Novel reporter constructs to accelerate antiviral and therapeutic discovery for Enterovirus-A71

Figures

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  Fig. 1.. Generation of fluorescent and luminescent EV-A71 replicon system (a) Schematic of the EV-A71 replicon system with an insertional handle positioned directly after the start codon, replacing the P1 region. This insertional handle enables the insertion of any reporter of interest at the target site. In vitro transcribed RNA generated from the replicon plasmid was transfected into NLS-BFP RD cells and measured using the BioTek Synergy Neo2. (b) Diagram showing the details of the insertional handle sequence (red). The BsaI restriction sites within the handle are highlighted in the DNA sequence. Cleavage of the sequence will remove and replace the 4 bolded residues in the amino acid residue sequence with an insert of interest, engineered with matching overhang sequences (shown with black lines). The reporter gene is followed by 30 base pairs (10AA) of sequence N-terminal to the P1–P2 cleavage site to ensure proper cleavage of the reporter from the replication proteins. (c) Time-course plot showing fluorescence and luminescence units as a function of time for the different replicons. The dynamic range (DR) is calculated by subtracting the minimum detected signal from the maximum signal.…

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  Fig. 2.. Engineering and characterization of genetically stable fluorescent and luminescent EV-A71 infectious systems (a) Schematic illustrating the EV-A71 infectious clone system with an insertional handle, enabling the insertion of reporter genes at the N-terminus of 2A. The schematic highlights the modularity of the system and the placement of the reporter insertion site. In vitro transcribed RNA generated from the infectious clones was transfected into RD cells to produce virus. (b) Diagram showing the details of the insertional handle sequence (red) engineered after the first residue of the 2A protein. The BsaI restriction sites within the handle are highlighted in the DNA sequence. Cleavage of the sequence will remove and replace the 4 bolded residues in the amino acid residue sequence with an insert of interest, engineered with matching overhang sequences. (c) Schematic of rescue and passage experiment assess fitness and stability. Viral RNA was transfected into RD cells and virus was collected after X hours (P0). 20% of viral lysate was passaged to the next plate, the process repeated for three passages (P1–3). (d) Bubble plot showing viral titers (color of circle) and reporter…

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  Fig. 3.. Characterization of the luminescent EV-A71 infectious system (a) Structure prediction of the NanoLuc-2A fusion protein. The active site of the 2A protease is shown in stick mode. Furimazine, the substrate for the NanoLuc luciferase, is shown as magenta sticks. (b) Schematic of the experimental outline for infection and measurement of luminescence. (c) Time-course plot showing the increase in luminescent units (yellow curve) over time. The black points represent uninfected cells or conditions treated with replication inhibitors. (d) Schematic showing the experimental pipeline for infection of human spinal organoids and measurement of luminescence. (e) Time-course plot comparing NanoLuc-2A virus (yellow points) luminescence with infected cells treated with Rupintrivir (grey points) and uninfected cells (black points).

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  Fig. 4.. Characterization of the fluorescent EV-A71 infectious system (a) Schematic of the experimental outline for infection, followed by imaging and image analysis, of NLS-BFP RD cells with fluorescent viruses. (b) Images from BFP, GFP, and RFP channels showing uninfected cells and cells infected with panel of engineered fluorescent viruses. The BFP channel shows the blue fluorescent protein with a nuclear localization signal, which is stably expressed in NLS-BFP RD cells. (c) Box plot showing the mean intensity of fluorescence in uninfected cells and cells infected with different fluorescent viruses. (d) Box plot showing the GFP/BFP ratio for the different conditions. (e) Box plot showing the RFP/BFP ratio for the different conditions. (f) Structure prediction of the mFAP10–2A fusion protein. DFHBI-1T is the substrate of mFAP10 (g) Frame captures from a time-lapse video showing NLS-BFP RD cells infected with the mNeonGreen-2A virus. (h) Experimental pipeline for infection of human spinal organoids with the mFAP10–2A virus. (i) GFP channel images of human spinal organoids showing non infected cells, cells infected with mFAP10, and cells infected with mFAP10 and treated with Rupintrivir.