HRB is Essential for Influenza A Virus Replication and Promotes

1

HRB is Essential for Influenza A Virus Replication and Promotes Genome Trafficking in 1

Late-Stage Infection 2

3

Amie J. Eisfeld1,*, Gabriele Neumann1, and Yoshihiro Kawaoka1,2,3,4,5,* 4

5

1University of Wisconsin-Madison, School of Veterinary Medicine, Department of 6

Pathobiological Sciences, Influenza Research Institute, Madison, Wisconsin, USA 7

8

2University of Tokyo, Institute of Medical Science, Division of Virology, Department of 9

Microbiology and Immunology, Tokyo, Japan 10

11

3Division of Virology, Department of Microbiology and Immunology, Institute of Medical 12

Science, University of Tokyo, Tokyo 108-8639, Japan 13

14

4Department of Special Pathogens, International Research Center for Infectious Diseases, 15

Institute of Medical Science, University of Tokyo, 108-8639, Japan 16

17

5ERATO Infection-Induced Host Responses Project, Saitama 332-0012, Japan 18

19

*Corresponding author20

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.05064-11 JVI Accepts, published online ahead of print on 13 July 2011

on January 11, 2019 by guesthttp://jvi.asm

.org/D

ownloaded from

http://jvi.asm.org/

2

Correspondence should be addressed to: 21

Amie J. Eisfeld, Ph.D. 22

Influenza Research Institute 23

University of Wisconsin-Madison 24

575 Science Drive 25

Madison, WI 53711 26

Phone: (608) 890-2908 27

Fax: (608) 890-2912 28

Email: [email protected]

30

or31

32

Yoshihiro Kawaoka, D.V.M., Ph.D. 33

Influenza Research Institute 34

University of Wisconsin-Madison 35

575 Science Drive 36

Madison, WI 53711 37

Phone: (608) 265-4925 38

Fax: (608) 262-9641 39

Email: [email protected]

41

Running title: HRB promotes influenza vRNP trafficking42

Abstract word count: 23443

Text word count: 514044


.org/D

ownloaded from

http://jvi.asm.org/

3

ABSTRACT 45

Influenza A virus uses cellular protein transport systems (e.g., CRM1-mediated nuclear 46

export and Rab11-dependent recycling endosomes) for genome trafficking from the nucleus to 47

the plasma membrane, where new virions are assembled. However, the detailed mechanisms of 48

these events have not been completely resolved and additional cellular factors are probably 49

required. Here, we investigated the role of the cellular human immunodeficiency virus (HIV) 50

rev-binding protein (HRB), which interacts with influenza virus nuclear export protein (NEP), 51

during the influenza virus life cycle. By using siRNAs and overexpression of a dominant-52

negative HRB protein fragment, we show that cells lacking functional HRB have significantly 53

reduced production of influenza virus progeny and that this defect results from impaired viral 54

ribonucleoprotein (vRNP) delivery to the plasma membrane in late-stage infection. Since HRB 55

co-localizes with influenza vRNPs early after their delivery to the cytoplasm, it may mediate a 56

connection between the nucleocytoplasmic transport machinery and the endosomal system, thus 57

facilitating the transfer of vRNPs from nuclear export to cytoplasmic trafficking complexes. We 58

also found an association between NEP and HRB in the perinuclear region, suggesting that NEP 59

may contribute to this process. Our results identify HRB as a second endosomal factor with a 60

crucial role in influenza virus genome trafficking, suggest cooperation between unique 61

endosomal compartments in the late steps of the influenza virus life cycle, and provide a 62

common link between the cytoplasmic trafficking mechanisms of influenza virus and HIV. 63


.org/D

ownloaded from

http://jvi.asm.org/

4

INTRODUCTION64

Influenza A virus is a highly contagious respiratory pathogen responsible for up to 0.5 65

million deaths annually (43). Seasonal epidemics are punctuated by rare but recurring 66

pandemics, and recently, highly pathogenic H5N1 avian influenza viruses have caused human 67

infections with high fatality (~60%) (42). These threats, along with the potential emergence of an 68

influenza virus strain with both high transmissibility and high pathogenicity, emphasize the 69

importance of controlling influenza viruses to promote global health. To achieve this, more 70

detailed knowledge of how viruses replicate and interact with their hosts is needed. Any essential 71

replication mechanism or host molecule interaction could be exploited for use in the 72

development of novel prevention or intervention strategies. Recent studies have revealed that 73

host molecules are required for influenza genome transport to newly forming virions (2, 10). 74

Here, we aimed to clarify the mechanisms of influenza genome trafficking further.75

Viral ribonucleoprotein (vRNP) complexes are the genetic elements of influenza virus, 76

and their incorporation into budding virions is a prerequisite for the formation of infectious 77

viruses. vRNPs are composed of individual negative-sense viral RNAs (vRNA) associated with 78

viral nucleoprotein (NP) and the heterotrimeric polymerase complex (PB2, PB1 and PA). A set 79

of eight vRNPs representing the eight unique genome segments is required for an influenza A 80

virus to be infectious. Upon infection, vRNPs within virions are released from endosomes and 81

transported to the nucleus where they serve as templates for the production of viral protein-82

encoding mRNAs and positive-sense complementary RNAs (cRNA). Subsequently, the cRNAs 83

serve as templates for vRNA synthesis. In late-stage infection, new vRNPs are assembled in the 84

nucleus and must undergo both nuclear export and transport across the cytoplasm to gain access 85

to the viral budding sites at the plasma membrane. 86


.org/D

ownloaded from

http://jvi.asm.org/

5

Influenza virus utilizes cellular protein trafficking systems to facilitate the journey of 87

vRNPs from the nucleus to the plasma membrane. vRNP nuclear export ensues through the 88

coordinated actions of the M1 matrix protein—which associates with vRNP—the cellular CRM1 89

nuclear export receptor, and the viral nuclear export protein (NEP) (6, 11, 22-24, 27, 31, 35, 38, 90

40). NEP encodes a nuclear export signal, binds CRM1 and M1, and is thought to bridge the 91

complex between M1-vRNP and the cellular nuclear export machinery (1, 27, 31). After nuclear 92

export, vRNPs accumulate at the microtubule organizing center (MTOC) (2, 24), where they 93

associate with the cellular Rab11 GTPase, a major component of recycling endosomes (2, 10). 94

vRNPs are later observed in punctate foci that co-localize with Rab11 in the peripheral 95

cytoplasm, and are transported to the cell surface in a manner dependent on Rab11 GTPase 96

activity (2, 10); microtubules also may be involved in this process (2, 24). Near the plasma 97

membrane, the vRNP foci coalesce and dissociate from Rab11 for their presumed incorporation 98

into budding virions (10). While it is clear that cellular protein trafficking systems are necessary 99

for influenza virus genome transport, additional unknown cellular factors likely contribute to this 100

process.101

Previously, the influenza virus NEP protein was shown to interact with the cellular 102

human immunodeficiency virus (HIV) rev-binding protein (HRB) in a yeast two-hybrid system 103

(31), but its role in the influenza virus life cycle was not examined. HRB contains a putative Arf 104

GTPase activating protein (GAP) domain at its N-terminus (7, 33), regulates cellular endocytic 105

processes (7, 8, 17, 33, 37), and may be a component of the nuclear pore complex (NPC) (12, 13, 106

29). We hypothesized that HRB contributes to influenza vRNP trafficking through effects on 107

either nucleocytoplasmic transport or vesicular transport systems. By using a combination of 108

siRNA-mediated protein knockdown and co-immunofluorescence analyses, we found that HRB 109


.org/D

ownloaded from

http://jvi.asm.org/

6

is critical for the efficient production of influenza virus progeny and is involved in mediating 110

vRNP transport to the plasma membrane. Our results highlight the complex nature of influenza 111

vRNP trafficking and suggest interplay between multiple endocytic compartments in vRNP 112

delivery to cell surface sites of influenza virus formation. 113


.org/D

ownloaded from

http://jvi.asm.org/

7

MATERIALS AND METHODS 114

Cells, viruses, and infections. Transformed human embryonic kidney cells (293), 115

human lung carcinoma cells (A549), Madin-Darby canine kidney cells (MDCK), human cervical 116

adenocarcinoma cells (HeLa), baby hamster kidney cells (BHK), and Cercopithecus aethiops117

kidney fibroblast cells (CV-1) were cultured at 37°C in 5% CO2 in the following media: 118

Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum 119

(FBS) (293 and CV-1), DMEM containing 5% FBS (BHK), a 1:1 mixture of DMEM and Ham’s 120

F12 nutrient mixture supplemented with 10% FBS (A549), minimum essential medium (MEM) 121

supplemented with 5% newborn calf serum (MDCK), or MEM supplemented with 10% FBS 122

(HeLa). All media contained 50 units/ml of penicillin and 50 μg/ml of streptomycin (Invitrogen, 123

Carlsbad, CA). 124

Influenza A/WSN/33 (H1N1; WSN) was generated by using plasmid-based reverse 125

genetics as described previously (28), and virus stock amplification and plaque assays were 126

performed in MDCK cells. Vesicular stomatitis virus (VSV, strain Indiana), vaccinia virus (VV, 127

strain Ankara; a kind gift from Dr. Paul Ahlquist, University of Wisconsin-Madison), and 128

adenovirus 5 (Ad5, strain McEwen; a kind gift from Dr. Paul Kinchington, University of 129

Pittsburgh) stock virus amplifications and plaque assays were performed in BHK, CV-1, or A549 130

cells, respectively. For multi-cycle growth experiments after siRNA treatment, cells grown in 24-131

well plates were infected with 100 plaque-forming units (PFU) of the indicated virus by direct 132

inoculation of the culture medium. Supernatants were collected from influenza virus- and VSV-133

infected cells at 48 h and 24 h, respectively, and virus titrations were performed in MDCK or 134

BHK cells. To harvest VV or Ad5, infected monolayers were scraped into the overlying media at 135

48 h, and the mixtures were subjected to three consecutive freeze-thaw cycles followed by 136


.org/D

ownloaded from

http://jvi.asm.org/

8

centrifugation to clear insoluble debris. The resultant supernatants were subjected to virus 137

titration in CV-1 or A549 cells for VV and Ad5, respectively. For similar experiments after 138

plasmid transfection, cells in 12-well plates were inoculated with 200 PFU of WSN and 139

supernatants were harvested at 72 h for plaque assays in MDCK cells. For immunofluorescence 140

and immunoblot experiments, cells were infected with WSN as described in the Figure Legends. 141

siRNAs, plasmids and transfections. siRNA transfections were performed as 142

previously described (10). The final concentration of all siRNAs in the culture medium was 20 143

nM, and siRNA transfections were allowed to proceed for 48 h before subsequent plasmid 144

transfections or virus infections. The following siRNAs were used: an extensively validated non-145

targeting siRNA (AllStars Neg; Qiagen, Valencia, CA; cat. no. 1027281); a previously described 146

siRNA targeting influenza NP mRNA (NP-1496; synthesized by Qiagen) (14); a previously 147

described siRNA targeting the human AGFG1 (HRB) gene (synthesized by Qiagen) (36, 45); 148

and a validated mixture of siRNAs targeting essential host survival genes (Death Control, Qiagen 149

cat. no. SI04381048). 150

To generate the HRB dominant-negative mutant, total RNA was isolated from A549 cells 151

using the RNeasy Mini Kit (Qiagen), and poly-A RNA was reverse transcribed using SuperScript 152

II reverse transcriptase and an oligo-dT primer (Invitrogen) according to the manufacturers’ 153

instructions. A segment of the AGFG1/HRB gene corresponding to amino acids 361–562 154

( N360) was amplified from the resultant cDNA using gene-specific primers with 5’ Xho I and 155

3’ Hind III overhangs and was inserted in-frame with enhanced green fluorescent protein (GFP) 156

in the pEGFP-N1 vector (Clontech, Mountain View, CA), producing the p N360-GFP plasmid. 157

The plasmid expressing NEP fused to enhanced yellow fluorescent protein (YFP) was created by 158

first PCR amplifying YFP (Clontech) with 5’ Not I and 3’ Bgl II overhangs and then inserting 159


.org/D

ownloaded from

http://jvi.asm.org/

9

the product into the pCAGGS/MCS (18, 30) backbone. The WSN NEP open reading frame 160

lacking the stop codon was then amplified by using 5’ Eco RI and 3’ Not I overhangs and 161

inserted upstream and in-frame with YFP in pCAGGS/MCS to produce pNEP-YFP. All 162

plasmids were sequenced to confirm proper fragment insertion. Primer sequences are available 163

upon request. For viral replication studies, 293 cells were transfected with plasmid DNAs by the 164

calcium phosphate precipitation method using a calcium phosphate transfection kit (Invitrogen) 165

according to the manufacturer’s recommendations. All other plasmid transfections were 166

performed with the TransIT LT1 transfection reagent (Mirus Bio, Madison, WI). 167

Cell viability assay. Cell viability was determined by assaying total intracellular ATP 168

with the CellTiter-Glo kit (Promega, Madison, WI) according to the manufacturer’s 169

recommendations, with some modifications. Briefly, at the times indicated in the Figure, 170

triplicate cultures of siRNA-treated 293 cells in 24-well dishes were lysed directly with 100 μl of 171

Glo Lysis Buffer (Promega) for 15 minutes at room temperature. Subsequently, 50 μl of the 172

resultant lysate was mixed with an equal volume of CellTiter-Glo reagent and luminescence was 173

read directly using a Tecan microplate reader (Tecan Group Ltd, Switzerland). 174

Mini-replicon assay. In vitro viral polymerase activity in siRNA or plasmid-transfected 175

293 cells was compared using a dual luciferase reporter assay system (Promega) according to the 176

manufacturer’s instructions. Briefly, 48 h after siRNA transfection, triplicate cultures in 24-well 177

plates were transfected with a plasmid expressing a firefly luciferase reporter under the control of 178

the influenza WSN NA terminal genome sequences (pPolWSNNA F-Luc; 0.025 μg), together 179

with the protein expression plasmids pCAGGS-PB2, pCAGGS-PB1, pCAGGS-PA, and 180

pCAGGS-NP, which express the WSN polymerase complex proteins (0.25 μg each) (20, 28). 181

Cells were also co-transfected with an internal control plasmid (0.025 μg) expressing Renilla182


.org/D

ownloaded from

http://jvi.asm.org/

10

luciferase regulated by the herpes simplex virus type 1 thymidine kinase promoter (pGL.74; 183

Promega), which exhibits basal transcriptional activation in mammalian cells. Firefly and Renilla184

luciferase activities were measured 48 h after plasmid transfection by using the Tecon microplate 185

reader. The level of viral gene expression was determined after normalization to the level of 186

cellular gene expression from the same sample (firefly luciferase light units/Renilla luciferase 187

light units). This produced a ratio indicative of specific effects on viral polymerase activity; the 188

average ratio for triplicate reads is reported.189

Immunological assays. Co-immunofluorescence and immunoblotting were performed 190

as described previously (10). The following primary antibodies were used: monoclonal mouse 191

anti-HRB (Santa Cruz Biotechnology, Santa Cruz, CA; catalog no. sc-166651; used at 1:100 for 192

immunofluorescence and 1:500 for immunoblotting); polyclonal rabbit anti-calnexin (Santa Cruz 193

Biotechnology cat. no. sc-11397, used at 1:5000 for immunoblotting); mouse anti-GFP antibody 194

(Clontech cat. no. 632375; used at 1:20,000 for immunoblotting); a previously described 195

polyclonal rabbit anti-serum against influenza vRNPs (R528; used at 1:5000 for immunoblotting 196

and 1:2500 for immunofluorescence) (26); polyclonal rabbit anti-actin (Santa Cruz 197

Biotechnology cat. no. sc-10731, used at 1:1000 for immunoblotting); a previously described 198

rabbit anti-serum against influenza NEP (R5023; used at 1:200 for immunofluorescence) (27); 199

and a previously described monoclonal mouse antibody that recognizes influenza NP in the form 200

of vRNP (MAb 3/1; used at 1:1000 for immunofluorescence) (10). For secondary detection in 201

immunoblot analysis, we used horseradish peroxidase-conjugated antibodies, including goat anti-202

rabbit (Invitrogen; 1:2000) and goat anti-mouse (Thermo Scientific, Rockford, IL; 1:2000). 203

Proteins were detected with the Lumi-Light PLUS western blotting substrate (Roche, 204

Indianapolis, IN), Kodak Biomax XAR film, and a Konica Minolta SRX-101A X-ray film 205


.org/D

ownloaded from

http://jvi.asm.org/

11

developer. Alexa Fluor (AF) 546-conjugated goat anti-mouse and AF 488-conjugated goat anti-206

rabbit antibodies were used for secondary detection in immunofluorescence analyses (Invitrogen; 207

1:1000). Where indicated in the Figure Legends, nuclei were stained with 0.4 μg/ml Hoechst 208

33258 (Invitrogen), or cells were treated with 20 ng/ml of leptomycin B (LMB) (Sigma-Aldrich, 209

St. Louis, MO) or an equivalent volume of dimethyl sulfoxide (DMSO). All fluorescence 210

images were captured with a Zeiss LSM 510 Meta point-scan laser microscope system, as 211

previously described (10). Images were exported as TIFF files and cropped using Adobe CS4 212

software, but were otherwise unaltered. 213


.org/D

ownloaded from

http://jvi.asm.org/

12

RESULTS 214

HRB expression and function are essential for efficient influenza virus production. 215

HRB is a critical host factor in the HIV life cycle, and both siRNA-mediated knockdown of HRB 216

expression and overexpression of a C-terminal fragment of the HRB protein ( N360) strongly 217

impair HIV replication (45). To determine whether HRB is involved in the influenza virus life 218

cycle, we examined the effect of reducing HRB expression or overexpressing the HRB N360 219

fragment on influenza A/WSN/1933 (H1N1; WSN) multi-cycle growth in 293 cells. HRB-220

specific siRNA treatment resulted in efficient protein knockdown without affecting a non-221

targeted cellular protein (calnexin; CANX). Parallel transfections with non-targeting control 222

siRNA (AllStars Neg) or siRNA targeting influenza NP mRNA did not result in knockdown of 223

either HRB or CANX (Figure 1A). Further, cells treated with siRNA targeting HRB exhibited 224

no major differences in cellular morphology (data not shown) or viability (Fig. 1B) compared to 225

cells treated with non-targeting or NP-specific siRNA controls over a 96-h time course. This was 226

in contrast to cells treated with death-inducing siRNAs, in which clear morphological changes 227

consistent with cytotoxicity (data not shown) and a sharp reduction in viability at the 96-h time 228

point (Fig. 1B) were observed. Because these data indicated that prolonged reduction in HRB 229

expression was not detrimental to cell viability, we next tested the effects of the HRB siRNA on 230

multi-cycle influenza virus growth. HRB siRNA induced a statistically significant (P = 0.0062), 231

287-fold reduction in WSN titer relative to cells treated with the AllStars negative control siRNA 232

(Fig. 1C). This result was highly reproducible and observed in three independent experiments. 233

Moreover, similar treatments did not cause a reduction in vesicular stomatitis virus (VSV) or 234

adenovirus type 5 (Ad5) replication and led to only a minor reduction in vaccinia virus 235


.org/D

ownloaded from

http://jvi.asm.org/

13

replication (Fig. 1D). Therefore, while cells exhibiting significantly reduced HRB expression 236

remained viable, they did not support the efficient production of progeny influenza viruses. 237

To corroborate these findings, we assessed multi-cycle growth in cells overexpressing the 238

HRB N360 protein fragment fused to green fluorescent protein (GFP), which was previously 239

shown to act in a dominant-negative manner (36). We used GFP alone as a negative control and 240

influenza NEP fused to yellow fluorescent protein (YFP) as a positive control, as our earlier 241

work indicated that NEP overexpression impairs influenza replication (data not shown). We 242

observed abundant levels of all plasmid-expressed proteins (Fig. 1E) and efficient transfection 243

efficiency, with fluorescence in at least 75% of cells under all conditions by 48 h post-244

transfection (data not shown). Plasmid-transfected cells were infected with influenza WSN at 245

this time, and at 72 h post-infection (hpi) cells expressing either NEP-YFP or N360-GFP246

exhibited greater than 200-fold reductions in WSN titers relative to cells expressing only GFP 247

(Fig. 1F). Taken together, our results show a critical dependence of influenza virus on HRB 248

expression and function for the generation of infectious virus particles. 249

HRB knockdown does not impair influenza virus entry or viral gene expression.250

HRB interacts with adaptor-related protein complex 2 (AP2), which is involved in clathrin-251

mediated endocytosis (37), and EPS15, a mediator of EGFR endocytosis (8) and is a regulator of 252

clathrin-dependent endocytosis and endocytic sorting (7, 17, 33). Because influenza virus enters 253

cells through a clathrin-dependent endocytosis mechanism (19) and virus entry depends on 254

EGFR signaling (9), we wondered whether HRB knockdown might impair influenza virus entry. 255

To test this, we assessed viral gene expression in WSN-infected cells after HRB or AllStars Neg 256

control siRNA treatments. Cells treated with either siRNA and subjected to immunofluorescence 257

staining with an NP-specific antibody exhibited a comparable number of infected cells and a 258


.org/D

ownloaded from

http://jvi.asm.org/

14

similar fluorescence signal at 6 hpi (data not shown). Consistent with this finding, we saw no 259

differences in influenza virus NP expression at 8 or 11 hpi (Fig. 2A). These results suggest that 260

HRB knockdown does not impair influenza virus entry or gene expression and that HRB 261

functions at a later stage in the influenza virus life cycle. 262

To further determine whether the disruption of HRB expression or function interferes 263

with viral polymerase activity, we evaluated the effects of HRB siRNA treatment or 264

overexpression of the N360-GFP fragment in a mini-replicon system, which provides an 265

overall representation of the efficiency of vRNA, cRNA and mRNA production. Cells were 266

treated with siRNAs or plasmids expressing fluorescently tagged proteins, as described above, 267

and were transfected with plasmids encoding the WSN polymerase complex proteins (PB2, PB1, 268

PA and NP) and a viral segment encoding a reporter firefly luciferase gene. Using this assay, we 269

detected a minimal effect on viral reporter gene expression with HRB-specific siRNA and no 270

effect with overexpression of N360-GFP (Fig. 2B). This was in contrast with strong reductions 271

in viral reporter gene expression observed with either NP-specific siRNA or NEP-YFP protein 272

expression. Together, these data imply that HRB functions in the late stage of influenza virus 273

infection and is not involved in regulating influenza virus entry, polymerase activity or protein 274

expression.275

HRB and NEP partially co-distribute in the cytoplasm during influenza virus 276

infection. Although HRB interacts with NEP in a yeast two-hybrid system, an association in the 277

context of influenza virus infection has not been demonstrated. In uninfected cells, HRB exhibits 278

juxtanuclear and punctate cytoplasmic localization, with occasional low-level accumulation in 279

the nucleus (7, 33). This distribution pattern is distinct from the generally diffuse nuclear and 280

cytoplasmic profile of NEP (6, 11, 22, 40). To determine whether influenza virus infection 281


.org/D

ownloaded from

http://jvi.asm.org/

15

induces changes in the HRB staining pattern, and to specifically address whether HRB and NEP 282

associate in influenza virus-infected cells, we performed co-immunofluorescence analysis of 283

WSN-infected A549 cells.284

As in previous reports (7, 33), HRB in mock-infected cells was observed in peripheral 285

cytoplasmic foci and accumulated in the perinuclear region, with occasional diffuse localization 286

in the nucleus (Fig. 3A). The overall HRB distribution pattern in WSN-infected cells was 287

comparable to that of mock-infected cells at all time points (Fig. 3B-D). At 5 hpi, NEP exhibited 288

prominent nuclear localization, with some diffuse staining in the cytoplasm and no co-289

localization with HRB (Fig. 3B). By 7 hpi, NEP began to accumulate in the perinuclear region of 290

the cytoplasm and partially co-localized with HRB immediately adjacent to the nucleus (Fig. 291

3C). Of note, NEP also exhibited a mesh-like cytoplasmic staining pattern that was reminiscent 292

of cellular cytoskeletal filaments (Fig. 3C); this localization pattern has not been described 293

previously. By 9 hpi, NEP nuclear levels were consistently reduced, perinuclear levels were 294

increased, and partial co-distribution with HRB occurred near the nuclear membrane (Fig. 3D). 295

Thus, our data reveal novel details about the localization of NEP in late-stage infected cells and 296

suggest that NEP and HRB may interact in the perinuclear region.297

HRB and vRNP co-localize at the MTOC in influenza virus-infected cells. We298

previously demonstrated that cytoplasmic influenza vRNPs undergo multi-stage trafficking 299

following nuclear export (10). Specifically, vRNPs initially accumulate at the MTOC, are co-300

transported with Rab11 through the cytoplasm in punctate foci, and accumulate at the plasma 301

membrane in late-stage infected cells. Since the HRB localization pattern in infected cells 302

mirrors that of vRNPs (Fig. 3), we hypothesized that HRB co-localizes with vRNPs to promote 303


.org/D

ownloaded from

http://jvi.asm.org/

16

trafficking. To test this hypothesis, we examined the HRB spatio-temporal distribution pattern in 304

relation to influenza vRNPs in WSN-infected A549 cells.305

To detect vRNPs by immunofluorescence analysis in influenza virus-infected cells, we 306

previously used a mouse monoclonal antibody (MAb 3/1) against influenza NP that exhibits a 307

punctate cytoplasmic staining pattern and nearly completely co-localizes with cytoplasmic 308

vRNA (10). Because we could not use MAb 3/1 with mouse anti-HRB (also a MAb) for indirect 309

co-immunofluorescence analyses, we needed a compatible antibody that could identify vRNPs. 310

The R528 rabbit polyclonal anti-serum (used in Fig. 2A) was generated against purified 311

influenza vRNPs and has been used to identify NP by immunofluorescence (26). Therefore, we 312

tested whether R528 could detect influenza vRNPs by performing co-immunofluorescence with 313

MAb 3/1. Both antibodies exhibited nearly identical staining patterns, recognizing NP in the 314

nucleus, accumulating at the MTOC and in punctate foci in the peripheral cytoplasm (Fig. 4). 315

Because MAb 3/1 is known to recognize vRNPs, we interpret these results to indicate that R528 316

also identifies trafficking vRNPs in the cytoplasm of influenza virus-infected cells.317

To assess the relationship between HRB and vRNP during infection, we next used R528 318

with mouse anti-HRB in co-immunofluorescence analysis. Similar to previous results (Fig. 3), 319

HRB was primarily juxtanuclear at 5 and 7 hpi (Fig. 5A and B). At 5 hpi, vRNPs were restricted 320

to the nucleus and, as such, did not co-localize with the predominantly cytoplasmic HRB (Fig. 321

4A). Upon export from the nucleus at 7 hpi, vRNPs co-localized with HRB in the MTOC region 322

(Fig. 5B, panel i). Some vRNPs could also be seen en route to the plasma membrane in close 323

proximity to HRB (Fig. 5B, panel vi). At 9 hpi, vRNPs were distributed throughout the 324

peripheral cytoplasm and near the plasma membrane, and no longer prominently co-localized 325

with HRB at the MTOC (Fig. 5C). By 11 hpi, vRNPs were observed in abundance near the 326


.org/D

ownloaded from

http://jvi.asm.org/

17

plasma membrane, and although some HRB was also dispersed in this region, no specific co-327

localization with vRNP was observed (Fig. 5D, panels ii and vi). These data suggest a potential 328

interaction between HRB and vRNP in the MTOC region and indicate that HRB is not a 329

component of transport complexes that deliver vRNPs to the plasma membrane. 330

HRB does not directly mediate vRNP nuclear export. HRB associates with the 331

cellular nuclear export factor CRM1 (29) and influenza NEP (31), both of which are required for 332

influenza vRNP nuclear export (27, 31). Indeed, the NEP nuclear export signal is required for its 333

association with HRB in the yeast two-hybrid assay (31). To clarify whether HRB participates in 334

vRNP nuclear export by facilitating an interaction between vRNP-M1-NEP complexes and 335

CRM1, we examined HRB, NEP, and vRNP distribution in cells treated with leptomycin B 336

(LMB), a specific inhibitor of CRM1. We reasoned that if HRB is required to bridge the 337

interaction between vRNP-M1-NEP and CRM1, then LMB treatment may result in HRB nuclear 338

accumulation and co-localization with vRNP or NEP.339

In mock-infected cells treated with LMB, we observed little to no accumulation of HRB 340

in the nucleus, and the HRB distribution pattern was similar to that observed in DMSO-treated 341

control cells, except that LMB treatment frequently resulted in more evenly distributed HRB 342

around the periphery of the nucleus (Fig. 6A). This indicates that HRB does not shuttle between 343

the nucleus and the cytoplasm in a CRM1-dependent manner. In influenza virus-infected cells, 344

LMB treatment induced nearly complete vRNP nuclear retention and significantly increased the 345

nuclear levels of NEP in most cells, consistent with previous findings (11, 22, 24, 40), but it did 346

not cause HRB nuclear accumulation (Fig. 6B and C). As a consequence of vRNP nuclear 347

accumulation and HRB cytoplasmic localization, vRNPs and HRB did not co-localize in the 348

presence of LMB (Fig. 6B). LMB treatment also reduced co-localization between HRB and 349


.org/D

ownloaded from

http://jvi.asm.org/

18

NEP in the perinuclear region, although some cytoplasmic forms of NEP persisted and low 350

levels of co-localization could be observed at the MTOC (Fig. 6D). These data argue against a 351

role for HRB in mediating interactions that promote influenza vRNP nuclear export and imply 352

that the HRB promotes cytoplasmic, rather than nuclear, activities of influenza virus.353

HRB knockdown provokes aberrant vRNP cytoplasmic trafficking in late-stage 354

infection. Our results excluded a role for HRB in influenza virus entry and polymerase activity, 355

and suggested that HRB may function in vRNP cytoplasmic transport but not vRNP nuclear 356

export. To directly test this possibility, we assessed vRNP trafficking in HRB siRNA-treated 357

cells, by using co-immunofluorescence of HRB and vRNPs. 293 cells were not suitable for these 358

studies because of their low cytoplasmic volume and weak adherence properties, and HRB 359

siRNA treatments in A549 cells induced insufficient knockdown of HRB protein expression to 360

impair influenza virus growth (data not shown). However, we observed efficient and specific 361

reduction in HRB protein expression in HeLa cells (Fig. 7A). Although influenza virus 362

undergoes abortive infection in this cell-type, the blocks occur principally at the levels of virus 363

entry and viral budding (15). Therefore, we used HeLa cells as an alternative model system to 364

examine influenza vRNP trafficking in the absence of abundant HRB protein expression. Of 365

note, non-targeting siRNA-treated mock-infected HeLa cells exhibited HRB distribution similar 366

to that observed in untreated A549 cells, with juxta- or perinuclear accumulation accompanied 367

by punctate foci in the peripheral cytoplasm (Fig. 7B). 368

By 6 h after influenza virus infection, cells in both siRNA treatments exhibited prominent 369

nuclear vRNP staining, despite appreciable differences in the level of HRB protein expression 370

(Fig. 7C). This result confirms our previous finding that the lack of HRB does not impair 371

influenza virus uptake or inhibit viral gene expression. At 9 hpi, HeLa cells treated with non-372


.org/D

ownloaded from

http://jvi.asm.org/

19

targeting siRNAs exhibited vRNPs in the perinuclear region and scattered throughout the 373

cytoplasm, with some vRNP accumulation near the plasma membrane (Fig. 7D). At the same 374

time point in HRB siRNA-treated cells, vRNPs accumulated in the perinuclear region, exhibiting 375

tight association with the nuclear membrane, and were only minimally observed in the peripheral 376

cytoplasm or at the plasma membrane. The effect of siRNA-mediated HRB knockdown was 377

even more pronounced at 15 hpi (Fig. 7E). No obvious differences in vRNP nuclear export were 378

observed for either siRNA transfection condition at any time point (Fig, 7C–E). These data 379

suggest that cells with minimal HRB expression remain competent for vRNP nuclear export, but 380

are impaired in their ability to transport vRNP complexes from the perinuclear region to the 381

plasma membrane. 382


.org/D

ownloaded from

http://jvi.asm.org/

20

DISCUSSION 383

Previous studies have implicated the cellular CRM1 nuclear export and Rab11 recycling 384

endosome pathways in influenza vRNP transport from the nucleus to plasma membrane sites of 385

virion formation (2, 10, 11, 22, 27, 40). However, the detailed mechanisms of vRNP trafficking 386

and the requirement for additional cellular co-factors remain to be illuminated. Here, we 387

identified a second cellular endosomal factor integral to vRNP intracellular transport, the HRB 388

Arf GAP protein. Our results clearly indicate that HRB expression and function are essential for 389

efficient production of influenza virus progeny and implicate HRB in regulating vRNP 390

trafficking from the perinuclear region to the plasma membrane. Since HRB has a role in both 391

nucleocytoplasmic and endosomal trafficking, we suggest that HRB operates as a linker between 392

these systems in influenza virus-infected cells, to facilitate vRNP cell surface delivery and 393

formation of infectious virus particles. 394

Upon its discovery, HRB was hypothesized to contribute to nucleocytoplasmic 395

trafficking due to its ability to bind HIV rev and facilitate rev function (3, 8, 13), its interaction 396

with the nuclear export receptor CRM1 (12, 29), and the presence of multiple nucleoporin-like 397

phenylalanine-glycine (FG) repeats in its C-terminus (13). Given these observations and the 398

identification of HRB as an interaction partner for influenza NEP (31)—a known associate of 399

CRM1 and an integral mediator of influenza vRNP nuclear export—we considered the potential 400

for HRB to affect vRNP nuclear export to be important. However, several lines of evidence 401

argue against this possibility. First, HRB exhibited exclusively cytoplasmic distribution in most 402

cells (infected and mock-infected), and did not consistently accumulate in the nucleus after 403

influenza virus infection. Second, LMB treatment did not increase HRB nuclear accumulation in 404

either mock-infected or infected cells, indicating that HRB does not traffic through the CRM1-405


.org/D

ownloaded from

http://jvi.asm.org/

21

dependent nuclear export pathway, which is required for influenza vRNP nuclear export (27). 406

Most importantly, siRNA-mediated knockdown of HRB protein expression had no affect on 407

vRNP nuclear export, and instead induced accumulation of vRNPs in the perinuclear region. 408

These observations strongly imply that HRB does not regulate vRNP nuclear export, but rather 409

participates in an early event in the vRNP cytoplasmic trafficking mechanism. 410

The tight association of vRNPs with the outer periphery of the nucleus in HRB siRNA-411

treated cells suggests that HRB may be involved in the release of vRNPs from CRM1-RanGTP 412

nuclear export complexes. CRM1 associates with cargo in the nucleus in a RanGTP-dependent 413

manner, and cargo-CRM1-RanGTP complexes are transported to the cytoplasm through the 414

nuclear pore complex (NPC) (32). On the cytoplasmic face of the NPC, RANBP1 and RANBP2 415

cooperate to facilitate RanGTP hydrolysis to RanGDP, thereby inducing the disassociation of 416

RanGDP and CRM1 and the release of cargo into the cytoplasm (16). Importantly, expression of 417

a mutant Ran protein deficient in GTP hydrolysis results in CRM1 accumulation at the nuclear 418

periphery (16). HRB preferentially accumulates at the periphery of the nucleus, contains a 419

putative GTPase activating protein (GAP) domain in its N-terminus (33) and interacts with 420

CRM1 (29). In influenza virus-infected cells, it is therefore conceivable for HRB to interact with 421

CRM1 and RanGTP-associated vRNP nuclear export complexes, possibly assisted by 422

interactions with NEP, to promote RanGTP hydrolysis following their transit across the NPC. A 423

lack of HRB expression would then result in the accumulation of unhydrolyzed RanGTP at the 424

nuclear periphery and the failure of CRM1 and vRNP to dissociate from the perinuclear region in 425

late-stage infection. This concept is consistent with our observations for HRB-specific siRNA 426

treated, influenza virus-infected cells, where vRNPs were abundantly retained at the periphery of 427

the nucleus. The HRB GAP domain is predicted to affect Arf GTPases, and to our knowledge no 428


.org/D

ownloaded from

http://jvi.asm.org/

22

Arf GAP protein is known to influence RanGTP. However, other GAP proteins exhibit broad 429

specificity against multiple small GTPases (4, 21, 39, 44). The verification of authentic HRB 430

GAP activity and identification of HRB-targeted small GTPases will be necessary to clarify the 431

potential role of HRB in modulating RanGTP at the nuclear membrane. 432

HRB may also facilitate other protein-protein interactions that promote vRNP recruitment 433

to cytoplasmic transport complexes. In addition to its N-terminal putative Arf GAP domain, 434

HRB contains multiple linear protein interaction motifs, including an established VAMP7 435

binding domain; a consensus clathrin binding motif and three consensus AP2 appendage binding 436

motifs; multiple FG repeats typical of nucleoporins; and four C-terminal asparagine-proline-437

phenylalanine (NPF) motifs, which mediate interactions with proteins containing an EPS15 438

homology (EH) domain (8, 33, 36). Several EH domain-containing proteins (e.g., EHD1, EHD3, 439

and EHD4) associate with endocytic recycling compartments containing Rab11 (25). Therefore, 440

HRB could recruit vRNPs to Rab11 cytoplasmic transport complexes through coordinate 441

interactions with NEP-associated vRNPs and an EH domain-containing protein. In support of 442

this idea, we observed co-localization between HRB and both vRNPs and NEP at the MTOC, the 443

site of initial vRNP-Rab11 association, immediately after vRNP nuclear export. Additional 444

studies are required to define the specific components of influenza vRNP nuclear export and 445

cytoplasmic trafficking complex intermediates. 446

In summary, we identified HRB as a novel host factor involved in an early step in 447

influenza virus cytoplasmic genome transport. HRB was previously implicated in the HIV life 448

cycle, facilitating the cytoplasmic trafficking of rev-directed viral RNAs by promoting their 449

release from the perinuclear region. Influenza virus and HIV genomes both use the CRM1 450

pathway for nuclear export (27, 29, 31); however, this is the first study to implicate a common 451


.org/D

ownloaded from

http://jvi.asm.org/

23

cytoplasmic trafficking strategy for the genomes of these two viruses. Since influenza virus, 452

unlike HIV, does not use the endosomal sorting complex required for transport (ESCRT) for new 453

virion production (5, 34, 41), it will be interesting to delineate the point of divergence between 454

the influenza virus and HIV genome trafficking mechanisms. 455


.org/D

ownloaded from

http://jvi.asm.org/

24

ACKNOWLEDGEMENTS 456

We thank Susan Watson for editing the manuscript. This work was supported by National 457

Institute of Allergy and Infectious Disease Public Health Service research grants; by a grant-in-458

aid for Specially Promoted Research from the Ministries of Education, Culture, Sports, Science, 459

and Technology; and by grants-in-aid from the Ministry of Health and by ERATO (Japan 460

Science and Technology Agency). 461


.org/D

ownloaded from

http://jvi.asm.org/

25

REFERENCES 462

1. Akarsu, H., W. P. Burmeister, C. Petosa, I. Petit, C. W. Muller, R. W. Ruigrok, and 463

F. Baudin. 2003. Crystal structure of the M1 protein-binding domain of the influenza A 464

virus nuclear export protein (NEP/NS2). EMBO J 22:4646-55.465

2. Amorim, M. J., E. A. Bruce, E. K. Read, A. Foeglein, R. Mahen, A. D. Stuart, and P. 466

Digard. 2011. A Rab11 and microtubule dependent mechanism for cytoplasmic transport 467

of influenza A virus vRNA. J Virol 85:4143-56.468

3. Bogerd, H. P., R. A. Fridell, S. Madore, and B. R. Cullen. 1995. Identification of a 469

novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins. Cell 470

82:485-94.471

4. Bowzard, J. B., D. Cheng, J. Peng, and R. A. Kahn. 2007. ELMOD2 is an Arl2 472

GTPase-activating protein that also acts on Arfs. J Biol Chem 282:17568-80.473

5. Bruce, E. A., L. Medcalf, C. M. Crump, S. L. Noton, A. D. Stuart, H. M. Wise, D. 474

Elton, K. Bowers, and P. Digard. 2009. Budding of filamentous and non-filamentous 475

influenza A virus occurs via a VPS4 and VPS28-independent pathway. Virology 476

390:268-78.477

6. Bui, M., E. G. Wills, A. Helenius, and G. R. Whittaker. 2000. Role of the influenza 478

virus M1 protein in nuclear export of viral ribonucleoproteins. J Virol 74:1781-6.479

7. Chaineau, M., L. Danglot, V. Proux-Gillardeaux, and T. Galli. 2008. Role of HRB in 480

clathrin-dependent endocytosis. J Biol Chem 283:34365-73.481

8. Doria, M., A. E. Salcini, E. Colombo, T. G. Parslow, P. G. Pelicci, and P. P. Di Fiore.482

1999. The eps15 homology (EH) domain-based interaction between eps15 and hrb 483


.org/D

ownloaded from

http://jvi.asm.org/

26

connects the molecular machinery of endocytosis to that of nucleocytosolic transport. J 484

Cell Biol 147:1379-84.485

9. Eierhoff, T., E. R. Hrincius, U. Rescher, S. Ludwig, and C. Ehrhardt. 2010. The 486

epidermal growth factor receptor (EGFR) promotes uptake of influenza A viruses (IAV) 487

into host cells. PLoS Pathog 6:e1001099.488

10. Eisfeld, A. J., E. Kawakami, T. Watanabe, G. Neumann, and Y. Kawaoka. 2011. 489

RAB11A is Essential for Influenza Genome Transport to the Plasma Membrane. J Virol 490

85:6117-26.491

11. Elton, D., M. Simpson-Holley, K. Archer, L. Medcalf, R. Hallam, J. McCauley, and 492

P. Digard. 2001. Interaction of the influenza virus nucleoprotein with the cellular 493

CRM1-mediated nuclear export pathway. J Virol 75:408-19.494

12. Floer, M., and G. Blobel. 1999. Putative reaction intermediates in Crm1-mediated 495

nuclear protein export. J Biol Chem 274:16279-86.496

13. Fritz, C. C., M. L. Zapp, and M. R. Green. 1995. A human nucleoporin-like protein 497

that specifically interacts with HIV Rev. Nature 376:530-3.498

14. Ge, Q., M. T. McManus, T. Nguyen, C. H. Shen, P. A. Sharp, H. N. Eisen, and J. 499

Chen. 2003. RNA interference of influenza virus production by directly targeting mRNA 500

for degradation and indirectly inhibiting all viral RNA transcription. Proc Natl Acad Sci 501

U S A 100:2718-23.502

15. Gujuluva, C. N., A. Kundu, K. G. Murti, and D. P. Nayak. 1994. Abortive replication 503

of influenza virus A/WSN/33 in HeLa229 cells: defective viral entry and budding 504

processes. Virology 204:491-505.505


.org/D

ownloaded from

http://jvi.asm.org/

27

16. Kehlenbach, R. H., A. Dickmanns, A. Kehlenbach, T. Guan, and L. Gerace. 1999. A 506

role for RanBP1 in the release of CRM1 from the nuclear pore complex in a terminal step 507

of nuclear export. J Cell Biol 145:645-57.508

17. Khwaja, S. S., H. Liu, C. Tong, F. Jin, W. S. Pear, J. van Deursen, and R. J. Bram.509

2010. HIV-1 Rev-binding protein accelerates cellular uptake of iron to drive Notch-510

induced T cell leukemogenesis in mice. J Clin Invest 120:2537-48.511

18. Kobasa, D., M. E. Rodgers, K. Wells, and Y. Kawaoka. 1997. Neuraminidase 512

hemadsorption activity, conserved in avian influenza A viruses, does not influence viral 513

replication in ducks. J Virol 71:6706-13.514

19. Lakadamyali, M., M. J. Rust, and X. Zhuang. 2004. Endocytosis of influenza viruses. 515

Microbes Infect 6:929-36.516

20. Li, C., M. Hatta, C. A. Nidom, Y. Muramoto, S. Watanabe, G. Neumann, and Y. 517

Kawaoka. 2010. Reassortment between avian H5N1 and human H3N2 influenza viruses 518

creates hybrid viruses with substantial virulence. Proc Natl Acad Sci U S A 107:4687-92.519

21. Liu, K., and G. Li. 1998. Catalytic domain of the p120 Ras GAP binds to RAb5 and 520

stimulates its GTPase activity. J Biol Chem 273:10087-90.521

22. Ma, K., A. M. Roy, and G. R. Whittaker. 2001. Nuclear export of influenza virus 522

ribonucleoproteins: identification of an export intermediate at the nuclear periphery. 523

Virology 282:215-20.524

23. Martin, K., and A. Helenius. 1991. Nuclear transport of influenza virus 525

ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import. 526

Cell 67:117-30.527


.org/D

ownloaded from

http://jvi.asm.org/

28

24. Momose, F., Y. Kikuchi, K. Komase, and Y. Morikawa. 2007. Visualization of 528

microtubule-mediated transport of influenza viral progeny ribonucleoprotein. Microbes 529

Infect 9:1422-33.530

25. Naslavsky, N., and S. Caplan. 2011. EHD proteins: key conductors of endocytic 531

transport. Trends Cell Biol 21:122-31.532

26. Neumann, G., M. R. Castrucci, and Y. Kawaoka. 1997. Nuclear import and export of 533

influenza virus nucleoprotein. J Virol 71:9690-700.534

27. Neumann, G., M. T. Hughes, and Y. Kawaoka. 2000. Influenza A virus NS2 protein 535

mediates vRNP nuclear export through NES-independent interaction with hCRM1. 536

EMBO J 19:6751-8.537

28. Neumann, G., T. Watanabe, H. Ito, S. Watanabe, H. Goto, P. Gao, M. Hughes, D. R. 538

Perez, R. Donis, E. Hoffmann, G. Hobom, and Y. Kawaoka. 1999. Generation of 539

influenza A viruses entirely from cloned cDNAs. Proc Natl Acad Sci U S A 96:9345-50.540

29. Neville, M., F. Stutz, L. Lee, L. I. Davis, and M. Rosbash. 1997. The importin-beta 541

family member Crm1p bridges the interaction between Rev and the nuclear pore complex 542

during nuclear export. Curr Biol 7:767-75.543

30. Niwa, H., K. Yamamura, and J. Miyazaki. 1991. Efficient selection for high-544

expression transfectants with a novel eukaryotic vector. Gene 108:193-9.545

31. O'Neill, R. E., J. Talon, and P. Palese. 1998. The influenza virus NEP (NS2 protein) 546

mediates the nuclear export of viral ribonucleoproteins. EMBO J 17:288-96.547

32. Pemberton, L. F., and B. M. Paschal. 2005. Mechanisms of receptor-mediated nuclear 548

import and nuclear export. Traffic 6:187-98.549


.org/D

ownloaded from

http://jvi.asm.org/

29

33. Pryor, P. R., L. Jackson, S. R. Gray, M. A. Edeling, A. Thompson, C. M. Sanderson, 550

P. R. Evans, D. J. Owen, and J. P. Luzio. 2008. Molecular basis for the sorting of the 551

SNARE VAMP7 into endocytic clathrin-coated vesicles by the ArfGAP Hrb. Cell 552

134:817-27.553

34. Rossman, J. S., X. Jing, G. P. Leser, and R. A. Lamb. 2010. Influenza virus M2 554

protein mediates ESCRT-independent membrane scission. Cell 142:902-13.555

35. Sakaguchi, A., E. Hirayama, A. Hiraki, Y. Ishida, and J. Kim. 2003. Nuclear export 556

of influenza viral ribonucleoprotein is temperature-dependently inhibited by dissociation 557

of viral matrix protein. Virology 306:244-53.558

36. Sanchez-Velar, N., E. B. Udofia, Z. Yu, and M. L. Zapp. 2004. hRIP, a cellular 559

cofactor for Rev function, promotes release of HIV RNAs from the perinuclear region. 560

Genes Dev 18:23-34.561

37. Schmid, E. M., M. G. Ford, A. Burtey, G. J. Praefcke, S. Y. Peak-Chew, I. G. Mills, 562

A. Benmerah, and H. T. McMahon. 2006. Role of the AP2 beta-appendage hub in 563

recruiting partners for clathrin-coated vesicle assembly. PLoS Biol 4:e262.564

38. Shimizu, T., N. Takizawa, K. Watanabe, K. Nagata, and N. Kobayashi. 2011. Crucial 565

role of the influenza virus NS2 (NEP) C-terminal domain in M1 binding and nuclear 566

export of vRNP. FEBS Lett 585:41-6.567

39. Tomoda, T., J. H. Kim, C. Zhan, and M. E. Hatten. 2004. Role of Unc51.1 and its 568

binding partners in CNS axon outgrowth. Genes Dev 18:541-58.569

40. Watanabe, K., N. Takizawa, M. Katoh, K. Hoshida, N. Kobayashi, and K. Nagata.570

2001. Inhibition of nuclear export of ribonucleoprotein complexes of influenza virus by 571

leptomycin B. Virus Res 77:31-42.572


.org/D

ownloaded from

http://jvi.asm.org/

30

41. Watanabe, R., and R. A. Lamb. 2010. Influenza virus budding does not require a 573

functional AAA+ ATPase, VPS4. Virus Res 153:58-63.574

42. World Health Organization (W.H.O.). April 21, 2011, posting date. Global Alert and 575

Response; Cumulative Number of Confirmed Human Cases of Avian Influenza 576

A/(H5N1) Reported to World Health Organization (WHO). 577

http://www.who.int/csr/disease/avian_influenza/country/cases_table_2011_04_21/en/inde578

x.html 579

43. World Health Organization (W.H.O). April 2009, posting date. Fact Sheet N°211. 580

http://www.who.int/mediacentre/factsheets/fs211/en/ 581

44. Xiao, G. H., F. Shoarinejad, F. Jin, E. A. Golemis, and R. S. Yeung. 1997. The 582

tuberous sclerosis 2 gene product, tuberin, functions as a Rab5 GTPase activating protein 583

(GAP) in modulating endocytosis. J Biol Chem 272:6097-100.584

45. Yu, Z., N. Sanchez-Velar, I. E. Catrina, E. L. Kittler, E. B. Udofia, and M. L. Zapp.585

2005. The cellular HIV-1 Rev cofactor hRIP is required for viral replication. Proc Natl 586

Acad Sci U S A 102:4027-32.587


.org/D

ownloaded from

http://jvi.asm.org/

31

FIGURE LEGENDS 588

Figure 1. HRB perturbation interferes with influenza virus growth. (A) 293 cells were 589

transfected with a validated, non-targeting commercial negative control siRNA (AllStars Neg, 590

referred to as ‘Neg’ on the figure), siRNA targeting influenza NP mRNA (NP) or siRNA 591

targeting HRB, and total cell lysates were subjected to immunoblot analysis with mouse anti-592

HRB or rabbit anti-calnexin (CANX; loading control). (B) Cell viability was measured using 593

the CellTiter-Glo assay. 293 cells were transfected with siRNAs as described for panel A, 594

except that a cell death-inducing siRNA mixture (Death) was also included. The data are 595

represented as an average luciferase reading ± standard deviation (SD) for triplicate 596

transfections. (C) Cells were transfected with siRNAs as described for panel A and were super-597

infected with influenza virus A/WSN/33 (WSN). At 48 h post-infection, supernatants were 598

assayed for infectious virus by plaque assay in MDCK cells. The data shown are a compilation 599

of three independent experiments, in which triplicate transfections were performed for each 600

siRNA, and are represented as an average ± SD. A paired Student’s t-test was used to compare 601

replication in AllStars Neg siRNA-treated cells versus either NP or HRB siRNA treatments, and 602

the P-value is indicated above the graph. (D) 293 cells were transfected with AllStars Neg or 603

HRB siRNA as described, and infected with VSV, Ad5 or vaccinia virus. Infectious viruses 604

from each condition were quantified as described in the Materials and Methods. Paired 605

Student’s t-tests were performed to compare replication between the siRNA treatment conditions 606

for each virus, and significant P-values are indicated above the graph. Data are represented as 607

means of triplicate transfections from two independent experiments ± standard error of the mean. 608

(E) 293 cells were transfected with plasmids expressing GFP, NEP-YFP or a N360-GFP and 609

expression levels were determined by immunoblot analysis of whole cell lysates with anti-GFP 610


.org/D

ownloaded from

http://jvi.asm.org/

32

antibodies at 48 h post-transfection. (F) At 48 h post-transfection, plasmid-transfected cells 611

were super-infected with influenza virus WSN, and the level of infectious virus in supernatants 612

was assayed by plaque assay in MDCK cells after 72 h. Data are represented as an average of 613

triplicate infections performed for each transfection condition ± SD. 614

615

Figure 2. HRB is not required for early events in the influenza virus life cycle. (A) siRNA-616

treated 293 cells were mock-infected or infected with influenza virus WSN at a multiplicity of 617

infection (MOI) of 3 PFU per cell, and total cell lysates were subjected to immunoblot analysis 618

with rabbit polyclonal antibody R528 (against influenza vRNP) or rabbit anti-actin (loading 619

control) at different times after infection. siRNA treatments are indicated to the left, time points 620

are shown at the top and antibodies are to the right. Duplicate samples were prepared for each 621

infection condition at each time point. (B) 293 cells were transfected with AllStars Neg 622

(referred to as ‘Neg’ on figure), NP or HRB-specific siRNAs; or plasmids expressing GFP, NEP-623

YFP or N360-GFP; and were subsequently transfected with plasmids for the influenza WSN 624

mini-replicon assay. Viral gene expression levels were quantified as described in the Materials 625

and Methods. Data are represented as average ratios of firefly (viral)/Renilla (cellular) gene 626

expression from triplicate transfections ± SD. 627

628

Figure 3. HRB and NEP distribution in influenza virus-infected cells. A549 cells were 629

mock-infected (A) or infected with influenza WSN (MOI 3) and fixed with 4% 630

paraformaldehyde at 5 (B), 7 (C) and 9 (D) hpi. Permeabilized cells were stained with rabbit 631

polyclonal anti-serum against influenza NEP (R5023) and a mouse monoclonal antibody against 632

HRB, combined with AF 488 goat anti-rabbit and AF 546 goat anti-mouse, and Hoechst 33258. 633


.org/D

ownloaded from

http://jvi.asm.org/

33

Individual NEP (green) and HRB (red) staining patterns are shown for each time point, along 634

with a merged panel including Hoechst nuclear staining. A 3X zoom highlighting the 635

perinuclear region from the merged panels is shown in (B-D). Nuc, nucleus 636

637

Figure 4. Polyclonal rabbit anti-vRNP (R528) identifies influenza vRNPs with similar 638

specificity to that of monoclonal antibody 3/1. A549 cells were infected with WSN at an MOI 639

of 3 PFU per cell and fixed with 4% paraformaldehyde at 9 hpi. Permeabilized cells were 640

stained with R528 and monoclonal antibody 3/1 (MAb 3/1), combined with AF 488 goat anti-641

rabbit (green) and AF 546 goat anti-mouse (red). Individual and merged staining patterns are 642

shown.643

644

Figure 5. vRNP and HRB spatio-temporal dynamics in influenza virus-infected cells. A549645

cells were infected with influenza WSN (MOI 3) and harvested at 5 (A), 7 (B), 9 (C) or 11 (D) 646

hpi. Cells were stained with rabbit anti-vRNP (R528) and mouse anti-HRB, followed by AF 488 647

goat anti-rabbit (green) and AF 546 goat anti-mouse (red). Representative staining profiles are 648

shown, with the specific time points indicated at the top. For each panel, (i) shows a merged 649

image of HRB and vRNP, and individual staining profiles are shown in (ii) and (iii), 650

respectively. In (ii), white traces indicate the plasma membrane boundaries. Individual and 651

merged staining is also shown for enlargements (3X) of boxed regions from (i): (iv, HRB), (v, 652

vRNP) and (vi, Merge). A staining key is shown in the lower right corner of each panel.653

654

Figure 6. Leptomycin B (LMB) does not induce HRB nuclear accumulation. A549 cells 655

were mock-infected or infected with influenza WSN (MOI 3), and at 4 hpi they were treated with 656


.org/D

ownloaded from

http://jvi.asm.org/

34

LMB and further incubated with LMB for 5 h, followed by fixation in paraformaldehyde. (A) 657

DMSO (control) and LMB-treated mock-infected cells were stained with monoclonal mouse 658

anti-HRB and AF 546 goat anti-mouse secondary antibodies. Infected, DMSO or LMB-treated 659

cells were stained as described in Fig. 5 (B) or Fig. 3 (C), respectively. All cells were 660

counterstained with Hoechst 33258. Individual HRB (red) and vRNP or NEP (green) panels are 661

shown, along with a merged panel including Hoechst. For (A-C), drug treatments are shown at 662

the top and specific stains are indicated to the left. (D) Enlarged images of the merged panels in 663

(C), highlighting HRB and NEP distribution around the periphery of the nucleus. A color key is 664

shown at the left and drug treatments are shown at the right. 665

666

Figure 7. HRB knockdown causes retention of vRNP in the peri-nuclear region. (A) Total 667

cell lysates from AllStars Neg (referred to as ‘Neg’ on the figure), NP and HRB siRNA-treated 668

HeLa cells at 48 h post-transfection were subjected to immunoblot analysis with mouse anti-669

HRB or rabbit anti-CANX (loading control). (B) Negative control siRNA-treated HeLa cells 670

were mock-infected and subjected to staining with mouse anti-HRB and rabbit anti-vRNP 671

(R528), followed by AF 546 goat anti-mouse and AF 488 goat anti-rabbit secondary antibodies. 672

(C-E) HeLa cells treated with either negative control (AllStars Neg) or HRB siRNA were 673

infected with influenza WSN (MOI 5) and fixed at 6, 9 and 15 hpi. Cells were stained as 674

described for panel (B). Individual HRB (red) and vRNP (green) staining profiles, as well as 675

merged images are shown for each condition. siRNA treatments are shown to the left and time 676

points and stains are indicated at the top of each panel. 677


.org/D

ownloaded from

http://jvi.asm.org/


.org/D

ownloaded from

http://jvi.asm.org/


.org/D

ownloaded from

http://jvi.asm.org/


.org/D

ownloaded from

http://jvi.asm.org/


.org/D

ownloaded from

http://jvi.asm.org/


.org/D

ownloaded from

http://jvi.asm.org/


.org/D

ownloaded from

http://jvi.asm.org/


.org/D

ownloaded from

http://jvi.asm.org/

Documents

HRB is Essential for Influenza A Virus Replication and Promotes