Technical Reference #1899

1899.

Genetic engineered; biarsenical labelled influenza virus allows visualization of viral NS1 protein in living cells Yang Li; Xinya Lu; Junwei Li; Nathalie Bérubé; Kerri-Lane Giest; Qiang Liu; Deborah H. Anderson; and Yan Zhou, University of Saskatchewan, Journal of Virology, 84(1899), (2010)
Link To Paper

Abstract:
Real-time fluorescence imaging of viral proteins in living cells provides a valuable means to study virus-host interactions. The challenge of generating replication-competent fluorescent influenza A virus is that the segmented genome does not allow fusion of a fluorescent protein gene to any viral gene. Here we introduced the tetracysteine (TC) biarsenical labeling system into influenza virus in order to fluorescently label viral protein in the virus life cycle. We generated infectious influenza A viruses bearing a small TC tag (CCPGCC) in the loop/linker regions of the NS1 proteins. In the background of A/Puerto Rico/8/34 (H1N1) (PR8) virus the TC tag can be inserted into NS1 after amino acid 52 (AA52) (PR8-410) AA79 (PR8-412) or AA102 (PR8-413) or the TC tag can be inserted and replace amino acids 79 to 84 (AA79-84) (PR8-411). Although PR8-410 PR8-411 and PR8-412 viruses are attenuated than the wild-type (WT) virus to some extent in multiple-cycle infection their growth potential is similar to that of the WT virus during a single cycle of infection and their NS1 subcellular localization and viral protein synthesis rate are quite similar to those of the WT virus. Furthermore labeling with membrane-permeable biarsenical dye resulted in fluorescent NS1 protein in the context of virus infection. We could exploit this strategy on NS1 protein of A/Texas/36/91 (H1N1) (Tx91) by successfully rescuing a TC-tagged virus Tx91-445 which carries the TC tag replacement of AA79-84. The infectivity of Tx91-445 virus was similar to that of WT Tx91 during multiple cycles of replication and a single cycle of replication. The NS1 protein derived from Tx91-445 can be fluorescently labeled in living cells. Finally with biarsenical labeling the engineered replication-competent virus allowed us to visualize NS1 protein nuclear import in virus-infected cells in real time.

Materials & Methods:
For visualizing NS1 nuclear import A549 cells seeded in glass-bottom 35 mm dishes (MatTek) were infected with PR8-411 at an MOI of 1. At 3 h.p.i. the media were changed to Opti-MEM containing FlAsH (0.2 μM) EDT (50 μM) and β- mercaptoethanol (10 μM). Between 4 and 7 h.p.i live-cell fluorescence images of infected A549 cells were recorded every 3 min using a Leica SP5 microscope equipped with a 40 × NA 1.25 oil objective an I3 filter cube a PB450-490 excitation filter and an LP515 emission filter. All images were acquired with 500-ms exposures under the same illumination conditions. Images were analyzed using Leica LAS AF 1.8.2 software

Microscopic Technique
Live-cell Fluorescence

Cell Type(s)
A549 cells