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.
Branco et al. Virology Journal 2010 7:279 doi:10.1186/1743-422X-7-279