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.
Highlights
Influenza A viruses are enveloped, negative-sense segmented RNA viruses of the familyOrthomyxoviridae
Influenza HA protein is a membrane protein expressed in a trimeric form on the host cell surface and influenza A virus (IAV) virion envelope
We chose sites for Tetra Cysteine tag (TC tag) insertion based on selected published reports (Table S2)
Summary
Influenza A viruses are enveloped, negative-sense segmented RNA viruses of the familyOrthomyxoviridae. The viral genome encodes more than a dozen proteins, of which Hemagglutinin is the major structural protein that binds to the sialic acid moieties on the host cell surface to facilitate viral entry. HA1 has the receptor binding site, which helps in entry by binding to the terminal α-2,3- or α-2,6-linked sialic acid moieties present on the host cell surface. Among influenza A virus (IAV) structural proteins, extensive research has been done on IAV HA structure and conformational changes during viral entry and HA post-translational modification. There is a dearth of literature on the aspects of HA trafficking through the ER-Golgi network to the virus budding sites on the cell membrane. Live imaging of HA in IAV infected cells can allow a detailed study of this aspect
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