A Comprehensive Roadmap Towards the Generation of an Influenza B Reporter Assay Using a Single DNA Polymerase-Based Cloning of the Reporter RNA Construct

Abstract

The mini-genome reporter assay is a key tool for conducting RNA virus research. However, procedural complications and the lack of adequate literature pose a major challenge in developing these assay systems. Here, we present a novel, yet generic and simple, cloning strategy for the construction of an influenza B virus reporter RNA template and describe an extensive standardization of the reporter RNP/polymerase activity assay for monitoring viral RNA synthesis in an infection-free setting. Using this assay system, we showed for the first time the effect of viral protein NS1 and host protein kinase C delta (PKCD) on influenza B virus RNA synthesis. In addition, the assay system showed promising results in evaluating the efficacy of antiviral drugs targeting viral RNA synthesis and virus propagation. Together, this work offers a detailed protocol for the standardization of the influenza virus minigenome assay and an excellent tool for screening of host factors and antivirals in a fast, user-friendly, and high-throughput manner.

Keywords: antiviral screening; influenza B; polymerase activity assay; reporter construct; ribonucleoprotein.

Figure 1

An overview of the cloning strategy of influenza B reporter plasmid and reporter assay. (A) Total RNA was isolated from amplified stocks of influenza B/Brisbane/60/2008 virus. (B) Total RNA was converted to cDNA by performing reverse transcription polymerase chain reaction (RT-PCR) and the 5′ and 3′ untranslated regions (UTRs) were amplified using specific primers containing overhangs. (C) The double-stranded 5′ and 3′ UTRs containing overlapping regions were used as primers to amplify the luciferase open reading frame (ORF). (D) The resulting PCR product was used as an insert for circular polymerase extension cloning (CPEC) assembly with the PCR amplified vector fragment. (E,F) The generated reporter and other protein expressing plasmids, upon co-transfection in human embryonic kidney 293T (HEK 293T) cells, reconstitute luciferase RNPs that express the luciferase enzyme under the control of the viral promoter. The quantification of the luciferase signal gives the measure of viral polymerase activity.

Figure 2

PCR amplification and CPEC reaction for the construction of a reporter construct: Agarose gel electrophoresis images of (A) PCR amplification products corresponding to the 5′ and 3′ UTRs of NA-NB segment. (B) PCR amplification product of the luciferase ORF using double-stranded PCR products corresponding to 5′ and 3′ UTRs as primers. (C) PCR amplification of the pHH21 vector. (D) CPEC products with different ratio of vector and insert.

Figure 3

Optimization of the reporter system: (A) An effect of Kozak sequence on the expression of viral polymerase proteins and influenza B RNP activity assay. (B) Reporter RNP activity assay with different ratios of the PA protein expression plasmid with respect to PB1 and PB2. (C) Reporter RNP activity assay with various amounts of NP expression plasmid. (D) Optimization of time for the reporter activity assay. (E) Optimization of incubation temperature for the influenza B RNP activity assay (n = 3 ± standard deviation (SD), ^*p < 0.05 one-way analysis of variance (ANOVA) with post-hoc Student's t-test when compared to the preceding set, for (E), a comparison was performed between two RNP positive sets, ns, not significant).

Figure 4

An effect of host and viral factors upon viral RNA synthesis: (A) effect of an increasing amount of viral NS1 protein on influenza B RNP activity assay and (B) effect of an increasing amount of constitutively active host protein kinase C delta (PKCD) protein on B RNP activity assay (n = 3 ± SD. ^*p < 0.05 one-way ANOVA with post-hoc student's t-test when compared to the preceding set.

Figure 5

An effect of antiviral drugs upon viral RNA synthesis in an infection-free and infection setting: (A,B) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine the cytotoxicity of Ribavirin and Favipiravir on HEK 293T cells. (C–E) The effect of Ribavirin and Favipiravir on influenza B virus RNP activity. Viral polymerase proteins in HEK 293T cells are expressed by transient transfection (C,D) or by infecting the cells with influenza B virus (E) (n = 3 ± SD, ^*p < 0.05 one-way ANOVA with post-hoc Student's t-test when compared to the preceding set, for (E), comparison was performed with control set, ns, not significant).