Biology Reference
In-Depth Information
packaged into new virions, and are not translated. Thus, the RNA level of a gene
transcript does not always reflect the level of proteins produced. As for any other
protein, translation of viral proteins is usually assessed by western blot, microscopy,
FACS, or ELISA.
10.2.6.3
Viral RNA replication assays
To assess the efficacy of viral RNA replication, indirect and direct methods for mea-
surements are commonly used. The level of RNA replication can be indirectly mea-
sured if the viral genome contains a reporter gene that is used as an indirect
measurement of the level of RNA replication. The two most common reporters
for HCV RNA replication are neomycin and luciferase. The first HCV clone that
was able to replicate in cell culture was a genotype 1 subgenomic replicon. Since
replication efficiency was relatively low, a neomycin phosphotransferase selection
gene enabled the selection of cells that contain the replicating HCV Replicon
RNA under G418 pressure, thus enhancing the level of RNA replication. Since then,
better replicating HCV genotype 2a clones have been identified. With those, RNA
replication can be measured indirectly through the expression of a luciferase reporter
gene in the viral genome.
Neomycin replicon assay
1.
One microgram of HCV replicon RNA containing a neomycin gene and
1
m
g of pGL3.5 (Firefly luciferase control plasmid, Promega) are transfected into
Huh7.5 cells as described above.
2.
After transfection, cells are added to 8 ml of medium, of which 100
m
l to 1 ml are
plated in 10 cm dishes for the replicon assay and 1 ml into 12-well plates for
the luciferase assay, and incubated at 37
C.
3.
To monitor for equal transfection efficiencies, cells for the luciferase assay are
lysed 24 h post-transfection in 1
Cell Culture Lysis Buffer (Promega).
Luciferase activity is measured using the Luciferase Assay Systems (Promega)
on a MonoLight 2010 Luminometer (Pegasus Scientific).
4.
For the HCV Replicon assay, 750-1000
m
g/ml of G418 is added to the medium.
5.
Cells are kept under selective pressure for 3-4 weeks until colonies have formed,
with medium changed twice a week.
6.
Cells are then washed with PBS, stained with Crystal Violet Working Solution
for 10 min at room temperature, and washed with water.
For biochemical studies of HCV RNA replication, often an HCV replicon cell line is
established by transfecting Huh7.5 cells with HCV Replicon RNA, kept under con-
tinuous G418 (800
m
g/ml) pressure, and split twice a week.
Luciferase replicon assay
1.
Ten micrograms of HCV Replicon RNA containing a luciferase gene are
transfected into Huh7.5 cells as described above.
2.
After transfection, cells are added to 8 ml of medium, and 1 ml is plated per well
in a 12-well dish, and incubated at 37
C.
