Biology Reference
In-Depth Information
10.2
METHODS
In this section, we describe our methods to study the interactions of viruses and LDs.
Since our area of expertise is HCV, we will mostly describe methods used to study
this virus. We will also detail other methods used in our lab pertaining to DENV.
10.2.1
Biochemical cell fractionation
10.2.1.1
LD isolation
Isolation and purification of LDs from cells is a powerful method to measure the
association of viral (and cellular) factors with LDs:
1.
Cells at 90% confluence in a 15 cm plate are scraped in 10 ml PBS and
centrifuged for 5 min at 1500 rpm.
2.
After resuspension in 400
m
l of hypotonic buffer containing protease
inhibitor, cells are incubated on ice for 15 min before being lysed with 40
passages in a tight-fitting dounce homogenizer (Wheaton).
3.
The lysate is then centrifuged at 500
g
for 5 min to remove the nuclei.
4.
The remaining supernatant is mixed with 400
m
l of 1.5 M sucrose isotonic buffer
(with 1 mM PMSF) and transferred into an ultracentrifuge tube. Isotonic
buffer containing PMSF is carefully added on top of the supernatant solution up
to 1 mm from the rim of the tube, taking precaution not to mix the solutions.
Centrifuge for 1 h at 100,000
g
(34,000 rpm) in an SW55 rotor.
5.
After ultracentrifugation, the tubes are frozen at
80
C for at least 1 h.
Isolate the top of the frozen solution, which should contain LDs and associated
proteins, by cutting it off with a razor blade and thawing in an eppendorf tube.
6.
For western blot analysis, TCA protein precipitation of the samples is usually
performed.
A representative example for an LD isolation performed in na¨ve or HCV replicon
cells is shown in
Fig. 10.3
. Details about the protocols and outcomes for each label-
ing method follow.
10.2.1.2
Cell fractionation
For cell fractionation, the previous protocol can be adapted to form a sucrose
gradient.
1.
The tubes are loaded as follows, from bottom to top: 2 M sucrose isotonic buffer,
the sample mixed with an equal volume of 2 M sucrose isotonic buffer, 0.75 M,
0.5 M, and 0.25 M sucrose isotonic buffers, and isotonic buffer without sucrose
on top.
2.
After centrifugation, fractions are carefully collected and analyzed.
As shown in
Fig. 10.1
A, most HCV proteins are indeed strongly associated with cel-
lular membranes, making it possible to identify their precise membrane distribution.
