Abstract
The influenza epidemic is a huge burden to public health. Current influenza vaccines provide limited protection against new variants due to frequent mutation of the virus. The continual emergence of novel variants necessitates the method rapidly monitoring influenza virus infection in experimental systems. Although several replication-competent reporter viruses carrying fluorescent proteins or small luciferase have been generated in previous studies, visualizing influenza virus infection via such strategy requires reverse genetic modification for each viral strain which is usually time-consuming and inconvenient. Here, we created a novel influenza A nucleoprotein (NP) dependent reporter gene transcription activation module using NP-specific nanobodies. Our results demonstrated the modular design allowed reporter genes (mNeonGreen fluorescent protein and Gaussia luciferase) specifically expressing to detect intracellular NP protein, and therefore acts as a universal biosensor to monitor infection of various influenza A subtypes in living cells. The new system may provide a powerful tool to analyze influenza A infections at the cellular level to facilitate new antiviral drug discovery. Moreover, this approach may easily extend to develop live-cell biosensors for other viruses.
Highlights
The influenza epidemic is a huge burden to public health
The expression of the reporter genes are dependent on the recruitment of the transactivation domain (AD) to the activating sequence upstream by DNA binding domain (DBD) via NP-specific nanobody mediated double-sandwich scaffold
In order to utilize intracellular NP protein as a dimerizer scaffolding DBD and AD domains, DBD-VHH and V16AD-VHH fusion constructs with various NP nanobodies report previously (NP121, NP52, NP77, NP135, NP170, NP296, NP35522 and NP5423) were screened in pair-wise combinations for NP-dependent activation of an upstream activating sequence-regulated EGFP-2A-Gaussia luciferase (Gluc) (UAS-EGFP-2A-Gluc, Fig. 1A) reporter in HEK293 cells (Fig. 1B)
Summary
The influenza epidemic is a huge burden to public health. Current influenza vaccines provide limited protection against new variants due to frequent mutation of the virus. The new system may provide a powerful tool to analyze influenza A infections at the cellular level to facilitate new antiviral drug discovery This approach may extend to develop live-cell biosensors for other viruses. A universal method for living cell sensing influenza A virus infection of various strains will be a robust tool to facilitate both basic researches and drug developments. By using single domain antibodies, we developed an NP responsive reporter module for sensitive sensing influenza A virus infection In this module, the expression of the reporter genes (green fluorescent protein and Gaussia luciferase) are dependent on the recruitment of the transactivation domain (AD) to the activating sequence upstream by DNA binding domain (DBD) via NP-specific nanobody mediated double-sandwich scaffold. We demonstrated successful living cell detections of infection and antibody neutralization of influenza A H1, H3, H5 and H7 by using this new system
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