Effects of untranslated-leader and gag-coding region mutations on viral RNA encapsidation. Equivalent amounts of virus derived from transfected 293T cells, based on levels of p27-CAantigen, were used to prepare viral RNA that was then used as template for quantitative RT-PCR to detect full-length viral RNA genome in an 18-cycle PCR reaction . Relative amounts of a 114-bp DNA product were quantified by molecular imaging, with wild-type values arbitrarily set at 1.0. Reactions run with RNA template, digested by DNase-free RNase, served as a negative control for each sample to exclude any potential DNA contamination. Relative amounts of viral RNA that were packaged were determined on the basis of four different experiments. A. RT-PCR vRNA packaging results of SD2 variants harboring compensatory mutations in the DIS (A423G), CA (K197R) and p6 (G49K) regions. B. RT-PCR vRNA packaging results of SD2 variants harboring mutations in the DIS (A423G), and NC (E18G and G31K) regions.