codon optimized t7 polymerase  (Addgene inc)

Journal: PLoS Pathogens

Article Title: Recombinant Lloviu virus as a tool to study viral replication and host responses

doi: 10.1371/journal.ppat.1010268

Figure Lengend Snippet: (A) Schematics of successfully rescued rLLOV comp full-length clones. Noncoding regions are indicated in light gray, LLOV ORFs are in gray, a ZsGreen-P2A reporter (ZsG) is in light green, red bars and green triangles indicate the GE and GS signals in the IR ins insertion in the VP24-L intergenic region, green indicates EBOV sequences (EBOV UTR+tr ), pink indicates short EBOV trailer sequences (LE 72 ) and blue indicates MARV sequences (MARV UTR+tr ). rLLOV comp clones are to scale except the T7 RNA polymerase promoter (P T7 ), hepatitis δ ribozyme (Rib HdV ), T7 RNA polymerase terminator sequences (T T7 ), and IR ins , which are enlarged for clarity. (B-D) Growth curve of rEBOV, rRESTV, and the indicated versions of rLLOV comp using an initial MOI of 0.1 in Vero E6 (B), SuBK12-08 (C), and Huh7 cells (D). (E) Proteomic analysis of LLOV proteins expressed in SuBK12-08 cells infected with rLLOV-ZsG-IR ins -EBOV UTR+tr at a multiplicity of infection (MOI) of 1 at two days post-infection (dpi). Signal intensities of viral proteins are plotted relative to L signal intensity within the same sample. (F) RNA FISH analysis of Vero E6 cells infected with rLLOV-IR ins -MARV UTR+tr at an MOI of 1 at 1 dpi. Red, negative sense genomic LLOV RNA clustered in viral inclusions. Cell nuclei were stained with DAPI (blue).

Article Snippet: A plasmid expressing a codon-optimized version of T7 RNA polymerase was synthesized (GeneArt) and the pMIR β-gal plasmid was a kind gift from Matthew Jones, Boston University.

Techniques: Clone Assay, Infection, Staining