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Background:  APOBEC3G provides an innate defense against lentiviruses. Its cytidine deaminase activity, if not fully counteracted by HIV-1 vif, causes G-to-A hypermutation of HIV-1 reverse transcripts. We previously showed that APOBEC3G is expressed at a higher level in CD4⁺ T lymphocytes than peripheral blood mononuclear cells, and that in vitro activation of cells by mitogen, interleukin-4, and T-cell receptor stimulation up-regulates APOBEC3G expression. This study characterized the variability in APOBEC3G RNA expression across different healthy humans and the stability of such expression over time within an individual.

Methods: Repeated samples were collected over time from 15 healthy adult donors (age 22 to 62 years; 6 males and 11 females). Cytoplasmic RNA was extracted immediately after drawing blood from CD4⁺ T cells selected by magnetic beads. APOBEC3G mRNA levels were quantified by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and normalized to gluceraldehvde-3-phosphate dehvdrogenase (GAPDH) RNA. Standard curves used RNA of known copy numbers transcribed in vitro from plasmid DNA. Longitudinal data were analyzed by one-way analysis if variance (ANOVA).

Results:  We first validated the real time RT- PCR assays for APOBEC3G and GAPDH over 420 replicate tests of a specimen by 5 different operators. Coefficient of variation was 28% for each assay. Inter-assay variability (for all variation, including different operators) was 2.7-fold for APOBEC3G and 2.3-fold for GAPDH. The mean level of APOBEC3G RNA was 26,986 copies/10⁶ GAPDH copies ± a standard deviation of 14,649 copies. The range across different individuals at a single time point was 10,488 to 69,111 copies /10⁶ GAPDH copies. This 6.6-fold variation across individuals was significantly above the technical variability of the assay. In contrast, analysis of multiple sequential samples from the same individuals showed that the variation in expression levels over time fell within the 2.7-fold inter-assay variability (p > 0.098, i.e., not significant).

Conclusion:  APOBEC3G RNA levels in primary CD4 lymphocytes vary 6.6 -fold across different humans in vivo, and expression levels are relatively stable over time within an individual. The observed stable inter-individual variation of APOBEC3G RNA across different humans suggests that HIV replication may differ for a given level of vif function. Further study of inter-individual variation in functional APOBEC3G protein, and potential genetic mechanisms underlying this variation, will provide new insight into HIV pathogenesis.

Keywords: Apobec3G; CD4+ lymphocytes; expression level