NFkB ONLY PARTIALLY MEDIATES LMP1 ACTIVATION OF B CELLS

1
Introduction
The latent membrane protein 1 (LMP1) of the
Epstein-Barr virus (EBV) is essential for the
transforming abilities of EBV. It induces
phenotypic changes similar to B cell activation,
such as ICAM-1 and CD71 expression and cell size
increase. Our purpose was to study the role NF-kB
has in mediating these effects.
2
Methods
For the luciferase assays, different cell lines
were transiently transfected with expression
plasmids for LMP1, a dominant inhibitor mutant,
IkBaDM, and a luciferase reporter gene under the
control of either p50/p65 or c-Rel responsive
promoters. The flowcytometric experiments were
carried out with either cell lines or human
primary B cells transfected with LMP1, IkBaDM and
ratCD2.
3
(No Transcript)
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LMP1 Activation of P50/p65 and C-rel Responsive
Kb-sites
2000
K562
luciferase units
1000
0
VC
VC
LMP1
LMP1
p50/p65-site
c-Rel-site
3000
DG75
2000
luciferase units
1000
0
VC
LMP1
VC
LMP1
c-Rel-site
p50/p65-site
5
Inhibition of LMP1-induced luciferase activity
from p50/p65 responsive kB sites.
7.5
K562
5.0
fold induction
2.5
0.0
VC LMP1 LMP1 LMP1
IkBa
IkBaDM
3
DG75
2
fold induction
1
0
6
Inhibition of Lmp1-induced Activity From C-rel
Responsive Kb-sites
15
K562
10
fold induction
5
0
VC LMP1 LMP1
IkBaDM
7.5
DG75
5.0
fold induction
2.5
0.0
VC LMP1 LMP1
IkBaDM
7
Flow Cytometric Analysis
8
Inhibition of LMP1-induced CD71 up-regulation
ratCD2
CD71
9
Inhibition of LMP1-induced ICAM-1 up-regulation
control
LMP1
LMP1IkBaDM
IkBaDM
DG75
K562
Primary B cells
10
Inhibition of LMP1-induced cell size increase
IkBaDM
LMP1IkBaDM
LMP1
control
DG75
K562
Primary B cells
11
DISCUSSION
LMP1 induced activation of NF-kB can be blocked
by expression of the dominant IkBaDM mutant.
ICAM1 and CD71 expression is still up-regulated
by LMP1 despite IkBaDM expression. LMP1 induced
cell size increase was also unaffected by IkBaDM
expression. Therefore we conclude that LMP1 is
able to activate B cells independently of NF-kB.
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Sara Liljeholm, Kate Hughes, Thomas Grundström
and Peter Brodin.
13
Department of Cell and Molecular Biology,Umeå
University, S-901 87 Umeå, Sweden
14
Present Address Department of Molecular
BiologyPreclinical Research
15
Development, Astra Hässle ABTvistevägen 48,
S-907 36 Umeå, Sweden
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NF-kB ONLY PARTIALLY MEDIATES
17
LMP1 ACTIVATION OF B CELLS