A reporter system for assaying influenza virus RNP functionality based on secreted Gaussia luciferase activity

Abstract

BackgroundInfluenza A virus can infect a wide variety of animal species including humans, pigs, birds and other species. Viral ribonucleoprotein (vRNP) was involved in genome replication, transcription and host adaptation. Currently, firefly luciferase (Fluc) reporter system was used in vRNP functional assay. However, its limitation for the testing by virus infection resulted in an increased need for rapid, sensitive, and biosafe techniques. Here, an influenza A virus UTR-driven gene reporter for vRNP assay based on secreted Gaussia luciferase (Gluc) activity was evaluated.ResultsBy measuring Gluc levels in supernatants, reporter gene activity could be detected and quantitated after either reconstitution of influenza A virus polymerase complex or viral infection of 293T and A549 cells, respectively. As compared with Fluc reporter, Gluc-based reporter was heat-tolerant (65°C for 30 min) and produced 50-fold higher bioluminescent activity at 24 h posttransfection. Signals generated by Gluc reporter gene could be detected as early as 6 h post-infection and accumulated with time. Testing by viral infection, stronger signals were detected by Gluc reporter at a MOI of 0.001 than that of 1 and the effects of PB2-627K/E or amantadine on influenza vRNP activity were elucidated more effectively by the Gluc reporter system.ConclusionsThis approach provided a rapid, sensitive, and biosafe assay of influenza vRNP function, particularly for the highly pathogenic avian influenza viruses.

Structural and Functional Characterization of K339T Substitution Identified in the PB2 Subunit Cap-binding Pocket of Influenza A Virus

“…180 -200 M protein in the cell was titrated with 2 mM m 7 GTP in the syringe, and 1 l was injected followed by 29 injections of 2 l. Data were fitted to a single binding site model and analyzed to obtain the parameter K a using the Origin 7.0 program. Polymerase Activity Assay-Polymerase activity of the ribonucleoprotein complex was detected and quantified using Gaussia luciferase (Gluc) system as described previously (31). The reporter plasmid polI-Gluc consisting of the Gluc ORF flanked by the noncoding regions of influenza NS segment was co-transfected in human A549 cells with PB1, PB2, PA, and NP that were cloned, respectively, into the bidirectional expression plasmid pHW2000.…”

“…However, the 2009 pandemic H1N1 isolates and approximately two-thirds of the human-isolated H5N1 viruses contain the avian-like Glu 627 (3,39). The polymerase activity of both wild-type Thr 339 -PB2 and mutant Lys 339 -PB2 from A/bar-headed goose/Qinghai/15c/ 2005 (H5N1) was quantitated using a Gluc system in human A549 cells (31). With either wild-type Lys 627 or avian-like Glu 627 , the polymerase activity of Thr 339 -PB2 was only ϳ50% of that of the Lys 339 mutant (Fig.…”

Adenovirus-mediated artificial MicroRNAs targeting matrix or nucleoprotein genes protect mice against lethal influenza virus challenge

“…Therefore, we hypothesized that NP-specific RNAi could not only contribute to target protein degradation, but also decrease viral RNA accumulation by inhibiting RNP activity. To assess this hypothesis, we constructed a luciferase-based mini-genome reporter assay system as previously described 21 . Luciferase activity was evaluated 24 hours post-transfecion.…”

“…A reporter plasmid driven by PolI promter was constructed as previously described 21 . Firefly luciferase open reading frame was flanked by influenza NP noncoding regions.…”

Screening for Novel Small-Molecule Inhibitors Targeting the Assembly of Influenza Virus Polymerase Complex by a Bimolecular Luminescence Complementation-Based Reporter System

“…A codon-optimized Gluc molecule has been widely used as a reporter in cultured mammalian cells (28). The sensitivity of Gluc is up to 2,000-fold higher than that of Renilla reniformis luciferase (Rluc) or firefly luciferase (Fluc), which is encoded by an important reporter gene (29). Features of PCA, including the detected interactions, are fully reversible, and the readout is easily detected (30).…”