Download emboj7601881-sup

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Supplementary information
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3
Supplementary table 1S
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and amino acid sequences corresponding to the trypsin-digested peptide molecular mass
5
database.
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(Shimazu) was done by Mascot search software (http://www.matrixscience.com/). A
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confirmation of the identity of each cysteine-containing peptide was made by reduction
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and alkylation of an equivalent sample.
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about 66 kDa present in the IREF-1 fraction (Figure 3A) was not subjected to the
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MALDI-TOF MS analysis since it was not found to have the IREF-1 activity (data not
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shown).
Molecular masses of trypsin-digested peptides from IREF-1
Assignment of observed ions from MALDI-TOF mass spectrometry
An unknown protein with molecule mass of
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1
1
2
3
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5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
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MCM2
Start-End
Observed
Mr(expt)
Mr(calc)
196-209
276-286
468-484
485-496
485-501
534-553
559-572
589-604
1662.9000
1305.8000
1917.0900
1172.6500
1625.9300
2011.1300
1543.8600
1788.9400
1661.8927
1304.7927
1916.0827
1171.6427
1624.9227
2010.1227
1542.8527
1787.9327
1661.8023
1304.7452
1916.0156
1171.6601
1624.8937
2010.0535
1542.8042
1787.8723
MCM4
Start-End
Observed
Mr(expt)
Mr(calc)
217-224
331-343
396-404
410-422
601-610
611-627
702-718
702-719
1042.5400
1577.8300
1021.6300
1630.9400
1115.6200
1825.9600
1924.9700
2053.0400
1041.5327
1576.8227
1020.6227
1629.9327
1114.6127
1824.9527
1923.9627
2052.0327
1041.5243
1576.7351
1020.6192
1629.8627
1114.5917
1824.9370
1923.9247
2052.0197
MCM5
Start-End
Observed
Mr(expt)
Mr(calc)
134-152
253-265
266-281
282-294
329-344
585-599
2087.1200
1316.8200
1838.9400
1416.9300
1670.9500
1688.9500
2086.1127
1315.8127
1837.9327
1415.9227
1669.9427
1687.9427
2086.0993
1315.6732
1837.8595
1415.8024
1669.8821
1687.8417
MCM6
Start-End
Observed
Mr(expt)
Mr(calc)
109-120
200-207
208-217
270-282
270-285
403-415
408-415
497-512
746-754
1471.8000
1038.5800
1184.6800
1381.7300
1707.9400
1603.9000
1000.5100
1685.9500
1151.7100
1470.7927
1037.5727
1183.6727
1380.7227
1706.9327
1602.8927
1999.5027
1684.9427
1150.7027
1470.7143
1037.5658
1183.6197
1380.6521
1706.8588
1602.8154
1999.4774
1684.8533
1150.6022
MCM7
Start-End
Observed
Mr(expt)
Mr(calc)
19-3011
31-4511
135-149
150-161
398-412
431-443
489-502
519-533
552-564
1433.8600
1826.0300
1706.9400
1317.8400
1653.0600
1589.9600
1556.8700
1745.9900
1473.9500
1432.8527
1825.0227
1705.9327
1316.8327
1652.0527
1588.9527
1555.8627
1744.9827
1472.9427
1432.6769
1824.9271
1705.8206
1316.6936
1651.9257
1588.8222
1555.7267
1744.8665
1472.8100
Delta Miss
-0.0905
-0.0475
-0.0672
-0.0174
-0.0290
-0.0693
-0.0485
-0.0604
1
0
1
0
1
0
0
0
Delta Miss
-0.0084
-0.0876
-0.0035
-0.0700
-0.0210
-0.0157
-0.0380
-0.0130
1
0
1
1
0
0
0
1
Delta Miss
-0.0134
-0.1395
-0.0733
-0.1203
-0.0606
-0.1010
0
0
1
0
1
0
Delta Miss
-0.0784
-0.0069
-0.0530
-0.0706
-0.0740
-0.0773
-0.0253
-0.0895
-0.1005
0
1
0
0
1
1
0
0
0
Delta Miss
-0.1759
-0.0956
-0.1121
-0.1391
-0.1270
-0.1305
-0.1361
-0.1162
-0.1328
0
1
0
0
0
0
1
1
0
Sequence
R.THVDSHGHNVFKER.I
R.ISHLPLVEELR.S
K.IFASIAPSIYGHEDIKR.G
R.GLALALFGGEPK.N
R.GLALALFGGEPKNPGGK.H
R.AIFTTGQGASAVGLTAYVQR.H
R.EWTLEAGALVLADR.G
R.TSIHEAMEQQSISISK.A
Sequence
K.SFDKNLYR.Q
R.CHTTHSMALIHNR.S
R.AVPIRVNPR.V
K.SVYKTHIDVIHYR.K
K.AGIICQLNAR.T
R.TSVLAAANPIESQWNPK.K
R.LSEEASQALIEAYVDMR.K
R.LSEEASQALIEAYVDMRK.I
Sequence
R.NTLTNIAMRPGLEGYALPR.K
R.VLGIQVDTDGSGR.S
R.SFAGAVSPQEEEEFRR.L
R.LAALPNVYEVISK.S
R.RGDINLLMLGDPGTAK.S
K.LQPFATEADVEEALR.L
Sequence
K.DFYVAFQDLPTR.H
R.FVDFQKVR.I
R.IQETQAELPR.G
R.VSGVDGYETEGIR.G
R.VSGVDGYETEGIRGLR.A
K.SQFLKHVEEFSPR.A
K.HVEEFSPR.A
R.TSILAAANPISGHYDR.S
R.SELVNWYLK.E
Sequence
R.SPQNQYPAELMR.R
R.RFELYFQGPSSNKPR.V
K.MQEHSDQVPVGNIPR.S
R.SITVLVEGENTR.I
R.SLEQNIQLPAALLSR.F
R.LAQHITYVHQHSR.Q
R.EAWASKDATYTSAR.T
R.MVDVVEKEDVNEAIR.L
R.TQRPADVIFATVR.E
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Supplementary figure 1S Interaction of MCM with NP Associated with vRNP but
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not that Free of vRNA.
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(mnRNP; lanes 4-6) was incubated with rMCM complex at 30ºC for 1 h. Micrococcal
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nuclease digests vRNA of vRNP and generates NP free of vRNA (Momose et al., 2001).
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After incubation, immunoprecipitation assays were carried out in the absence (lanes 2
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and 5) or presence (lanes 3 and 6) of goat anti-MCM2 antibody, and then
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immunoprecipitated proteins were visualized by western blotting assays with rabbit
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anti-NP antibody.
The vRNP (lanes 1-3) or micrococcal nuclease-treated vRNP
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Supplementary figure 2S
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at G1 and S Phases of Cell Cycle. (A) The scheme for experiments.
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were synchronized at the G1/S boundary in growth medium containing 2 mM thymidine,
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and then released by washing with fresh growth medium.
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the cells were infected with influenza virus at an M.O.I. = 10 and further incubated for 2
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h. (B)
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release, cells were trypsinized, and fixed in 70% ethanol.
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cells were stained with propidium iodide, and subjected to FACS analyses to determine
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the cell proportion in S (2 h post release) and G1 (11 h post release) phases of the cell
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cycle by measuring DNA content.
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in S phase, while 85% of cells stayed in G1 phase at 11 h post release.
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synthesis level of cRNA and mRNA in the S and G1 phases.
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purified from infected cells, and the synthesis level of cRNA and mRNA was
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semi-quantitatively analyzed by RT-PCR with primer sets specific for segment 5 cRNA
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and NP mRNA as described in Supplementary methods. Products were separated
The Level of cRNA and mRNA Synthesis in Cells Staying
HeLa cells
At 2 h and 11 h post release,
Fluorescence-activated cell sorter (FACS) analysis. At 2 h and 11 h post
After RNase A treatment,
At 2 h post release, 75% of cells were synchronized
(C) The
Total RNAs were
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1
through 7% PAGE and visualized by ethidium bromide. To quantitatively evaluate,
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mock-treated sample (lane 1), 10%, 30%, and 100% of infected S phase sample (lanes
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2-4), and 100% of infected G1 phase sample (lane 5) were subjected to RT-PCR.
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5
Supplementary figure 3S
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MCM2 KD Cells.
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HeLa cells were mock-transfected or transfected with random siRNA (lane 2) and
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MCM2 siRNAs (lane 3). At 60 h post transfection, cell lysates were prepared, and then
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subjected to western blotting analyses with goat anti-MCM2 and mouse anti--actin
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antibodies. (B) The level of virus genome replication in MCM2 knock-down cells.
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At 60 h post siRNA transfection, cells were transfected with viral protein expression
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plasmids encoding PB1, PB2, PA, NP, and pHH21-vNS-Luc plasmid, in which the
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luciferase gene of reverse orientation sandwiched with 23 nucleotide-long 5′- and 26
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nucleotide-long 3′-terminal promoter sequences of the influenza virus segment 8 is
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placed under the control of human Pol I promote (a gift from F. Momose).
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system, we can detect virus genome replication independent of viral transcription since
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viral proteins involved in virus genome replication are expressed stably from plasmids
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(Fodor et al., 2002).
After additional incubation for 24 h, total RNAs were purified,
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and
subjected
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5′-TATGAACATTTCGCAGCCTACCGTAGTGTT-3′, corresponding to the luciferase
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coding region between nucleotide sequence positions 351 to 380 for reverse transcription
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of vRNA, 5′-AGTAGAAACAAGGGTGTTTTTTAGTA-3′, which is complementary to
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the 3′ portion of the segment 8 cRNA for synthesizing cDNA of cRNA, or oligo(dT)20 for
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the synthesis of cDNA for mRNA.
then
The Level of Steady-state Synthesis of vRNA and cRNA in
(A) Expression level of MCM2 in MCM2 knock-down cells.
to
reverse
transcription
With this
with
These single-stranded cDNAs were subjected to
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1
real-time
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5′-TATGAACATTTCGCAGCCTACCGTAGTGTT-3′ corresponding to the luciferase
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coding
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5′-CCGGAATGATTTGATTGCCA-3′ complementary to the luciferase coding region
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between nucleotide sequence positions 681 to 700.
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(left panel) and cRNA (right panel) relative to that of mRNA are shown. The synthesis
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level of vRNA and cRNA in MCM KD cells reduced to around 50% of that in control
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cells.
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of luciferase mRNA synthesized from vNS-Luc model vRNA and NP mRNA
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transcribed from its expression plasmid were measured using real-time quantitative
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PCR with specific primer sets.
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that of the NP mRNA is shown.
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mRNA synthesis between control and MCM KD cells.
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replication on mRNA synthesis.
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the plasmid-based influenza virus replication system at 12, 16, and 20 h post
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transfection. At 24 h post transfection, total RNAs were purified, and subjected to
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real-time quantitative RT-PCR with primer sets specific for vNS-Luc model vRNA and
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luciferase mRNA.
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reduced, whereas that of mRNA was less affected.
quantitative
region
between
PCR
analyses
nucleotide
with
sequence
two
positions
specific
351
to
primers,
380
and
The ratio of the amount of vRNA
(C) The level of mRNA synthesis in MCM2 knock-down cells. The amounts
The ratio of the amount of luciferase mRNA relative to
There was no significant difference in the level of
(D) The effect of vRNA
CHX (100 g/ml) was added to cells transfected with
By the addition of CHX, the amount of vRNA was significantly
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Biological materials
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HeLa and WI-38 cells were grown in minimal essential medium (MEM) (Nissui)
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containing 10% fetal bovine serum. Rabbit anti-MCM2, 3, 4, 5, 6, and 7 antibodies were
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kind gifts from H. Nojima and N. Yabuta. Goat anti-MCM2 antibody (N-19) and Goat
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control IgG were purchased from SANTA CRUZ BIOTECHNOLOGY.
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expressing full-length PB1 (pCAGGS-PB1), PB2 (pCAGGS-PB2), PA (pCAGGS-PA),
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NP
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PB2-FLAG (pCAGGS-PB2cFLAG), and PA-FLAG (pCAGGS-PAcFLAG) were
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prepared as previously described (Naito et al., 2007). A plasmid for expression of
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(pCAGGS-NP),
FLAG-tagged
PB1
(PB1-FLAG;
Plasmids
pCAGGS-PB1cFLAG),
Myc-tagged NP (pCAGGS-NP-Myc) was kindly provided from F. Momose.
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Purification of IREF-1
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The purification scheme started with nuclear extracts prepared from uninfected HeLa
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cells (Dignam et al., 1983).
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phosphocellulose column (P11, Whatman) equilibrated with buffer H (50 mM
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HEPES-NaOH [pH 7.9], 1 mM dithiothreitol, and 20% [vol/vol] glycerol) containing 50
17
mM KCl. The column was washed with buffer H containing 50 mM KCl, and proteins
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adsorbed to the column were eluted with buffer H containing 0.2 M KCl. (NH4)2SO4
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was added to the fraction eluted by 0.2 M KCl, and the concentration was adjusted to 0.5
20
M (NH4)2SO4.
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Pharmacia). After washing the column with buffer H containing 0.5 M (NH4)2SO4,
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proteins adsorbed to the column were eluted with buffer H containing 0.25 M (NH4)2SO4.
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This eluate was dialyzed against buffer H containing 0.1 M KCl and then applied to a
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Mono Q PC 1.6/5 column (Amersham Pharmacia) equilibrated with buffer H containing
Nuclear extracts (40 mg of proteins) were loaded onto a
The fraction was loaded onto a phenyl-Sepharose (Amersham
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1
0.1 M KCl. The IREF-1 activity was eluted with a linear gradient of 0.1 to 0.6 M KCl.
2
3
Preparation of recombinant MCM complex
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We purified recombinant MCM complex by procedure described previously with slight
5
modification (You et al., 1999).
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baculoviruses carrying MCM2/7, MCM3/5, and MCM4/6 genes (kind gifts from Y.
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Ishimi) at multiplicity of infection of approximately 5 and then collected at 60 hr post
8
infection. The recombinant MCM complex was purified by Ni-nitrilotriacetic acid
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(NTA) affinity column chromatography.
In brief, Sf9 insect cells were co-infected with
Partially purified MCM complex was
10
concentrated with a Vivaspin 500 apparatus (Sartorius).
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complex was loaded onto a gel filtration column (Superose 6 PC 3.2/30; Amersham
12
Pharmacia Biotech). The fractions containing MCM complex were pooled and then
13
concentrated by a Mono Q column equilibrated with buffer H containing 0.1 M KCl.
The concentrated MCM
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15
Immunoprecipitation
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Infected or transfected cells crosslinked with 0.5 mM Dithiobis(succinimidylpropinate)
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(DSP; Pierce) and 0.5% formaldehyde for 10 min at room temperature were lysed by
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sonication in RIPA buffer (20 mM Tris-HCl [pH 7.9], 150 mM NaCl, 0.1% SDS, 1%
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NP-40, and 1% deoxycholic acid). The lysates were subjected to centrifugation at
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12,000 xg, and the supernatant fraction was subjected to immunoprecipitation with
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antibodies indicated in each figure legend and protein A agarose beads (Amersham
22
Pharmacia). After incubation, the beads were washed three times with RIPA buffer.
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Proteins bound to the bead were eluted by boiling in an SDS-PAGE loading buffer, and
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then subjected to 7.5% SDS-PAGE. To identify viral proteins and MCM proteins, rabbit
7
1
anti-PB1, -PB2, -PA, -NP, -MCM2, -MCM3, -MCM4, -MCM5, -MCM7, and goat
2
anti-MCM2 antibodies were used for the western blotting analysis.
3
immunoprecipitated from infected cells using anti-MCM2 antibody were subjected to
4
reverse-crosslinking in a buffer containing 50 mM Tris-HCl (pH 7.9), 5 mM EDTA, 50
5
mM DTT, and 1% SDS for 45 min at 70ºC. After reverse crosslinking, RNAs were
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purified, and then semi-quantitatively analyzed by RT-PCR with primers specific for
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segment 5 vRNA, 5′-GACGATGCAACGGCTGGTCTG-3′ (for reverse transcription and
8
PCR) and 5′-AGCATTGTTCCAACTCCTTT-3′ (for PCR).
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through 7% PAGE and visualized by ethidium bromide.
vRNAs
Products were separated
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11
siRNA-mediated gene silencing
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HeLa and WI-38 cells were transfected with stealth RNAi duplex oligonucleotides
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(Invitrogen) targeting MCM2 mRNA (5′-GGUCAACAUGGAGGAGACCAUCUAU-3′,
14
5′-GCGAAGUCGCAGUUUCUCAAGUAUA-3′,
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5′-AGGACACUAUUGAGGUCCCUGAGAA-3′)
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(5′-CCAUGGGCAUGACUAUGUCAAGAAA-3′,
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5′-GAGGCGUGGUUUGCAUUGAUGAAUU-3′,
18
5′-CCUUGAGACAGAAUAUGGCCUUUCU-3′)
19
(Invitrogen). At 60 h post transfection, cells were infected with influenza virus at an
20
M.O.I. = 10.
21
extraction followed by DNase I treatment. Purified RNA was then reverse-transcribed
22
with 5′-AGTAGAAACAAGGGTATTTTTCTTTA-3′, which is complementary to the 3′
23
portion of the segment 5 cRNA for synthesizing cDNA of cRNA, or oligo(dT)20 for the
24
synthesis of cDNA for mRNA.
and
and
MCM3
mRNA
and
using
Lipofectamine
2000
Total RNA was isolated after further incubation for 8 hr by phenol
These single-stranded cDNAs were subjected to
8
1
real-time quantitative PCR analyses (Thermal Cycler DiceTM Real Time System TP800;
2
TaKaRa)
3
5′-GACGATGCAACGGCTGGTCTG-3′ corresponding to the NP mRNA between
4
nucleotide sequence positions 424 to 444 and 5′-AGCATTGTTCCAACTCCTTT-3′
5
complementary to the NP mRNA between nucleotide sequence positions 595 to 614.
6
-actin
7
5′-ATGGGTCAGAAGGATTCCTATGT-3′ corresponding to the -actin mRNA
8
between
9
5′-GGTCATCTTCTCGCGGTT-3′ complementary to the -actin mRNA between
10
nucleotide sequence positions 343 to 360. The relative amounts of cRNA and mRNAs
11
were calculated by using the 2nd derivative maximum method. The ratio of the amount
12
of cRNA and mRNA relative to the -actin mRNA are shown.
with
mRNA
SYBR
was
nucleotide
Premix
also
Ex
Taq
amplified
sequence
and
with
positions
two
two
139
specific
specific
to
161
primers,
primers,
and
13
14
Thin-layer chromatography
15
The [-32P]GTP-labeled 12 nt-long products synthesized in the limited elongation RNA
16
synthesis assay were separated by 15% PAGE containing 8 M urea, and then eluted from
17
the gel. The purified RNA products were mock-treated or treated with an E. coli alkaline
18
phosphatase. Treated products were digested with either RNase T2 or snake venom
19
phosphodiesterase. Digested products were separated by a polyethylenimine-cellulose
20
thin-layer chromatography (Merck) with 1.6 M LiCl, and visualized by autoradiography.
21
22
9
1
References
2
3
Dignam, J.D., Lebovitz, R.M. and Roeder, R.G. (1983) Accurate transcription initiation
4
by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
5
Nucleic Acids Res, 11, 1475-1489.
6
Fodor, E., Crow, M., Mingay, L.J., Deng, T., Sharps, J., Fechter, P. and Brownlee, G.G.
7
(2002) A single amino acid mutation in the PA subunit of the influenza virus
8
RNA polymerase inhibits endonucleolytic cleavage of capped RNAs. J Virol, 76,
9
8989-9001.
10
Momose, F., Basler, C.F., O'Neill, R.E., Iwamatsu, A., Palese, P. and Nagata, K. (2001)
11
Cellular splicing factor RAF-2p48/NPI-5/BAT1/UAP56 interacts with the
12
influenza virus nucleoprotein and enhances viral RNA synthesis. J Virol, 75,
13
1899-1908.
14
Naito, T., Momose, F., Kawaguchi, A. and Nagata, K. (2007) Involvement of Hsp90 in
15
assembly and nuclear import of influenza virus RNA polymerase subunits. J
16
Virol, 81, 1339-1349.
17
You, Z., Komamura, Y. and Ishimi, Y. (1999) Biochemical analysis of the intrinsic
18
Mcm4-Mcm6-mcm7 DNA helicase activity. Mol Cell Biol, 19, 8003-8015.
19
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