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. 2015 Mar 18;10(3):e0119625. doi: 10.1371/journal.pone.0119625

Fig 2. The singly-spliced RNA encodes a 21.5 kD viral protein that inhibits HBV replication.

Fig 2

(A) Top panel, schematic of the structure and open reading frames of the singly-spliced RNA. Lower panel, schematic of the plasmid constructs. pShSS/R, a plasmid expressing full-length singly-spliced RNA. pShSScore-/R, a plasmid expressing singly-spliced RNA with a nonsense mutation within the precore/core ORF. pShPC-1/R, a plasmid expressing singly-splicedRNA with 3' UTR truncation. pShSS_PCmut/R, a plasmid derived from pShPC-1/R in which the ATG of precore ORF was mutated to GTG. pShSS_Cmut/R, a plasmid derived from pShPC-1 in which the ATG of core ORF was mutated to GTG. (B) and (C) Huh7 cells were transfected with the indicated plasmids for three days, and total cell lysate was harvested. In the top two panels, the expression levels of nucleocapsid were analyzed by particle blot analysis. The encapsidated nucleic acid was revealed by hybridizing with an HBx-specific probe. In the lower two panels, the expression levels of HBV core protein and GFP protein were examined by western blot analysis. (C) Lane 1: p1.3HBcl/Hyg and pShSS/R; lane 2: p1.3HBcl/Hyg and pShPC-1/R; lane 3: p1.3HBcl/Hyg and pShShuttle/R (Mock); lane 4: p1.3HBcl/Hyg and pShSS_PCmut/R; lane 5: p1.3HBcl/Hyg and pShSS_Cmut/R.