Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging

The U⁸⁵CUG⁸⁸ stretch plays a crucial role in FIV gRNA packaging. (A) Schematic drawing of the previously published secondary structure of FIV packaging signal RNA (Kenyon et al. 2011). Position of LRI, U⁸⁵CUG⁸⁸ and its complementary strand sequences are enlarged and shown as an inset. (B) Description of mutations introduced in U⁸⁵CUG⁸⁸ and its complementary strand. (C) Gel images showing PCR amplification of cytoplasmic and viral cDNAs with appropriate controls. PCR amplifications using FIV-specific primers (210 bp) on panel I: cyt and panel II: viral cDNAs. Panel III: Multiplex PCR amplifications performed on cytoplasmic cDNAs using primers/competimer for 18S rRNA (324 bp) and unspliced β-actin mRNA (200 bp). (D) Relative RPE of the mutant transfer vector RNAs using quantitative real-time RT-PCR. (E) The relative RNA propagation efficiencies of the mutant RNAs are shown as Hyg^R CFU/mL normalized to the transfection efficiency. The data shown in the histograms are from a minimum of three independent experiments (shown as open circles) performed in triplicate (±SD). Mock transfection cocktail contained only WT transfer vector (TR394) and no packaging construct. Statistical significance: ns, not significant; (*) 0.01 ≤ P < 0.05, (**) 0.001 ≤ P < 0.01, (***) 0.0001 ≤ P < 0.001.