sf 9 insect cells  (ATCC)

Journal: Nucleic Acids Research

Article Title: Nodavirus protein A’s interdomain elbow controls RNA replication organelle formation and function

doi: 10.1093/nar/gkag151

Figure Lengend Snippet: Global alanine-scanning mutagenesis of the protein A elbow identifies amino acid contributions to RNA replication. ( A–C ) Alanine substitutions were introduced across the 17-amino acid elbow (aa 379–395) as blocks (5 alanines, A), pairs (2 alanines, B), or single residues (1 alanine, C). Top panels: western blot detecting protein A, with tubulin as a loading control. Middle panels: northern blot analysis of RNA1 and RNA3 replication following co-transfection of mutant protein A plasmids with the RNA1 fs template. Bottom panels: bar graphs summarizing RNA3 replication relative to wt control across three or more experimental replicates. ( D ) Summary diagram mapping replication values from block, pair, and single alanine substitutions onto the elbow sequence. Color-coded gradients indicate functional impact: white represents mutations that abolish RNA3 replication (0%), and blue represents full replication comparable to wt (100%), allowing visualization of residues and segments critical for RNA replication. ( E ) Structure mapping of elbow (aa 379–395) characteristics. The surface diagrams use the indicated color gradients to show RNA3 replication levels (% of wt) induced by single alanine substitutions [gradient as in panel (D)], electrostatic potential (negative: red, positive: blue), and hydrophobicity (hydrophilic: blue, hydrophobic: yellow) to highlight functional features. Gray shading of the arrows indicates amino acids facing the back, while white shading indicates amino acids facing the front.

Article Snippet: Transfection mixtures were prepared by complexing 1.5 μg of total plasmid DNA (1 μg of RNA1 template plasmid and 0.5 μg of protein A expression plasmid[s]), 6 μl of Trans IT-Insect Transfection Reagent (Mirus Bio), and 100 μl of Opti-MEM (Gibco/ThermoFisher).

Techniques: Mutagenesis, Western Blot, Control, Northern Blot, Cotransfection, Blocking Assay, Sequencing, Functional Assay

Journal: Nucleic Acids Research

Article Title: Nodavirus protein A’s interdomain elbow controls RNA replication organelle formation and function

doi: 10.1093/nar/gkag151

Figure Lengend Snippet: Plasmid-based trans -RNA replication assay. ( A ) Schematic of the trans -RNA replication assay in Drosophila S2 cells. Co-transfection of two plasmids separates protein A expression from RNA replication template functions: the left protein A plasmid expresses functional protein A from a nonreplicable mRNA lacking viral 5′ and 3′ replication signals, and the right RNA1 fs plasmid expresses a full-length RNA1 template containing an early frameshift (fs) to prevent translation of protein A. This approach largely stabilizes expression levels of different protein A mutants and allows more direct assessment of their effects on RNA replication, measured by genomic RNA1 and subgenomic RNA3 accumulation. Color coding follows Fig. . ( B ) Northern blot analysis validating the trans -RNA replication assay, with RNA and protein collected ∼65 h post-transfection. The top panel shows a western blot detecting protein A, with tubulin as a loading control. The bottom panel shows the northern blot: Lanes 1 and 2 show no RNA1 or RNA3 signals when either the protein A plasmid or RNA1 fs plasmid is transfected alone. Lane 3 shows strong replication of both genomic RNA1 and RNA3 when wtwt protein A is co-expressed with the RNA1 fs template, confirming robust replication in trans . Lane 4 shows that co-expression of the RNA1 fs template with a protein A deletion mutant lacking the 17–amino acid elbow (Δ379–395) completely abolishes RNA replication, confirming that the elbow region is essential for FHV RNA replication.

Article Snippet: Transfection mixtures were prepared by complexing 1.5 μg of total plasmid DNA (1 μg of RNA1 template plasmid and 0.5 μg of protein A expression plasmid[s]), 6 μl of Trans IT-Insect Transfection Reagent (Mirus Bio), and 100 μl of Opti-MEM (Gibco/ThermoFisher).

Techniques: Plasmid Preparation, Cotransfection, Expressing, Functional Assay, Northern Blot, Transfection, Western Blot, Control, Mutagenesis