Journal: Journal of Virology

Article Title: Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes

doi: 10.1128/jvi.00488-22

Figure Lengend Snippet: Design and validation of rotavirus (RV) VP4 and nonstructural protein 3 (NSP3) plasmid constructs used to generate mutant viruses. (A) Schematic showing overall topology of SARS-CoV-2 spike protein in gray boxes: N-terminal domain (NTD), receptor binding domain (RBD), which contains the receptor binding motif (RBM), fusion peptide (FP), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and the intracellular domain (IC) (adapted from Lan et al. ). Dashed lines represent selected regions of the spike protein (colored boxes) that were inserted into the hypervariable region of the SA11 viral protein 4 (VP4) gene (top) and the C terminus of the RF NSP3 gene (bottom). SA11 VP4 was edited to incorporate either NTD, RBM.1, RBM.2, or HR2 spike peptide sequences. RF NSP3 was fused with either the RBD or RBM sequence with or without Thosea asigna virus 2A (T2A) (yellow box), represented by +/− sign. Both gene segments were flanked by the T7 promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (“HDV Rib,” green boxes) followed by T7 terminator (T7T). *Stop codons. Schematic not to scale. (B) Ribbon representation of VP4 (adapted from Settembre et al. ). Two orthogonal views are shown. The VP8* fragment is in magenta extending into the VP5* foot domains (in blue), the hypervariable region of VP4 is in gray and the region where SARS-CoV-2 peptides (omitted for clarity) were inserted is in green. VP5* β-barrel domains are in cyan and purple. (C and D) Coupled in vitro transcription and translation reactions of mutated SA11 VP4 (C) and RF NSP3 (D) segments were carried out using the TnT rabbit reticulocyte lysate system supplemented with [ 35 S]methionine. Samples were analyzed using SDS-PAGE and autoradiography. The molecular weight marker and the expected product sizes of each segment (in brackets) are indicated (kDa). Empty pCDNA 3.1 vector was used as a negative control. In panel D, black asterisks indicate T2A read-through product and red asterisks identify separated products. WT, wild type.

Article Snippet: Coupled in vitro transcription and translation reactions were carried out using the Promega TnT Coupled Reticulate Lysate System labeled with radioactive [ 35 S]methionine (PerkinElmer Inc.) according to the manufacturer’s protocol.

Techniques: Plasmid Preparation, Construct, Mutagenesis, Binding Assay, Sequencing, In Vitro, SDS Page, Autoradiography, Molecular Weight, Marker, Negative Control