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. 2007 Apr 18;1(2):147–157. doi: 10.1016/j.chom.2007.03.003

Figure 7.

Figure 7

Expression of GFP by rsT3D/S4-GFP

(A) Schematic of pT7-S4GFP. The GFP ORF is flanked by S4 gene nucleotides 1–149 and 769–1196.

(B) RT-PCR analysis of rsT3D and rsT3D/S4-GFP. Viral dsRNA was extracted from purified virions and subjected to RT-PCR using primers specific for T3D S4 and GFP sequences. Numbers delineate the S4 RNA nucleotide position corresponding to the 5′ end of S4-specific primers.

(C) Viral protein expression in cells infected with rsT3D/S4-GFP. L cells were infected with rsT3D or rsT3D/S4-GFP at an MOI of 1 PFU per cell and incubated for 24 hr. Cell lysates were analyzed by immunoblotting using antibodies specific for μNS, σNS, and σ3 proteins.

(D) Image analysis of cells infected with rsT3D/S4-GFP. L cells were infected with rsT3D/S4-GFP, stained with antibodies specific for μNS (blue) and σ3 (red) proteins at 24 hr postinfection, and imaged by confocal laser scanning microscopy. Scale bar, 10 μM.