Download VVZ ADAR1 -E3L

*** * VV-E3L
** ** *
eKrevNkaLYdlqRsamvyssddiPPrWfm
sKheaNrqLYrlldsdevccedgnPPrWfv ORF-E3L
pKkeiNrvLYslaKkgklqkeagtPPlWki hADAR-Z
eKkriNhfLYtfetkgllcrypgtPPlWrv xADAR-Z
pKktlNqvLYrlkKedrv-sspe-PatWsi DLM-1-Z
K keiNr LY l
hrv q
rtl h
f
v
l
Pp W v
a
Consensus.
i
m
a k
Figure 1. The amino terminus of E3L shows homology to a family of Z-DNA binding proteins.
The amino terminus of VV-E3L shows homology to ORF-E3L and the Z-DNA binding domains (Z)
of ADAR1 and DLM-1. Shown here are the Z domains of human and xenopus ADAR1. The
conserved residues and the consensus sequence are shown in green. The asterisk denotes residues in
ZADAR1 which contact Z-DNA in vitro.
VVE3L
N
Lac Z
VVE3L
VVE3L83N
VVZADAR1-E3L
C
C
Z
C
Figure. 2. Schematic representation of virus constructs.
VVE3L represents wild type VV, which encodes a full length E3L protein with
amino and carboxy terminal domains. VVE3L represents virus deleted of the E3L
gene, which expresses Lac Z from the E3L locus. VVE3L83N virus encodes an
amino terminal deletion mutant of E3L, which contains an intact carboxy terminus.
VVZADAR1-E3L represents the chimeric virus in which the amino terminus of E3L
is replaced by the Z domain of ADAR1, attached to the carboxy terminus of E3L.
VVZADAR1-E3L
VVE3L37N
VVE3L
Mock
VVZADAR1-E3L
VVE3L37N
Wt VV
VVE3L
Mock
Wt VV
CHASE
PULSE
p25
p20
p25
p20
Figure 3. Confirmation of protein expression and stabilities.
Confluent RK13 cells were either mock infected or infected in duplicate with VVE3L, Wt VV,
VVE3L37N and VVZADAR1-E3L viruses at a multiplicity of infection of 5. Following infection,
cells were radio-labeled with [35S] methionine between 3-4 hours post infection. At 4 hours post
infection one set of dishes were harvested (pulse) and NP40 lysates were prepared. The second set of
dishes were overlaid with non-radioactive media (chase) for two hours, and NP40 lysates were prepared
at 6 hours post infection in the same way as before. The lysates were incubated with pre-washed
poly(rI).poly(rC)-agarose resin for one hour at 4C. Following incubation, the resin was washed with
buffer A to remove excess unbound protein and radioactivity. The proteins that were bound to the resin
were resolved on a 12% SDS-PAGE gel and subjected to autoradiography.
-
+
-
+
-
+
-
+
-
+
IFN
Figure 5. Protein synthesis in the presence of interferon.
RK13 cells were pre-treated with 100 U/mL of rabbit interferon and either mock infected or infected with
VVE3L, Wt VV, VVZADAR1-E3L, and VVE3L83N viruses at a multiplicity of infection of 5. The cells
were radio-labeled with [35S] methionine between 5-6 hours post infection. Cells were harvested at 6 hours
post infection and lysates were prepared. The lysates were resolved on a 12% SDS-PAGE gel and
subjected to autoradiography.
Percent plaque reduction
120
100
80
Wt VV
VVE3L 83N
VV E3L
VVZADAR1-E3L
60
40
20
0
0
0.1
1
10
100
1000
Units of interferon/mL
Figure 4. Interferon resistance assay.
RK13 cells were pre-treated with increasing doses of interferon (unit/mL) for 16 hours followed by
infection with 1x102 pfu of Wt VV, VVE3L83N, VVE3L and VVZADAR1-E3L viruses. Cells were
stained 36 hours post infection with crystal violet and the number of plaques were counted. The reduction
in the number of plaques was recorded as a percentage of plaques in the absence of interferon and was
plotted against increasing units of interferon.
Efficiency of plaquing
0.81
Wt VV
< 0.001
VVE3L
VVE3L83N
VVZADAR1-E3L
0.66
0.56
Figure 6. Determination of host range of VVZADAR1-E3L in HeLa cells.
HeLa and RK13 cells were infected with increasing dilutions of Wt VV, VVE3L, VVE3L83N and
VVZADAR1-E3L viruses and stained with crystal violet 36 hours post infection. A sample of the infected
HeLa cells is shown in the panel on the left. The panel on the right shows the efficiency of plaquing,
which is determined by dividing the virus titer on HeLa monolayers by the titer on RK13 monolayers.
VVZADAR1-E3L
VVE3L83N
VVE3L37N
VVE3L
Wt VV
Mock
eIF2-P at
6 hours post
infection
eIF2-P at
12 hours post
infection
Figure 7. eIF2 phosphorylation assay.
HeLa cells were either mock infected or infected with Wt VV, VVE3L, VVE3L37N,
VVE3L83N, and VVZADAR1-E3L viruses at a multiplicity of infection of 5. Cell lysates were
prepared at 6 and 12 hours post infection using RIPA lysis buffer. The proteins were resolved on a
12% SDS PAGE gel followed by western blotting using antibodies to the phosphorylated form of
eukaryotic translation initiation factor eIF2.
VVE3L83N
VVZADAR1-E3L
Wt VV
VVE3L
Mock
VVE3L83N
VVZADAR1-E3L
Wt VV
VVE3L
Mock
Figure 8. DNA fragmentation analysis for the detection of apoptosis
HeLa cells were either mock infected or infected with VVE3L, Wt VV, VVZADAR1-E3L and
VVE3L83N viruses at a multiplicity of infection of 5. Cytoplasmic DNA was prepared at 24 and
32 hours post infection, resolved through a 1.5% agarose gel, and visualized by ethidium bromide
staining.
120
Percent survival
100
80
Wt VV
VVE3L 83N
VVZADAR1 -E3L
60
40
20
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
0
virus dose (pfu)
Figure 9. Percent survival of mice infected following intranasal infection.
Groups of 4-6 mice were injected intranasally with increasing doses of Wt VV, VVE3L83N and
VVZADAR1-E3L viruses. Mice were monitored for 14 days after injections. Percentage of mice
that survived at the end of 14 days were plotted against dose of virus administered
1.00E+09
pfu/gm of tissue
1.00E+08
1.00E+07
1.00E+06
1.00E+05
Wt VV
VVE3L83N
br a
in
lung
nos
e
br a
in
lung
nos
e
br a
in
lung
nos
e
1.00E+04
VVZADAR1-E3L
1.00E+09
1.00E+08
pfu/gm of tissue
1.00E+06
1.00E+05
1.00E+07
1.00E+06
1.00E+05
1.00E+04
VVE3L83N
VVZADAR1-E3L
Wt VV
VVE3L83N
lung
br a
in
nos
e
lung
br a
in
nos
e
lung
br a
in
nos
e
br a
in
br a
in
lung
nos
e
br a
in
lung
Nos
e
lung
br a
in
Nos
e
lung
br a
in
Nos
e
Wt VV
lung
1.00E+03
1.00E+04
nos
e
pfu/gm of tissue
1.00E+08
1.00E+07
VVZADAR1-E3L
Figure 10. Tissue distribution.
Groups of three mice were infected with 1x105 pfu of Wt VV, VVE3L83N and VVZADAR1-E3L viruses
intranasally. At 2,4 and 8 days post infection nasal turbinates, lungs and brains were harvested from the
animals and homogenized. Plaque assays were performed to detect titers of virus in pfu/gm of the tissue.
Average virus titers are shown at days 2 (top), 4 (bottom left), and 8 (bottom right) post infection.
120
100
% Survival
80
Wt VV
VVZADAR1 -E3L
VVE3L 83N
60
40
20
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
0
Virus Dose (pfu)
Figure 11. Percent survival of mice following intracranial infection.
Groups of 4-6 mice were injected intracranially with increasing doses of Wt VV, VVZADAR1-E3L
and VVE3L83N viruses. Mice were monitored for 14 days after injections. Percentage of mice
that survived at the end of 14 days were plotted against dose of virus administered
Virus titer (pfu/gm) of brain
1.00E+09
1.00E+08
1.00E+07
1.00E+06
1.00E+05
Wt VV
VVE3L83N
VVZADAR1-E3L
Figure 12. Neurovirulence.
Groups of 2 mice were injected intracranially with 1x102 pfu of Wt VV, VVE3L83N
and VVZADAR1-E3L viruses. Brains were harvested at 6 days post infection and
homogenized. The amount of virus was determined by titrations in RK13 cells. Shown
above are viral titers per gram of brain.
VVZADAR1-E3L
Wt VV
Mock
VVZADAR1-E3L
VVE3L83N
Wt VV
Mock
VVE3L83N
I/C
I/N
eIF2-P
Figure 13. Detection of eIF2 phosphorylation in vivo.
Mice were either uninfected or infected with 1x107 pfu of Wt VV, VVE3L83N and
VVZADAR1-E3L viruses by intranasal (I/N) or intracranial (I/C) routes, in separate infections.
At 4 days post infection noses were harvested from mice infected by intranasal route, while
brains were harvested from mice infected by intracranial route. The organs were homogenized
and cell lysates were prepared using RIPA buffer. The proteins were resolved on a 12% SDS
PAGE gel followed by western blotting using antibodies to the phosphorylated form of
eukaryotic translation initiation factor eIF2.