Hepatitis C virus (HCV) is a global health problem affecting an estimated 170 million people worldwide. Chronic infection can lead to the development of cirrhosis resulting in end-stage liver failure or hepatocellular carcinoma. There is no vaccine for HCV and the current therapy is only effective in around 50 % of individuals. Therefore, improved understanding of the viral life cycle and systems for screening potential viral inhibitors are required.
The development of a subgenomic replicon system, encoding only the viral non-structural proteins required for RNA replication, allows the study of replication of the virus, without the release of infectious particles and the necessary containment facilities required for this work. Recently a subgenomic replicon has been generated from the efﬁciently replicating genotype 2a strain of HCV (JFH1), incorporating the ﬁreﬂy luciferase gene under the control of the HCV internal ribosome entry site element.
This has allowed monitoring of early stage replication events of HCV in the absence of any selective pressure, and furthermore investigation of the effects of compounds on these events.
This application note describes a method used for directly assessing the effects of potential inhibitors simultaneously on both the RNA replication of HCV and overall translation within human hepatoma cells using a BMG LABTECH microplate reader. As an indicator for overall translation in the cell, a capped RNA transcript from the pRLTK plasmid was used. This plasmid contains a Renilla luciferase gene, preceded by a T7 promoter.
Materials & Methods
- BMG LABTECH microplate reader with injectors
- MEGAscript® T7 Kit, Ambion (Applied Biosystems)
- Cap Analog (m7G(5’)ppp(5’)G), Ambion (Applied Biosystems)
- Bio-rad Gene Pulser Xcell Eukaryotic System electroporator
- Gene Pulser/MicroPulser Cuvettes, 0.4 cm gap, Bio-rad
In addition, tissue culture consumables, pipette tips, etc were used as needed from various manufacturers.
Dual luciferase Replication and Translation Assay
In vitro transcribed (IVT) replicon RNA was made from a linearised template of the JFH1 luciferase subgenomic replicon (SGR-JFH1-Luc) DNA using the MEGAscript® T7 Kit. In vitro transcription of linearised pRLTK plasmid DNA was as above but in the presence of a cap analogue. Four million PBS washed human hepatoma (Huh-7.5) cells were electroporated in 0.4 cm cuvettes with 5 μg of SGR-JFH1-Luc RNA and 5 μg of RTLK RNA simultaneously at 270 V and 950 μF.
Cells were then resuspended in standard cell media and seeded at a density of 1000 cells per well in 96 well plate. Cells were incubated as normal at 37°C. Cells were lysed directly in 96 well plates by carefully removing media and washing twice in PBS before adding 20 μL of 1 x Passive Lysis Buffer (Promega), and placing on a rocking platform (with gentle rocking to ensure even coverage of the cell monolayer) at room temperature for 15 minutes.
The 96 well plate containing the lysed cells was then either stored at -20°C (for up to a month) or read directly in the microplate reader using the following parameters:
|Read mode:||Luminescence, well mode kinetics|
|Positioning delay:||0.2 sec|
|Measurement start time:||0.0 sec|
|No of intervals:||48|
|Emission Filter:||empty/lens (depending on reader)|
|Injection speed:||260 μL/sec|
|Injection start time:||1 and 13 sec|
For data calculation, the relative luminescence units were averaged over two ranges:
Range 1 – Fireﬂy luminescence (cycles 5-26 or 2 -12.5 secs)
Range 2 – Renilla luminescence (cycles 29-48 or 14-23.5 secs)
Results & Discussion
Figure 1 shows a typical signal curve of the experiment.
Cells were harvested at 4, 8, and 24 hours post-electroporation, and analysed for Fireﬂy and Renilla luciferase activity. Readings taken at 4 hours allowed establishment of input levels of RNA (Figs. 2 and 3).
In order to assess the effects of various compounds on replication rather than translation, compounds were added 8 hours after electroporation, and cells were incubated for a further 16 hours. Untreated cells were harvested at 4 hours (for normalisation of input RNA) and both treated and untreated cells were harvested at 24 hours to detect effects on replication (data not shown).
This assay allows evaluation of the effects of compounds on early stage replication events and on cellular translation. The 96 well plate format allows a number of compounds of varying concentrations to be tested simultaneously against cells derived from a single electroporation event.
The assay was successfully performed on the BMG LABTECH microplate reader. All BMG LABTECH microplate readers with luminescence detection and injectors are certiﬁed by Promega for the Dual Luciferase Reporter (DLRTM) gene assay.