Dual-Luciferase (Firefly-Renilla) reporter assay to study the Interferon signalling pathways in virology
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The firefly experimental reporter is correlated with the effect of specific experimental conditions, while the activity of the co-transfected renilla control reporter provides an internal control that serves as the baseline response. Normalisation of the firefly activity to the renilla activity minimises any experimental variability caused by differences in cell viability and/or transfection efficiency. Other sources of variabilities such as differences in pipetting volumes, cell lysis efficiency and assay efficiency are also effectively eliminated. Normalisation increases data accuracy by minimising experimental variability providing more reliable interpretation of the experimental data. Reading both luciferases simultaneously from the same samples reduces plates and time without compromising on the quality of data.
DLR to study interferon (IFN) system
The DLR is very widely used to study the interference of specific proteins on various signalling pathways. For example, DLR has been adapted by virologists to test whether specific viral proteins have an antagonistic role on the Interferon system. The immune response to viral infection consists of both the innate and the adaptive defences. The adaptive response consists of antibodies and lymphocytes referred to as the humoral response and the cell-mediated response, respectively. Both humoral and cell-mediated responses are essential for antiviral defence. However, many viral infections are halted by the quick innate immune response; specifically the interferon system, particularly the type I Interferon system. The Interferon system is considered the primary antiviral response and is the first line of defence against viruses.
Anti-viral response: Activation of type I Interferon (IFN) system
The detection of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) on or in the infected host cell leads to the activation of downstream signalling pathways leading to an induction of type I IFN. Viral PAMPs are nucleic acids with structures generated by viruses and not found in normal uninfected host cells, such as double-stranded RNA (dsRNA) or exposed/uncapped short dsRNA with 5’-triphosphate (Randall and Goodbourn, 2008). Secreted IFN can then act in an autocrine and/or paracrine manner by binding to its receptor on the infected cell and/or on the neighbouring cells, leading to the activation of the Janus kinase (JAK)/ signal transducers and activators of transcription (STAT) signalling pathway (Collins and Mossman, 2014). Activation of the JAK/STAT signalling pathway leads to the transcription of hundreds of IFN stimulated genes (ISGs). ISGs encode proteins that mediate antiviral effects such as cell death, mRNA degradation, translational arrest and up-regulation of antigen presentation in preparation for the adaptive immune response (Audsley and Moseley, 2013).
Anti-immune response: Evasion of type I Interferon (IFN) system
To survive and replicate within their hosts, viruses must be able to evade the interferon system. The viruses can evade the IFN system by interfering with the induction pathway or the signalling pathway of IFN; the JAK/STAT pathway which leads to the induction of ISGs. Therefore, viral proteins that modulate virus-induced IFN response should be considered as candidates for managing virus infection (Randall and Goodbourn, 2008).
DLR to study evasion of IFN induction pathway
For assaying modulation of IFN induction pathway, the cells are co-transfected with a reporter containing the promoter region from IFN upstream of a firefly luciferase gene, the transfection control renilla reporter and an expression vector encoding the viral gene of interest (left image). After 32 hours the cells are stimulated with synthetic dsRNA; polyinosinic:polycytidylic acid (poly (I:C)). The cells are lysed and the firefly and renilla luciferase activities are read after 16 hours of stimulation.
DLR to study evasion of JAK/STAT signalling pathway
For assaying modulation of JAK/STAT signalling pathway, the cells are co-transfected with a reporter containing the promoter region from ISG upstream of a firefly luciferase gene, the transfection control renilla reporter and an expression vector encoding the viral gene of interest (right image). After 42 hours the cells are stimulated with IFN. The cells are lysed and the firefly and renilla luciferase activities are read after 6 hours of stimulation.
In virology, the DLR system is also widely used to study the replications of viruses, refer to application note: Dual luciferase assay to assess the replication of the hepatitis C virus subgenomic replicon
Randall, R. E. and Goodbourn, S. (2008) ‘Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures’, Journal of General Virology. Microbiology Society, 89(1), pp. 1–47. doi: 10.1099/vir.0.83391
Collins, S. E. and Mossman, K. L. (2014) ‘Danger, diversity and priming in innate antiviral immunity’, Cytokine & Growth Factor Reviews, 25(5), pp. 525–531. doi: 10.1016/j.cytogfr.2014.07.002
Audsley, M. D. and Moseley, G. W. (2013) ‘Paramyxovirus evasion of innate immunity: Diverse strategies for common targets.’, World journal of virology. Baishideng Publishing Group Inc, 2(2), pp. 57–70. doi: 10.5501/wjv.v2.i2.57