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: allison.abendroth@sydney.edu.au 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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efficient gene delivery by mRNA electroporation in human hematopoietic 648

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2011. Varicella Zoster Virus Immediate Early Protein ORF61 abrogated IRF3-667

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Virol. 669

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

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40

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(E)

(F)

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(A)

Figure 2

NFκB p65/VZV gE/DAPI ( )

(B)

(C)

100

120

kB p

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60

80

100

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40

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Figure 3

1 2

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1.8

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Figure 4

phos IκBα(A)

IκBα

α-Tubulin

IκBα(B)

1.2

1.4

1.6

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1 2y

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Parental ORF61 ORF64

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n>3 ORF47 ORF2 ORF49 ORF21 ORF23

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Figure 6

TNFα + +

IκBα

(A)

α-Tubulin

IκBα

(B) 2.5

of I

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κ

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1.5

2

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Parent + TNFα

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ORF61 + TNFα

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(A)

Figure 7

( )

3.0

3.5

NF

kB ** *

(B)

1 0

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2.0

2.5

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Parent ORF61 ORF6Q62_ST

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61TOP

ORF61Q139_STOP

ORF61Q62_START

ORF61 C19G

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