC3G preferentially introduces mutations at consensus target sites, and  APOBEC3G-induced mutations are maintained in the integrated provirus plus-strand.

Keywords: APOBEC3G; Vif; deamination