Biology Reference
In-Depth Information
3.
Cells are harvested at different times post-transfection, such as 4, 24, 48, and
72 h.
4.
Luciferase activity is measured as described above and normalized to the 4 h time
point (at which no viral RNA replication has occurred).
The most accurate and direct method to determine the efficiency of HCV RNA rep-
lication is to measure viral RNA levels. This can be done using classical tools to
quantify RNA levels, such as real-time RT-PCR.
10.2.6.4
Viral RNA measurement at LDs
Viral RNA can be measured in different cellular fractions, including the LD fraction
(see below for cell fractionation assays):
1.
20
m
g of carrier RNA (Qiagen) is added to 150
m
l of each fraction.
2.
RNA is extracted from each fraction with the Quick-RNA MiniPrep kit (Zymo)
according to the manufacturer's recommendations.
3.
RNA is eluted using 35
m
l water.
4.
cDNA is synthesized as described above, using 11
m
l of RNA and an HCV
antisense probe located in the 5'UTR, followed by RNase A (Thermo Scientific)
digestion (2.5
m
g/reaction for 30 min at 37
C).
For the generation of RNA standards, cDNA is generated for a twofold serial dilution
of
in vitro
transcribed HCV RNA by the above protocol. For real-time quantitative
PCR, two HCV-specific primers in the 5'UTR are used. The cDNA from the twofold
serial dilution of HCV RNA is used to create a standard curve. Absolute amounts of
viral RNA in fractions are calculated using this standard curve.
10.2.6.5
Viral assembly and release assays
The late phases of viral replication seem to be more associated with LDs, especially
for HCV. Here, we will describe the main protocols used to investigate the role
of factors into the assembly or the release of new virions, and how to differentiate
between these discreet steps.
When studying the late phase of viral replication, it is common to directly trans-
fect the viral genome in its physiological form into cells with the adapted methods
(electroporation for viral RNAs, for example, for HCV or DENV). The amount of
virus produced from the transfected cells is then measured, usually after 24 h to pre-
vent reinfection of cells in the culture. Viruses released into the supernatant are usu-
ally harvested and quantified by ELISA or specific assays to assess their infectivity.
Viruses assembled but not released from the cells can also be isolated and tested. This
can give insights on the assembly versus release efficiency of the virus. The infec-
tivity of the viruses can be measured using a reporter gene (luciferase or fluorescent
for example), but if none is available, staining of the infected cells can be performed
and analyzed by FACS (or microscopy). The following is an example of a protocol
that can be used for a thorough investigation of the assembly and release of HCV
