Figure 5

Analysis of RNA content in LASV VLP and corresponding transfected HEK-293T/17 cells. RNA was isolated from the total VLP fraction generated in a single 10 cm cell culture dish (~ 6 ×10⁷ cells), and the entire nucleic acid pellet was resolved on denaturing glyoxal agarose gels. RNA from Z3'HIS, Z3'HIS+GPC, Z3'HIS+NP, Z3'HIS+GPC+NP, and Z+GPC+NP (lanes 2, 4, 6, 8, and 10, respectively [V]), and 5 μg of total RNA isolated from the corresponding transfected HEK-293T/17 cells (lanes 1, 3, 5, 7, and 9, respectively [C]) were resolved per lane of a 1.5% gel. Untransfected HEK-293T/17 cell RNA was run alongside test samples as a control (lane 11 [C]). All VLP samples were devoid of rRNAs (28S ~5.5 kbp; 18S ~ 1.8 kbp), but all contained low molecular weight RNA species corresponding in size to tRNAs, approximately 50 - 100 nucleotides in length (lanes 2, 4, 6, 8, 10). Transfected cells producing LASV VLP showed a significant reduction in the 28S rRNA species (lanes 1, 3, 5, 7, 9) when compared to untransfected control cells (lane 11). Ratios of 18S/28S RNA in transfected and untransfected cells, determined by densitometry, are shown below panel A. Molecular weight sizes ranging from 0.5 - 6 kbp are noted to the left of the gel. The positions of cellular 28S and 18S ribosomal RNAs, and tRNA are noted to the right of the gel.