Role of SL1 in FIV gRNA packaging and propagation. (A) Schematic drawing of the previously published secondary structure of FIV packaging signal RNA (Kenyon et al. 2011). The SL1 region with its nucleotide sequences is enlarged and shown as an inset. (B) Description of mutations introduced in SL1. (C) Gel images showing polymerase chain reaction (PCR) amplification of cytoplasmic and viral cDNA with appropriate controls. Panels I and II: PCR amplifications using FIV-specific primers (210 bp). 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 RNA packaging efficiency (RPE) of the mutant transfer vector RNAs using quantitative RT-PCR. (E) The relative RNA propagation efficiency of the mutant RNAs is given as HygR 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) except for the RPE of AD108 where two values were used. Mock transfection cocktail contained only WT transfer vector (TR394) and no packaging construct. Statistical significance: ns, not significant; (*) 0.01 ≤ P < 0.05, (***) 0.0001 ≤ P < 0.001.
