Live Visualization of Hemagglutinin Dynamics during Infection by Using a Novel Reporter Influenza A Virus

Abstract

Live visualization of influenza A virus (IAV) structural proteins during viral infection in cells is highly sought objective to study different aspects of the viral replication cycle. To achieve this, we engineered an IAV to express a Tetra Cysteine tag (TC tag) from hemagglutinin (HA), which allows intracellular labeling of the engineered HA protein with biarsenic dyes and subsequent fluorescence detection. Using such constructs, we rescued a recombinant IAV with TC tag inserted in HA, in A/Puerto Rico/8/1934(H1N1) background (HA-TC). This recombinant HA-TC tag reporter IAV was replication-competent; however, as compared to wild type PR8 IAV, it was attenuated in multicycle replication. We confirmed expression of TC tag and biarsenical labeling of HA by immunofluorescence assay in cells infected with an HA-TC tag reporter IAV. Further, we used this reporter virus to visualize HA expression and translocation in IAV infected cells by live confocal imaging. We also tested the utility of the HA-TC IAV in testing chemical inhibitors of the HA translocation. Overall, HA-TC IAV is a versatile tool that will be useful for studying viral life cycle events, virus-host interactions, and anti-viral testing.

REPLICAID: replicon vaccines to combat avian infectious diseases

“…Additionally, we demonstrated that the TMUV replicon was capable of expressing viral (glyco)proteins of AIV (HA), IBV (Spike), and IBDV (pVP2). HA was detected in the perinuclear region within electroporated cells, indicating that this viral glycoprotein was correctly routed to the cell surface as previously described for a reporter Influenza A virus (dos Anjos Borges et al, 2020). Indeed, HA was also detected at the cell surface.…”

Infectious clone of a contemporary Tembusu virus and replicons expressing reporter genes or heterologous antigens from poultry viruses

“…HA was detected in the perinuclear region within electroporated cells, indicating that this viral glycoprotein was correctly routed to the cell surface as previously described for a reporter Influenza A virus. [ 35 ] Indeed, HA was also detected at the cell surface. For the TMUV replicon expressing pVP2, a cytosolic distribution was observed which is in agreement with the literature.…”

Influenza A Virus: Host–Virus Relationships

“…Research labs around the globe are devising newer and more robust techniques in order to identify novel ways to perform new antiviral screens. Using live confocal imaging, Anjos Borges et al [ 3 ] have used an innovative Hemagglutinin (HA)-Tetra Cysteine tag reporter IAV in an assay to screen chemical inhibitors of HA translocation.…”