Abstract 1461 A Two-Assay Analysis of Mutational Activity of APOBEC3G, an Anti-HIV Cellular Restriction Factor

APOBEC3G (A3G) is a human cellular restriction factor that inhibits HIV replication. Its endogenous expression in immune cells leads to its packaging during virion assembly after HIV infection, where A3G then interferes with productive reverse transcription in target cells. A3G catalyzes cytidine to uridine mutations in viral reverse transcripts, ultimately rendering the virus noninfectious. A saturating alanine scanning mutagenesis strategy has generated a library of A3G variants; the catalytic activity of these variants was evaluated via a bacterially-based antibiotic-resistance screening assay. In the rifampicin-resistance assay, A3G-mediated mutations in the E. coli rpoB gene lead to antibiotic resistance. Additionally, the antiviral activity of the variants was quantified in a tissue culture-based infectivity assay. This study aimed to elucidate the connection between the two described assays and whether the antibiotic-resistance assay could be used to predict the antiviral activity of A3G in the infectivity assay. To accomplish this, the rpoB gene, where rifampicin-resistant mutations are conferred, and segments of the HIV genome from the human cell culture assay were sequenced following the infectivity assay and antibiotic-resistance assays of the variants. This sequencing data from both assays was compared first to examine the frequencies of mutations made by A3G. Data indicate a significant difference in A3G mutation rate between these two systems, suggesting that an intrinsic difference between the assays affects variant catalytic activity. Furthermore, examination of the nucleotides flanking each cytidine mutation site, or mutational context, revealed that the preferred context for mutation varied between the two systems. Taken together, the results of this study demonstrate that the human cell culture assay provides a better model of overall endogenous A3G function, and the bacterial assay, by contrast, is best utilized as a screening tool for A3G catalytic activity. We thank the biology department at the College of the Holy Cross for their funding and support.