FIG 8.

Analysis of nonspecific RNA packaging by capsids assembled in mammalian and bacterial cells. The indicated WT and mutant HBc expression constructs were transfected into HEK293 cells. (A) Cytoplasmic lysates (5 μl) from transfected cells were resolved on an agarose gel and transferred to a nitrocellulose membrane. Packaged HBc mRNA was detected by ³²P-labeled antisense HBV riboprobe (top) and the capsid (Ca) by the anti-HBc NTD MAb (bottom). (B) Total RNA packaged in capsids in the HEK293 lysates (15 μl), along with the capsid standard purified from E. coli, was also detected by Sypro gold staining and the capsid protein signal detected by Sypro ruby staining following treatment of the lysate with micrococcal nuclease and proteinase K (see Materials and Methods for details) and agarose gel electrophoresis (gel images). Green fluorescent protein (GFP) (lane 2) represented a negative control for the staining that contained the lysate from cells transfected with a GFP-expressing plasmid. Note that duplicate 3E (lanes 7 and 8), 3A (lanes 9 and 10), and 7A (lanes 5 and 6) samples from two separate transfections were loaded. The RNA staining signals normalized to the protein signals are presented in the graph. (C) Capsids purified by sucrose gradient centrifugation from transfected HEK293 cells (lanes 1 to 5) or E. coli (lanes 6 to 12) were resolved on an agarose gel and detected by Sypro ruby staining (top) and their associated nucleic acids by Sybr gold staining (bottom). (D) Nucleic acid isolated from the purified capsids from HEK293 cells (3A, lane 2; 3E, lane 3) or E. coli (lane 4) was isolated and resolved on an agarose gel and detected by Sybr gold staining. The RNA marker and tRNA were also loaded as size standards (lanes 1 and 5, respectively). Ca, capsids; kb, sizes of RNA markers.