Genetically Engineered, Biarsenically Labeled Influenza Virus Allows Visualization of Viral NS1 Protein in Living Cells

Replication-Competent Influenza A Virus That Encodes a Split-Green Fluorescent Protein-Tagged PB2 Polymerase Subunit Allows Live-Cell Imaging of the Virus Life Cycle

“…An alternative to our approach is the biarsenical-tetracysteine (TC) labeling system which has already been used to label the NS1 protein of an infectious influenza virus (18). However, applications of the TC labeling method are limited by toxicity and a high background fluorescence due to nonspecific labeling of cellular proteins, which do not occur with the split-GFP labeling method.…”

“…Several previous attempts that involved partial or complete replacement of a viral protein by green fluorescent protein (GFP) (e.g., neuraminidase [40], hemagglutinin [22], or NS1 [21]) thus resulted in either replication-deficient virions and/or severe impairment of viral protein function. Another approach involved incorporation of a tetracysteine (TC) biarsenical labeling tag into NS1 (18) but has the drawback that it requires a potentially perturbing chemical labeling step after cells are infected. To circumvent these limitations, we applied the split-GFP technique to the PB2 gene, which allowed us to fluorescently label PB2 although only a short additional sequence was added to the PB2 gene.…”

Identification of RNA Helicase A as a Cellular Factor That Interacts with Influenza A Virus NS1 Protein and Its Role in the Virus Life Cycle

“…These rescued mutant viruses possessing the TC tag allowed us to monitor the NS1 movement in live cells (29). Motivated by these results, we extended these findings to rescue mutant viruses possessing the optimized Strep-Tag II motif (WSHPQFEK) in NS1, which would enable us to pull down the proteins associated with NS1 in the context of virus infection.…”

“…Consistent with the smaller-plaque phenotype, PR8-442 grew much more slowly than the WT virus. A previous study with TC-tagged viruses showed that although the TC-tagged virus was attenuated in multiple-cycle replication, the mutant virus was not significantly attenuated during single-cycle growth assays (29). Therefore, we were interested in assessing the replication potential of the Strep-tagged viruses in a single-cycle growth assay.…”

Visualization of Oxidative Stress Induced by Experimental Periodontitis in Keap1-Dependent Oxidative Stress Detector-Luciferase Mice

“…A polyclonal antibody against Nrf2 (Santa Cruz Biotechnology Inc., Dallas, TX, USA) was diluted to 1:200 in phosphate-buffered saline [30]. Alexa Fluor 594-conjugated anti-rabbit IgG (1:250) (Thermo Fisher Scientific K.K., Kanagawa, Japan), which produces a red fluorescence with an excitation maximum at 561 nm and emission at about 594 nm, was used as a secondary antibody [31]. Afterwards, a mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (ImmunoSelect Antifading Mounting Medium; Dianova, Hamburg, Germany), which produces a blue fluorescence with an excitation maximum at 365 nm and emission at about 460 nm, was used to cover the slides [32].…”