Biology Reference
In-Depth Information
required if a flow cytometer is available within the BSL3 facility, and thus, cells do
not need to be transported out of the BSL3 facility). The mean fluorescence intensity
can provide information about the viral replication and translation levels into the
cells. If the viral construct contains a luciferase reporter gene, the infected cells
can be lysed and relative light units (RLU) can be measured. Although it does not
give an exact count of the amount of infected cells, the RLU can be directly corre-
lated with the percentage of infected cells when normalized to the whole protein con-
centration of the cell lysate.
Titration of DENVs is performed using the plaque assays described above.
10.2.7
Drug treatments
DGAT1 inhibitor (2-((1
s
,4
s
)-4-(4-(4-amino-7,7-dimethyl-7
H
-pyrimido[4,5-
b
][1,4]
oxazin-6-yl)phenyl)cyclohexyl)acetic acid) is kept at a stock concentration of
10 mM in DMSO. This stock solution is diluted 10-fold in DMSO (1 mM) before
a final 1/500 dilution in cell culture medium (final concentration of 20
m
M).
Although inhibition of DGAT1 function is significant after only a few hours of treat-
ment, DGAT1 inhibition must be maintained for about 2 days when looking at the
loading of viral factors on LDs as the half-life of these factors on the already existing
LDs is quite long. Inhibiting DGAT1 does not disrupt the already formed LDs.
DGAT1 inhibitor 4a, (1
R
,2
R
)-2-[[4'-[[(phenylamino)carbonyl]amino] [1,1'-
biphenyl]-4-yl]carbonyl]cyclopentanecarboxylic acid (TOCRIS Bioscience) is
resuspended in DMSO at a stock concentration of 37.5 mM before being diluted
1/500toaworkingconcentrationof75
m
M in cell culture medium. See below for
advice about treatment with this inhibitor.
Triacsin C is used at 6
m
M concentration. The stock solution is at 6 mM
in DMSO.
Oleic acid-albumin (Sigma) is used at a final concentration of 2 mg/ml. Cells are
usually loaded with oleate for no longer than 20 h.
Rapamycin is an mTOR inhibitor that causes an increase in cellular autophagy.
Rapamycin is solubilized in DMSO and generally used at a 200 nM final concentra-
tion, although this must be optimized for each cell line and experiment.
10.3
DISCUSSION
We describe here the various methods used to examine the interplay between viruses
and LDs. Most general methods for studying viral replication can be used to study the
roles of LDs in viral replication. For example, viral RNA replication can be measured
under treatment with an inhibitor of a triglyceride-synthesizing enzyme, such as
DGAT1. Similarly, strategies used to study cellular LDs, such as identification of
LD-associated proteins after LD isolation, can be applied to viral components.
The various methods presented here can be beneficial for cell biologists and virol-
ogists studying viral propagation or LD metabolism.
