Varicella zoster virus inhibition of the NFκB pathway during infection of human 1
dendritic cells: role for ORF61 as a modulator of NFκB activity 2
3
Elizabeth Sloan1,2, Rodney Henriquez1,2, Paul Kinchington3, Barry Slobedman1,2*, 4
Allison Abendroth#1,2* 5
6 1Centre for Virus Research, Westmead Millennium Institute, 2Dept Infectious 7
Diseases and Immunology, University of Sydney, 3Departments of Ophthalmology & 8
of Molecular Microbiology and Genetics, University of Pittsburgh, Pittsburgh PA. 9
10
11
12
13 #Address for correspondence: 14
Associate Professor Allison Abendroth 15
Department of Infectious Diseases and Immunology 16
The University of Sydney, New South Wales, Australia 2006. 17
Ph: +61-2-93516878 18
Fax: +61-2-93514731 19
Email: [email protected] 20
21
22
Running Title: Modulation of NFκΒ activity by VZV ORF61 23
24
Word count for abstract: 229 25
Word count for text: 5171 26
Figures: 7 27
Tables: 0 28
29
* These authors contributed equally towards this work. 30
31
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.06400-11 JVI Accepts, published online ahead of print on 16 November 2011
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32
ABSTRACT 33
Dendritic cells (DC) are antigen presenting cells essential for initiating primary 34
immune responses and therefore an ideal target for viral immune evasion. Varicella 35
zoster virus (VZV) can productively infect immature human DCs and impair their 36
function as immune effectors by inhibiting their maturation, evident by expression 37
modulation of functionally important cell-surface immune molecules CD80, CD86, 38
CD83 and MHC I. The NFκB pathway largely regulates expression of these immune 39
molecules and therefore we sought to determine whether VZV infection of DCs 40
modulates the NFκB pathway. Nuclear localization of NFκB p50 and p65 indicates 41
pathway activation; however, immunofluorescence studies revealed cytoplasmic 42
retention of these NFκB subunits in VZV infected DCs. Western blotting revealed 43
phosphorylation of the inhibitor of κBα (IκBα) in VZV infected DC, indicating the 44
pathway is active at this point. We conclude VZV infection of DC inhibits the NFκB 45
pathway following protein phosphorylation but before translocation of NFκB subunits 46
into the nucleus. An NFκB reporter assay identified VZV ORF61 as an inhibitor of 47
TNFα induced NFκB reporter activity. Mutational analysis of ORF61 identified the 48
E3 Ubiquitin ligase domain as a region required for NFκB pathway inhibition. In 49
summary, we provide evidence that VZV inhibits the NFκB signaling pathway in 50
human DCs, and that the E3 Ubiquitin ligase domain of ORF61 is required to 51
modulate this pathway. Thus, this work identifies a mechanism by which VZV 52
modulates host immune function. 53
54
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INTRODUCTION 55
Varicella zoster virus (VZV) is an α-herpesvirus causing chickenpox (varicella) 56
during primary infection and shingles (herpes zoster) following reactivation from a 57
latent infection. Following initial exposure to the virus, there is a 10-21 day 58
incubation period before the appearance of the varicella rash. During this time it has 59
been proposed that VZV actively evades immune recognition in this period, as 60
development of adaptive immunity is delayed (reviewed in (1). We have postulated 61
that VZV infection of DCs and/or modulation of the immune function of these potent 62
antigen presenting cells would provide a strategy that would enhance the capacity of 63
the virus to be transported from the site of inoculation to the draining lymph nodes to 64
infect T cells whilst also evading immune detection. 65
66
We have previously shown that VZV can productively infect human dendritic cells in 67
vitro and in vivo (2, 16, 22). These studies included demonstration that productively 68
infected immature monocyte derived DCs (MDDCs) are unable to upregulate 69
functionally important immune molecules CD80, CD83, CD86, MHC-I or CCR7, 70
which are required for DC maturation and induction of an effective anti-viral immune 71
response (2). The expression of the immune molecules inhibited by VZV are largely 72
regulated by the nuclear factor kappa B (NFκB) signal transduction pathway (4, 6, 12-73
14). The NFκB signal transduction pathway is an important regulator of innate 74
immunity and inflammation that is triggered by a wide variety of stimuli, including 75
virus infection, TNFα, and other cytokines and pathogens (26, 29). Activation of the 76
NFκB pathway via pattern recognition receptors (PRRs) results in the 77
phosphorylation of inhibitor of κB kinase complex (IKK) which in turn 78
phosphorylates IκB, targeting it for ubiquitination and degradation, allowing NFκB 79
proteins (p50 and p65) to translocate into the nucleus and bind to promoters 80
containing NFκB response elements, initiating transcription of target genes (reviewed 81
in (26, 29)). 82
83
Herpesviruses encode multiple proteins that function in immune evasion, and several 84
herpesvirus proteins target and disrupt the NFκB pathway. Viral genes encoded by 85
Epstein Barr virus (19, 27, 28), cytomegalovirus (23, 34), and herpes simplex virus 86
type-1 (3, 9, 24) have been identified to regulate the NFκB pathway in a cell type 87
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dependent manner. Jones and Arvin (2006) reported that VZV inhibits the NFκB 88
pathway in human fibroblasts in vitro and in vivo following phosphorylation and 89
ubiquitination of IκBα, but prior to the translocation of NFκB proteins into the 90
nucleus (17). 91
92
In this study, we sought to extend these studies and examine the effect of VZV on the 93
NFκB pathway within VZV infected human MDDCs. Using flow cytometry, 94
immunofluorescent (IFA) staining and western blotting, we establish the point where 95
VZV impacts the NFκB pathway in VZV antigen positive DCs. In addition, using a 96
transient transfection approach and flow cytometry, we identified the E3 Ubiquitin 97
ligase domain of VZV ORF61 as responsible for inhibition of TNFα-induced NFκB 98
reporter activity. In summary, this study provides evidence that VZV inhibits the 99
NFκΒ signaling pathway in human DCs and defines a role for ORF61 as a modulator 100
of this pathway. 101
102
103 104
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MATERIALS AND METHODS 105
Viruses and cell culture. Peripheral blood mononuclear cells were isolated from 106
healthy adult donors by Ficoll-Hypaque density gradient (Amersham Pharmacia 107
Biotech, Sweden) in accordance with University of Sydney Human Ethics Approval. 108
Monocytes were isolated by CD14 magnetic bead separation (MACS Miltenyi Biotec, 109
Germany) and resuspended at 5 x 105 cells/ml in RPMI (Gibco, Gaithersburg, Md.) 110
containing 10% heat-inactivated Foetal Bovine Serum (FBS, CSL, Australia) 111
supplemented with interleukin 4 (IL-4) 500U/ml (Schering Pleogh, Germany), and 112
granulocyte-macrophage-colony-stimulating factor (GM-CSF, 400U/ml) (Schering 113
Pleogh, Germany). Cells were cultured for 6 days. Typically >90% of the monocyte 114
derived dendritic cells (MDDC) were shown by flow cytometry analysis to be 115
CD1a+/CD14- and have an immature dendritic cell phenotype as we have previously 116
described (2). 117
118
Human foreskin fibroblasts (HFFs) (ATCC) and human embryonic kidney cells 119
(293FT) (Invitrogen, USA) were cultured in Dulbecco’s modified Eagle’s medium 120
(DMEM; Gibco, Gaithersburg, Md.) containing 10% heat-inactivated FBS and 1% 121
PenStrep. Recombinant OKA (rOKA) VZV, kindly provided by Prof Arvin, Stanford 122
University, was propagated in HFFs and was used for cell-associated infections when 123
60-80% of cells were infected. HSV-1 strain (CW) was kindly provided by Prof 124
Cunningham, Westmead Millennium Institute (20). 125
126
TNFα treated HFFs, MDDCs and 293FT transfected cells were included as controls 127
for flow cytometry, western blot analysis and NFκB reporter assay. HFFs and 128
MDDCs were treated with 20 nM TNFα (R&D Systems, USA) 5 mins prior to 129
harvest. 293FT transfected cells were treated with 20 nM TNFα 24 hours prior to 130
analysis. 131
132
VZV infection of DCs. Dendritic cells were infected using VZV rOKA as previously 133
described (2). Mock infected dendritic cells were prepared in parallel using uninfected 134
HFFs as an inoculum. 135
136
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Antibodies. Antibodies for human polyclonal NFκB p50 and NFκB p65 were 137
obtained from Santa Cruz Biotechnology (USA) and used for immunofluorescence 138
staining. Human IκBα, phosphorylated IκBα, NFκB p50 and NFκB p65 antibodies 139
were obtained from Cell Signaling Technologies (USA) and used for western blot 140
analysis. Monoclonal α-Tubulin used for western blot analysis was obtained from 141
Millipore (USA) and Topoisomerase II (Ab-1) from Merck (Australia). Monoclonal 142
TLR3-fluorescein isothiocyanate (FITC) conjugated (clone 40C1285.6), TLR8-FITC 143
(clone #44C143) and TLR9-FITC (clone 26C593.2) antibodies were obtained from 144
Imgenex (USA). Monoclonal CD1a-Allophycocyanin (APC) conjugated and CD14-145
FITC conjugated antibodies were obtained from BD Biosciences (Australia). CD120a 146
(TNF-Rec p55) (clone #MR1-2) and CD120b (TNF-Rec p75) were obtained from 147
Biodesign (USA) and Caltag (USA), respectively. Anti-HA-Alexafluor ® 647 148
antibody was obtained from Cell Signaling Technologies (USA). Antibodies used to 149
detect VZV antigens were VZV glycoprotein E (gE) (Chemicon, Australia), VZV gB 150
(Biodesign, USA) and VZV-mixed epitope-FITC (Biodesign, USA). Isotype control 151
antibodies included mouse monoclonal IgG1-APC (BD Biosciences, USA), mouse 152
monoclonal IgG2aκ-PE (Pharmingen, USA), mouse monoclonal IgG2a-FITC (Caltag, 153
USA) and normal rabbit IgG (Santa Cruz Biotechnology, USA). 154
155
Flow cytometry. MDDC were washed twice in 1xPBS and fixed using 1% 156
paraformaldehyde (PFA) for 15mins. Cells were washed twice in 1xPBS before 157
permeabilization using 0.5% Saponin (Sigma-Aldrich, Australia) in 1xPBS containing 158
1% FBS at 105 cells/100μl for 15mins at room temperature. Primary antibodies were 159
diluted 1:50 for anti-human TLR3-FITC, TLR8-FITC, and TLR9-FITC, and 1:200 for 160
VZV gB in 0.5% Saponin/PBS. Secondary antibody for gB, goat-anti mouse-PE was 161
diluted 1:200 in 0.5% Saponin/PBS. Primary and secondary antibody incubations 162
were for 20mins at room temperature in the dark. Cells were washed twice in 0.5% 163
Saponin/PBS between each incubation with a final resuspension in FACS buffer 164
(1xPBS containing 1% FBS and 0.2% sodium azide) before acquisition using 165
FACScanto (Beckton Dickinson, USA) and analysis using FlowJo analysis software. 166
Cells were incubated with appropriate isotype control antibodies in parallel. A signal 167
exceeding the level of 98% of the isotype control cell sample was considered to be 168
positive for the cell specific antibody staining. 169
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170
TNFα stimulated 293FT cells co-transfected with HA tagged expression constructs 171
and pNFκB-hrGFP reporter (Stratagene, Agilent Technologies, Australia) were 172
washed twice in 1xPBS and fixed using 1xPBS containing 1% PFA. Cells were 173
washed twice in 1xPBS before permeabilization using 100% ice-cold methanol 174
incubated at -20˚C for 30mins. Cells were washed twice in 1xPBS and resuspended in 175
FACS buffer. Anti-HA-Alexafluor ® 647 antibody was diluted 1:50 in FACS buffer 176
and incubated for 30mins at room temperature. Cells were washed twice in FACS 177
buffer and resuspended in 400μl FACS buffer prior to analysis with FACScanto and 178
Flow Jo software. 293FT cells not exposed to TNFα and no plasmid DNA 179
transfection controls were performed in parallel. 180
181
Fluorescence activated cell sorting (FACS). Cells were washed twice in 1xPBS and 182
resuspended in 500μl FACS buffer per 2.5 x 106 cells. Anti-CD1a-APC (20μl/2.5 x 183
106 cells) and anti-VZV-mixed epitope-FITC (3.5μl/2.5 x 106 cells) primary 184
antibodies were incubated for 20 mins at 4˚C in the dark. Cells were washed three 185
times in FACS buffer, resuspended in FACS buffer (3 x 106 cells/ml) and sorted 186
immediately using FACS Vantage (Becton Dickinson, USA). Cells were sorted to 187
positively select for CD1a+VZV+ cells. To establish non-specific background staining, 188
cells were incubated with appropriate isotype control antibodies and a signal 189
exceeding the level of 98% of the isotype control cell sample was considered to be 190
positive for the cell surface specific antibody staining. Mock infected cells were 191
stained in parallel using CD1a-APC antibody only and sorted for CD1a+ cells. Purity 192
of DCs post sorting was typically >98% for mock infected DCs and >93% for VZV 193
infected DC population. Sorted DCs were used for subsequent immunofluorescence 194
staining or western blot analyses. 195
196
Western Blot. Cells were incubated for 10 mins in lysis buffer (50mM Tris pH 7.4, 197
240mM NaCl, 0.5% NP-40, 10% glycerol, and 0.1 mM EDTA) containing a protease 198
inhibitor cocktail (Roche, Australia). Cell lysates for immunoblotting using phospho-199
specific antibodies were lysed using PhosphoSafe extraction reagent (Merck, 200
Australia) according to manufacturer’s instructions. Proteins were resolved by 12% 201
polyacrylamide gels (BioRad, Australia), transferred to Hybond-P polyvinylidene 202
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difluoride (PVDF) membrane (Amersham, UK), with non-specific sites blocked using 203
5% skim milk powder in 1xTBST (Tris-buffered saline containing 0.1% Tween 20) 204
for at least 1 hour. Primary antibodies were incubated for 1 hour or O/N diluted in 5% 205
skim milk powder - or 5% BSA/TBST, respectively. Membranes were washed in 206
1xTBST for 3 x 5mins intervals and secondary antibody conjugated to horseradish 207
peroxidase was incubated for 1 hour, followed by a 3 x 5mins TBST wash and 208
exposed using ECL Plus detection system (GE Healthcare, Australia). Densitometry 209
was performed using Kodak Image Station 4000MM (Carestream Health INC. USA) 210
to quantify relative protein amounts detected by western blotting. 211
212
Immunofluorescence staining and confocal microscopy. Immunofluorescence 213
staining was performed as previously described (15). At least 2 x 105 cells were 214
spotted onto glass slides. Primary antibodies were incubated for 1 hour at room 215
temperature, and secondary antibodies for 45mins. Slides were mounted using 216
Prolong slowfade Gold with 4'-6-Diamidino-2-phenylindole (DAPI) (Invitrogen, 217
USA) and confocal analysis performed using Olympus FV1000 confocal microscope. 218
Percentage of cells with NFκB p50 or p65 nuclear localized staining was determined 219
by counting 100 cells per slide. 220 221
Plasmids and transfection. A series of plasmids was used, developed in the vector 222
pGK2-HA (parental construct) (11), designated pGK2-ORF61-HA, pGK2-ORF64-223
HA, pGK2-ORF47-HA, pGK2-ORF2-HA, pGK2-ORF21-HA, pGK2-ORF23-HA 224
and pGK2-ORF49-HA depending on the designated ORF. Each was generated by 225
PCR amplification of the complete ORF from VZV, using primers designed with 226
flanking restriction sites (either EcoRI or mfeI at the amino end, and bclI, bamhI or 227
BglII at the 3’ end) for ligation into the pGK2-HA construct. All ORFs contain an 228
amino terminal HA tag for antibody recognition. Plasmid pIRES2-ORF61-DsRED2 229
was generated by PCR amplification of the complete ORF61 sequence from VZV S 230
strain, using primers designed with flanking EcoRI and BamHI sites to facilitate 231
cloning into EcoRI and BamHI sites of pIRES2-DsRED2 (Clontech, USA). Fugene 232
HD (Promega) was utilized for transfection of 293FT cells as per manufacturer’s 233
instructions using a DNA to Fugene ratio of 2:6. Cells were seeded 24 hours prior to 234
transfection so they were 30% confluent at time of transfection. Transfected cells 235
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were harvested at 48 hours post transfection and analyzed by flow cytometry or 236
western blot analysis. 237
238
Site-directed mutagenesis. QuikChange II XL site-directed mutagenesis kit 239
(Stratagene, Agilent Technologies, Australia) was used to construct pIRES2-ORF61 240
C19G (C19G; disrupt RING finger) (31), pIRES2-ORF61 Q62_START (M1T, 241
M23T, M33T, Q62M; express following RING finger domain), pIRES2-ORF61 242
Q62_STOP (Q62Stop; express RING finger domain) and pIRES2-ORF61 243
Q139_STOP (Q139Stop; express E3 Ubiquitin ligase domain) as per manufacturers 244
instructions. Primers were designed using PrimerX software, with sequences available 245
upon request. All mutations were confirmed by sequencing using Australian Genome 246
Research Facility (AGRF). Protein expression was confirmed by flow cytometry and 247
western blot analysis. 248
249
NFκB reporter Assay. The day prior to transfection 293FT cells were seeded into 6-250
well plates so they were 30% confluent at time of transfection. Cells were transfected 251
using Fugene HD with 2μg pNFκB-hrGFP (Stratagene, Agilent Technologies, 252
Australia) reporter construct and 1μg DsRED2 or HA tagged expression construct. 253
TNFα (20 nM) was added following 24 hours post transfection and cultured for a 254
further 24 hours before cells were harvested and analyzed by flow cytometry using 255
FACScanto and Flow Jo software as described above. Percentage of VZV ORF+GFP+ 256
cells were normalized to the respective parental plasmid, pIRES2-DsRED2 or pGK2-257
HA. 258
259
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RESULTS 260
VZV infection prevents NFκB p50 and p65 translocation into the nucleus of DCs 261
VZV infection of immature DCs inhibits their maturation in vitro (2). It has also been 262
reported that VZV actively inhibits NFκB signal transcription pathways within 263
infected fibroblasts in vitro and in vivo (17). Given the importance of NFκB signal 264
transduction pathways in regulating expression of functionally important immune 265
molecules expressed by DCs, we sought to determine whether VZV modulated NFκB 266
signaling within human DCs. 267
268
We first investigated the cellular localization of the NFκB subunits, p50 and p65, via 269
immunofluorescent staining and confocal microscopy, within VZV infected DCs. 270
Human MDDCs were infected using an established cell-associated method of 271
infection by using VZV rOKA infected fibroblasts (HFFs) to infect immature DCs at 272
a DC:fibroblast ratio of 2:1 (2, 22). DCs were differentiated from the HFF inoculum 273
using CD1a immunostaining, a marker for DCs (2). Since our infection protocol does 274
not result in 100% of DCs becoming infected (2), cells were also stained with anti-275
VZV antigen antibody (VZV-mixed epitope-FITC), and subjected to fluorescence 276
activated cell sorting (FACS) to select for VZV+CD1a+ DCs. Purity of VZV+/CD1a+ 277
sorted populations was typically 93-98%. Mock infected MDDCs were 278
immunostained in parallel and CD1a+ cells isolated by FACS. Figure 1 shows 279
localization of p50 and figure 2 shows distribution of the p65 subunit. 280
281
VZV infected DCs and mock infected DCs were spotted onto glass microscope slides, 282
fixed, permeabilized and stained with anti-NFκB p50 or p65 antibody (Red) along 283
with an anti-VZV gE antibody (Green). In parallel, this staining was also performed 284
on TNFα treated uninfected HFFs and uninfected DCs (as positive controls for 285
activation of the NFκB pathway), uninfected HFFs (as a negative control for 286
activation of the NFκB pathway), as well as VZV infected HFFs, as a control for 287
inhibition of NFκB activation (17). In parallel duplicate cell spots were stained with 288
isotype control antibodies to establish background fluorescence. All immunostained 289
slides were then analyzed by confocal microscopy. 290
291
Mock infected DCs did not stain for VZV antigens and the majority of cells showed 292
predominant cytoplasmic localization of NFκB p50 and p65 subunits, indicating an 293
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inactive NFκB pathway (Figure 1A and 2A). An average of 31% and 29% of mock 294
infected DCs showed nuclear staining for NFκB p50 and p65, respectively. NFκB p50 295
and p65 localized to the nucleus in 100% of DCs treated with TNFα (Figures 1B and 296
2B), consistent with activation of the NFκB pathway. In stark contrast, VZV+ CD1a+ 297
DCs showed an average of only 8% of cells with NFκB p50 localized to the nucleus 298
(Figure 1C). NFκB p65 within VZV+CD1a+ DCs localized predominantly to the 299
cytoplasm with no detectable nuclear location (Figure 2C). This indicates that VZV 300
prevents NFκB subunits from translocating into the nucleus of VZV antigen positive 301
cells. In a side by side comparison, the NFκB pathway within unstimulated HFFs was 302
predominantly inactive as demonstrated by cytoplasmic NFκB p50 and NFκB p65 303
staining within 67% and 100% of uninfected cells, respectively (Figures 1D and 2D), 304
and active within TNFα treated HFFs with NFκB p50 localized to the nucleus of 83% 305
of cells and NFκB p65 localized to the nucleus of 100% of cells within this group 306
(Figures 1E and 2E). In VZV infection of HFFs, typically 80% of HFFs were positive 307
for VZV antigen, and an average of 20% of VZV antigen positive HFFs showed 308
NFκB p50 localized to the nucleus, with 4% of cells with nuclear localized NFκB p65 309
(Figure 1F and 2F), suggesting that the NFκB pathway is predominantly inactive 310
within these cells. We conclude VZV efficiently prevents nuclear localization of 311
NFκB subunits in DCs. 312
313
Four replicate experiments were quantified for the percentage of cells with nuclear 314
localization of NFκB proteins (Figure 1G and 2G). The VZV infected DCs had a 315
greatly reduced ability to activate the NFκB pathway, as indicated by the predominant 316
cytoplasmic localization of both NFκB p50 and p65 subunits. Taken together, these 317
results establish VZV modulation of the NFκB pathway by sequestering NFκB 318
protein subunits, p50 and p65 in the cytoplasm of infected DCs. 319
320
VZV does not down-modulate expression of NFκB pathway stimulatory 321
receptors; TLR-3, 8, 9 or TNFR-1 or 2 within infected DCs 322
The NFκB pathway was subsequently examined for upstream events known to trigger 323
NFκB p50 and p65 nuclear translocation. Immune receptors that stimulate the NFκB 324
pathway include toll-like receptors (TLRs) and tumour necrosis factor receptors 325
(TNFRs) (26), so mock infected or VZV infected DCs at 48 hours post infection were 326
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costained for VZV antigen (VZV gB) and TLR3, TLR8, TLR9, TNFR-1 or TNFR-2 327
in conjunction with the DC cell surface marker CD1a. We have previously 328
demonstrated that VZV infection of DCs does not affect the cell surface expression of 329
this molecule on DCs (2). Additional controls consisted of mock infected and VZV 330
infected DCs immunostained with isotype control antibodies. Cells were then 331
analyzed by flow cytometry to determine the level of expression of each cellular 332
marker on VZV antigen positive DCs. Mean fluorescence intensity of immune 333
molecule expression by VZV antigen positive DCs from 3 independent replicate 334
experiments were averaged and normalized to mock infected DCs (Figure 3). There 335
was no significant difference in level of expression of TLR 3, 8, or 9 and TNFR-1 or 336
TNFR-2 when comparing VZV infected DCs to mock infected DCs. These results 337
demonstrate that VZV infection does not inhibit the expression of NFκB signaling 338
receptors, TLR-3, 8, 9 or TNFR-1 or 2 by DCs, suggesting the NFκB pathway may be 339
modulated downstream of these signaling receptors. 340
341
VZV infection of DCs induces phosphorylation of IκBα 342
The activation of the NFκB pathway routinely involves phosphorylation of IκB, 343
leading to its ubiquitination and degradation (14, 26). To assess IκB phosphorylation, 344
DCs were mock infected or VZV infected and 48 hours post infection were first 345
immunostained for VZV antigen and CD1a. VZV+CD1a+ DCs from infected DC 346
cultures and CD1a+ DCs from mock infected cultures were separated from any 347
remaining HFF inoculum by FACS, lysed and protein extracts examined by SDS-348
PAGE and western blot using antibodies against IκBα or phosphorylated IκBα (phos 349
IκBα). Relative amounts of each protein were determined by densitometry with all 350
values normalized to expression of α-tubulin, which was included as a protein loading 351
control (Figure 4). In 7 independent replicate experiments, it was observed that while 352
unstimulated HFFs showed basal levels of phosphorylation and mock CD1a+ DCs 353
demonstrated the presence of low levels of phosphorylated IκBα (Figure 4A), 354
VZV+CD1a+ DCs showed the presence of high levels of phosphorylated IκBα (Figure 355
4A). Average densitometry measurements of 7 replicate experiments, normalized to 356
α-tubulin and then to mock infected DC levels (Figure 4B) revealed VZV+CD1a+ 357
DCs demonstrated a statistically significant increase in expression of both IκBα and 358
phosphorylated IκBα (p< 0.05). These data demonstrate that impairment of the NFκB 359
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pathway in VZV infected DCs is not a consequence of inhibited phosphorylation of 360
IκBα as the pathway remains active at this step. 361
362
363
VZV ORF61 inhibits TNFα induced NFκB pathway activation 364
The inhibition of the NFκB pathway in VZV infected cells argues in favour of a viral 365
gene product which encodes this function. To identify viral gene products that 366
modulate the NFκB pathway, we used a transient transfection approach to assess the 367
ability of individual VZV gene products to inhibit NFκB activity. Due to the low 368
transfection efficiency of DCs (30), 293FT cells were co-transfected with pNFκB-369
hrGFP reporter along with a plasmid expressing, from the complete CMV IE 370
promoter, one of several HA tagged VZV open reading frames (ORFs): 2, 21, 23, 47, 371
49, 61 or 64. After 48 hours, co-transfected cells were stimulated with TNFα to 372
activate the NFκB pathway 24 hours prior to harvest. Cells were also stained with 373
anti-HA antibody prior to analysis by flow cytometry. The parental pGK2-HA 374
construct without any VZV ORF expressed a level of GFP expression indicative of 375
NFκB pathway activation that was set as a value of one. Three replicate experiments 376
were averaged and normalized to values from the cells transfected with the pGK2-HA 377
control and treated with TNFα (Fig 5). Relative to the control, plasmids expressing 378
VZV ORFs-64, 47, 2, 49, 21 and 23 did not significantly alter TNFα stimulated NFκB 379
reporter activity compared to the parental control. However, pGK2-HA VZV ORF61 380
showed a striking reduction in TNFα stimulated NFκB reporter activity, with 381
statistical significance (p< 0.00005). This suggested this viral gene product is at least 382
in part, responsible for inhibiting the NFκB pathway. 383
384
VZV ORF61 inhibits TNFα induced IκBα degradation 385
Phosphorylation of IκBα stimulates its ubiquitin-induced degradation, allowing 386
NFκB subunits to translocate into the nucleus for transcription of target genes (5). 387
However, no decrease in IκBα was seen following VZV infection of DCs and 388
phosphorylation of IκBα was readily detected. With the identification of ORF61 as an 389
inhibitor of NFκB pathway activity, we sought to determine if ORF61 functions to 390
inhibit IκBα degradation, preventing subsequent NFκB p50 and p65 translocation 391
into the nucleus. 293FT cells transiently transfected with pGK2-ORF61-HA and 392
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cultured for 48 hours followed by TNFα (20 nM, 5 mins) stimulation showed IκBα 393
protein was detected within all transfected 293FT cells following TNFα stimulation 394
(Figure 6A). However densitometric quantification of 3 replicate experiments, 395
normalized to α-Tubulin and then to levels in cells transfected with the parent 396
construct (Figure 6B), revealed ORF61 expression construct (pGK2-ORF61-HA) had 397
significantly more IκBα protein present following TNFα stimulation compared to the 398
parental construct control (p<0.05). These findings indicate that either ORF61 399
protects IκBα protein from TNFα-induced degradation, further defining the 400
mechanism of ORF61-mediated NFκB pathway inhibition, or that ORF61 upregulates 401
the expression of IκBα, as is consistent with previous cell specific transcriptional 402
activation. 403
404
The E3 Ubiquitin ligase domain of ORF61 is responsible for inhibiting TNFα 405
induced NFκB pathway activation 406
To explore the ORF61 inhibition of TNFα stimulated NFκB reporter activity further, 407
ORF61 was expressed using a vector, pIRES2-DsRED2, in which DsRed2 is a second 408
reporter of activity of expression. In the NFκB reporter assay, TNFα stimulated 409
pIRES2-DsRED2-ORF61 expressing 293FT cells reproducibly showed an average 410
65% reduction in NFκB reporter activity compared to pIRES2-DsRED2 parental 411
control, with statistical significance (p<0.005) (Figure 7B). Subsequent site-directed 412
mutagenesis was performed to produce mutations within functionally important 413
regions of ORF61 coding sequence within the pIRES2-DsRED2-ORF61 vector 414
(Figure 7A). These ORF61 mutant constructs were co-transfected along with pNFκB-415
hrGFP reporter into 293FT cells, stimulated with TNFα 24 hours post transfection, 416
and cultured for a further 24 hours. Transfected cells were analyzed by flow 417
cytometry, gating for GFP+DsRED+ cells, with GFP expression indicative of an active 418
NFκB pathway. Results from three replicate experiments were averaged and 419
normalized to cells co-transfected with the full length ORF61 construct (Figure 7B). 420
421
As shown for HA-tagged ORF61 expressing cells (Figure 5), cells expressing the full 422
length ORF61 displayed significantly decreased NFκB reporter activity compared to 423
the parental control, pIRES2-DsRED2 (p< 0.005). In contrast, the ORF61 mutant 424
constructs ORF61 Q62_STOP, ORF61 Q62_START and ORF61 C19G lost their 425
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capacity to inhibit the TNFα stimulated NFκB reporter activity. Only cells 426
transfected with the ORF61 Q139_STOP construct, which encompasses the E3 427
Ubiquitin ligase domain, retained the capacity to inhibit the NFκB pathway to the 428
level of inhibition seen for full length ORF61. Based on the protein regions expressed, 429
these results indicate the E3 Ubiquitin ligase domain of ORF61 is required to mediate 430
an inhibition of the NFκB pathway. Of note is that a single point mutation in the 431
ORF61 RING finger domain, known to disrupt the E3 ubiquitin like activity (31), lost 432
the capacity to inhibit the TNFα stimulated NFκB reporter activity. 433
434
DISCUSSION 435
This study identifies VZV encoded modulation of the NFκB signaling pathway within 436
infected DCs and identifies the viral gene product of ORF61 as functioning to inhibit 437
this important signal transduction pathway. Given that DCs are proposed as the first 438
immune cells to encounter VZV following inoculation, these results indicate that 439
ORF61 plays an important role in immune evasion of the NFκB signal transduction 440
pathway. This pathway controls transcription of many immune molecules required to 441
initiate an immune response to foreign pathogens, and so disruption of this pathway is 442
likely to suppress critical immune effector capacity of the host cell. 443
444
Other herpesviruses modulate the NFκB pathway in a cell type dependent manner (3, 445
9, 19, 23, 24, 27, 28, 34). Our work indicates VZV modulates the NFκB pathway 446
within DCs. The cytoplasmic retention of NFκB protein subunits p50 and p65 indicate 447
that the pathway is modulated prior to their translocation into the nucleus as shown by 448
immunofluorescence and confocal microscopy. These results are distinct from those 449
reported for HSV-1 infection where NFκB proteins are translocated into the nucleus 450
of HSV-1 infected C33-A cells (24). The NFκB pathway in VZV infected DCs was 451
not modulated by down-regulation of TLR-3, 8, 9 or TNFR-1 or 2, which signal the 452
activation of the NFκB pathway, as these were unaffected by VZV infection. This 453
finding is consistent with VZV interfering with NFκB signaling at a point 454
downstream of cell surface (TNFR-1 and TNFR-2) or intracellular (TLR-3, 8, 9) 455
receptors which trigger this pathway. 456
457
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A critical component of activation of the NFκB pathway is phosphorylation of IκBα 458
(5). We therefore explored the phosphorylation state of IκBα in infected DCs. The 459
increase in phosphorylated IκBα within VZV infected DCs indicated that the NFκB 460
pathway was able to be activated at this level following infection. In addition, IκBα 461
protein levels within DCs did not decrease following infection with VZV. Thus, IκBα 462
can be phosphorylated and is not necessarily degraded following VZV infection of 463
DCs. The NFκB pathway is therefore apparently inhibited following phosphorylation 464
of IκBα but prior to translocation of NFκB p50 and p65 into the nucleus of VZV 465
infected DCs. The identification of this step is under further consideration. 466
467
We identified VZV ORF61 as a gene responsible for inhibiting TNFα stimulated 468
NFκB reporter expression. VZV ORF61 is transcribed within 1 hour of infection, 469
however is not a component of the virion, supporting reports of UV-inactivated VZV 470
being unable to inhibit the NFκB pathway within fibroblasts and that de novo gene 471
synthesis is required (17, 18, 33). ORF61 has also been implicated in the modulation 472
of other cellular pathways, such as the MAPK pathways where a significant increase 473
in phosphorylation of JNK/SAPK and decrease in p38/MAPK phosphorylation was 474
observed in MeWo cells transfected with ORF61 (25). ORF61 also down-modulates 475
the IRF3-mediated IFNβ pathway by its direct binding to and degradation of IRF3 476
(35). We demonstrate that when ORF61 is expressed alone, it is sufficient to inhibit 477
TNFα-induced IκBα degradation, further defining the role of ORF61 in NFκB 478
pathway inhibition. We also showed that the E3 Ubiquitin ligase domain of ORF61 as 479
essential for NFκB pathway inhibition as mutations within this domain rendered 480
ORF61 unable to inhibit NFκB reporter activity. HSV-1 ICP0 and VZV ORF61 share 481
homology within their RING finger domains, which are involved in E3 Ubiquitin 482
ligase activity (21). ICP0 modulates the NFκB pathway in a cell type dependent 483
manner, as shown by transfection of ICP0 expression constructs into SHSY-5Y and 484
HEK293 cells. Within HEK293 cells stably expressing either TLR2 or TLR4, ICP0 485
causes inhibition of TLR-mediated NFκB signaling (9). ICP0 inhibits the NFκB 486
pathway within these cells by binding and transporting the host cell protein USP7, a 487
ubiquitin specific protease, to the cytoplasm where it de-ubiquitinates TRAF6 and 488
IKKγ. The USP7 binding site on ICP0 is within the C terminal region, due to the 489
homology of ICP0 and ORF61 being restricted to the RING finger domain in the N 490
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terminus, it is unlikely ORF61 encodes this USP7 binding region and therefore 491
inhibits the NFκB pathway in a different manner to its HSV-1 homologous protein in 492
HEK293/TLR cells. Expression of ICP0 within SHSY-5Y cells stimulated NFκB 493
reporter activity through the E3 Ubiquitin ligase action of ICP0 resulting in 494
ubiquitination and subsequent degradation of IκBα (10). Our demonstration that the 495
E3 Ubiquitin ligase domain of ORF61 is responsible for the inhibition of NFκB 496
reporter activity indicate that it may be the process of ubiquitination causing the 497
affect, however protein expression analysis of IκBα within VZV infected DCs 498
suggests IκBα is not degraded. The Vpu protein of HIV, however, blocks proteasome-499
dependent degradation of IκBα by binding to βTRCP in the E3 ubiquitin ligase 500
complex that is involved in the regulated degradation of IκBα (7). This suggests that 501
the E3 ubiquitin ligase activity of ORF61 may have an indirect effect on NFκB 502
proteins. 503
504
While we have shown that ORF61 is a viral factor that downregulates NFκB, we have 505
not yet verified if it is the only viral factor able to do this. This requires the 506
construction of VZV that lack the ORF61 protein, particularly in the amino terminal 507
ring finger domain. Such mutants have proven very difficult to develop. Using a 508
VZV bacterial artificial chromosome (BAC) system we found that VZV BAC 509
constructs engineered to contain the amino acid substitution within the RING finger 510
domain of ORF61 (C19G) or a stop codon inserted directly after the RING finger 511
domain, or a stop codon inserted directly following the E3 Ubiquitin ligase domain all 512
resulted in either failure to obtain any recombinant VZV or resulted in the reversion 513
of the point mutation to wild type ( MB Yee and PR. Kinchington, unpublished 514
studies). This mirrors previous reports of ORF61 playing an important role in virus 515
replication (8, 33). Thus while ORF61 is identified as functioning to inhibit the 516
NFκB pathway, it remains possible that other VZV gene products may also encode a 517
similar function. In addition, whether the E3 Ubiquitin ligase activity of ORF61 518
exerts a direct effect on NFκB proteins or whether it impacts on the cellular regulators 519
of these transcription factors will be an important focus of future work to delineate the 520
mode of action of ORF61 in suppressing NFκB signaling. The continued presence of 521
phosphorylated IκBα within VZV infected DCs indicates it is not being degraded, 522
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therefore it would be useful to also investigate ubiquitin/protease pathways which 523
control degradation of IκBα following NFκB pathway activation. 524
525
In summary, this work identifies inhibition of the NFκB signal transduction pathway 526
in VZV infected human monocyte derived DCs, which occurs following 527
phosphorylation of IκBα, but prior to the translocation of NFκB p50 and p65 into the 528
nucleus. We also demonstrate that the immediate early gene ORF61 inhibits TNFα 529
stimulated NFκB reporter expression indicating this viral gene can inhibit NFκB 530
pathway activation. Furthermore, we show that the region of ORF61 important for its 531
inhibitory affects on TNFα stimulated NFκB reporter activity is the E3 Ubiquitin 532
ligase domain indicating it is the process of ubiquitination that causes this pathway 533
inhibition. During the preparation of this manuscript Wang et al (2011) presented 534
findings that ORF61 binds SUMO-1 via three SUMO-interacting motifs (SIMs) and 535
that these SIMs were required for ORF61 binding to and disrupting of PML nuclear 536
bodies (32). Their work also found ORF61 can act as an inhibitor of TNFα induced 537
NFκB reporter activity in a transient transfection assay within a melanoma cell line, 538
supporting the results presented in this manuscript. VZV encoded modulation of the 539
NFκB pathway may be the mechanistic basis for the observed down-regulation of 540
immune molecules in VZV infected DCs, an important immune evasion strategy of 541
VZV. 542
543
544
ACKNOWLEDGEMENTS 545
This work was supported by Australian National Health and Medical Research 546
(NHMRC) Project Grant awarded to A.A. and B.S. E.S. was the recipient of an 547
Australian Postgraduate Award and Westmead Millennium Foundation Stipend 548
Enhancement Award. PRK was supported by NIH grants EY08098 and NS064022, 549
by unrestricted funds from the Eye and Ear Institute of Pittsburgh and by Research to 550
Prevent Blindness Inc. 551
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2011. Varicella Zoster Virus Immediate Early Protein ORF61 abrogated IRF3-667
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670
671
672
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FIGURE LEGENDS 673
674
Figure 1: Cellular localization of NFκB p50 determined by immunofluorescent 675
staining and confocal microscopy. NFκB p50 subunit cellular localization was 676
determined within DCs (A-C) and HFFs (D-F) that were either mock infected (A, D), 677
TNFα (20 nM 5 mins) stimulated (B, E) or VZV infected (C, F). Cells were harvested 678
48 hours post infection. DCs were fluorescence activated cell sorted for CD1a+ cells 679
within the mock infected population or VZV+CD1a+ cells from the infected 680
population. Sorted cells were stained with an antibody against NFκB p50 (red), VZV 681
gE (green) and DAPI nuclei (blue). Three-colour images are shown on the left, with 682
magnified inserts of NFκB p50 alone on the upper right and three-colour magnified 683
inserts on the lower right. Nuclear localization of NFκB p50 is indicative of an active 684
NFκB pathway. Percentages of cells with NFκB p50 localized to the nucleus were 685
determined by analysis of 100 cells per slide (G). Statistical significance was 686
determined using t-test (* p<0.05, **** p<0.00005). 687
688
689
Figure 2: Cellular localization of NFκB p65 protein subunits determined by 690
immunofluorescence staining and confocal microscopy, as detailed in the legend to 691
Figure 1. Cellular localization of NFκB p65 in DCs (A-C) and HFFs (D-F), either 692
mock infected (A, D), TNFα treated (20 nM 5 mins) (B, E) and VZV infected (C, F). 693
NFκB p65 (red), VZV gE (green) and DAPI nuclei (blue). Percent of cells with NFκB 694
p65 localized to the nucleus were determined by analysis of 100 cells per slide (G). 695
Statistical significance was determined using t-test (* p<0.05). 696
697
Figure 3: Mean fluorescence intensity (MFI) of pattern recognition receptors, TLR-3, 698
8, or 9, or of surface TNFR-1 or 2 on VZV infected DCs. VZV infected DCs were 699
harvested 48 hours post infection and dual stained for intracellular TLR-3, 8, 9, or 700
surface stained for TNFR-1 or 2 along with VZV gB. MFI’s were normalized to the 701
respective mock infected DCs. CD1a was included as a control as its expression 702
should remain unchanged following infection. 703
704
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Figure 4: Analysis of IκBα phosphorylation following VZV infection of DCs. 705
Western blot analysis was performed on total cell lysates obtained from uninfected 706
HFFs, mock infected CD1a+ DCs and VZV antigen positive CD1a+ DCs (A) 707
separated on a 12 % polyacrylamide gel and transferred to PVDF membrane. Blots 708
were probed using antibodies specific for IκBα or phosphorylated IκBα. All blots 709
were also probed using an antibody against α-Tubulin as a protein loading control. 710
Results from seven independent replicate experiments were averaged and normalized 711
to α-Tubulin by densitometry (B), statistical significance was obtained using t-test 712
(*p<0.05). 713
714
Figure 5: Identification of a viral gene product responsible for inhibiting TNFα 715
stimulated NFκB pathway activation. 293FT cells were co-transfected with pNFκB-716
hrGFP reporter construct and either pGK2-HA-ORF61, -64, -47, -2, -49, -21, or -23. 717
Cells were stimulated with TNFα (20 nM) 24 hours post transfection and analyzed by 718
flow cytometry 48 hours post transfection. Level of GFP expression within HA 719
expressing cells was then determined with GFP expression representing NFκB 720
pathway activity. Replicate experiments were averaged and normalized to the parental 721
control, pGK2-HA, with statistical significance obtained using t-test (**** 722
p<0.00005). 723
724
Figure 6: Impact of ORF61 on TNFα-induced levels of IκBα protein. Western blot 725
analysis performed on total cell lysates from 293FT cells transfected with pGK2-726
ORF61-HA cells (A) and stimulated with TNFα 48 hours post transfection. Proteins 727
were separated by SDS-PAGE and membranes probed with an antibody against IκBα. 728
pGK2-HA (parent construct) and no DNA transfected cells were included as controls. 729
Densitometry was performed to determine relative levels of protein expression with 730
results averaged and normalized to α-Tubulin, which was included as a protein 731
loading control (B). Results are presented relative to parental control. Statistical 732
significance was determined using t-test (* p<0.05). 733
734
Figure 7: Mutation of ORF61 to identify domains mediating NFκB inhibition. 735
Depiction of ORF61 mutants generated using site-directed mutagenesis of pIRES2-736
DsRED2-ORF61 constructs (A). ORF61-DsRED2 full length shows functionally 737
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important regions of ORF61. ORF61 Q62_STOP truncates ORF61 following its 738
RING finger domain. ORF61 Q139_STOP is truncated following the E3 Ubiquitin 739
ligase domain, ORF61 Q62_START is transcribed following the RING finger domain 740
and ORF61 C19G contains an amino acid substitution within its RING finger domain 741
reported to disrupt its function. NLS, nuclear localization signal. Mutant ORF61-742
DsRED2 expressing constructs were co-transfected along with pNFκB-hrGFP 743
reporter construct into 293FT cells, stimulated with TNFα (20 nM) 24 hours post-744
transfection and cultured for a further 24 hours before being analyzed by flow 745
cytometry for GFP expression within DsRED2 positive cells (B). GFP expression is 746
indicative of NFκB pathway activity. Replicate experiments were averaged and 747
normalized to full length ORF61. Statistical significance was determined by t-test (* 748
p<0.05, ** p<0.005). 749
750
751
752
753
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NFκB p50/VZV gE/DAPI
(A)
Figure 1
(A)
(B)
(C)
(G)100
120
B p
50
60
80
100
ge o
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NF
kBal
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20
40
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ive
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Re
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(E)
(F)
* ****
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Mock DC VZV infected DC
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(A)
Figure 2
NFκB p65/VZV gE/DAPI ( )
(B)
(C)
100
120
kB p
65
(G)
60
80
100
geof
nuc
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NF
kal
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ion
20
40
Rel
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e fo
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han
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R
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(D) NFκB p65/ VZV gE/DAPI
(D)
(E)
(F)
*
n=4
Mock DC VZV infected DC
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Figure 3
1 2
1.4
1.6
1.8
e of
MF
I
0.6
0.8
1.0
1.2
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0.0
0.2
0.4
TNFR1 TNFR2 TLR3
Rel
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e
TNFR1 TNFR2 TLR3
MockVZV
TLR8 TLR9 CD1aTLR8 TLR9 CD1a
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Figure 4
phos IκBα(A)
IκBα
α-Tubulin
IκBα(B)
1.2
1.4
1.6
1.8
otei
n ex
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sion *
( )
0.4
0.6
0.8
1
e fo
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Mock VZV+CD1a+
Rel
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Mock VZV+CD1a+
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1 2y
Figure 5
0.8
1.0
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NFk
B p
athw
ay
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0.4
0.6
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of
activ
ity
0.0
0.2
Parental ORF61 ORF64
Rel
ativ
e f
n>3 ORF47 ORF2 ORF49 ORF21 ORF23
on March 22, 2018 by guest
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Dow
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Figure 6
TNFα + +
IκBα
(A)
α-Tubulin
IκBα
(B) 2.5
of I
Bα
κ
*
1.5
2
nge
of e
xpre
ssio
n o
0
0.5
1
Rel
ativ
e fo
ld c
han
Parent + TNFα
+
n=3
ORF61 + TNFα
on March 22, 2018 by guest
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Dow
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(A)
Figure 7
( )
3.0
3.5
NF
kB ** *
(B)
1 0
1.5
2.0
2.5
ve fo
ld c
hang
e of
pa
thw
ay a
ctiv
ity
0.0
0.5
1.0
Parent ORF61 ORF6Q62_ST
Rel
ativ
n=3
* **
61TOP
ORF61Q139_STOP
ORF61Q62_START
ORF61 C19G
on March 22, 2018 by guest
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Dow
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