JGV Papers in Press. Published February 9, 2015 as doi:10.1099/vir.0.000082

Journal of General Virology Role of Stem Glycans Attached to Haemagglutinin on the Biological Characteristics of H5N1 Avian Influenza Virus --Manuscript Draft-Manuscript Number:

JGV-D-14-00015R1

Full Title:

Role of Stem Glycans Attached to Haemagglutinin on the Biological Characteristics of H5N1 Avian Influenza Virus

Short Title:

Stem Glycans on HA protein of H5N1 AIV

Article Type:

Standard

Section/Category:

Animal - Negative-strand RNA Viruses

Corresponding Author:

Daxin Peng Yangzhou University Yangzhou, Jiansu CHINA

First Author:

Xiaojian Zhang

Order of Authors:

Xiaojian Zhang Sujuan Chen Da Yang Xiao Wang Jingjing Zhu Daxin Peng Xiufan Liu

Abstract:

There are three conserved N-linked glycosites, namely, Asn10, Asn23, and Asn286, at the stem region of hemagglutinin (HA) in H5N1 avian influenza viruses (AIVs). To understand the effect of glycosylation in the stem domain of HA on the biological characteristics of H5N1 AIVs, we used site-directed mutagenesis to generate different patterns of stem glycans on the HA of A/Mallard/Huadong/S/2005. The results indicated that these three N-glycans were dispensable for the generation of replicationcompetent influenza viruses. However, when N-glycans at Asn10 plus either Asn23 or Asn268 were removed, the cleavability of HA was almost completely blocked, leading to a significant decrease of the growth rates of the mutant viruses in MDCK and CEF in comparison with that of the wild-type (WT) virus. Moreover, the mutant viruses lacking these oligosaccharides, particularly the N-glycan at Asn10, revealed a significant decrease in thermostability and pH stability compared with the WT virus. Interestingly, the mutant viruses induced a lower level of neutralizing antibodies against the WT virus, and most of the mutant viruses were more sensitive to neutralizing antibodies than the WT virus. Taken together, these data strongly suggest that the HA stem glycans play a critical role on HA cleavage, replication, thermostability, pH stability, and antigenicity of H5N1 AIVs.

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1 2

Role of Stem Glycans Attached to Haemagglutinin on the Biological Characteristics of H5N1 Avian Influenza Virus

3

Xiaojian Zhang§, Sujuan Chen§, Da Yang, Xiao Wang, Jingjing Zhu, Daxin Peng*,

4

Xiufan Liu

5

(College of Veterinary Medicine, Yangzhou University; Jiangsu Co-Innovation Centre

6

for the Prevention and Control of Important Animal Infectious Disease and Zoonoses;

7

Jiangsu Research Centre of Engineering and Technology for the Prevention and

8

Control of Poultry Disease; Yangzhou, Jiangsu, 225009, P.R. China)

9

Running title: Stem Glycans on HA protein of H5N1 AIV

10

§

11

*Corresponding author: Daxin Peng

12

48 East Wenhui Road

13

Yangzhou, Jiangsu 225009

14

PR China

15

Tel: 86-514-87979386

16

Email: [email protected]

These authors contributed equally to this work.

17

Abstract

18

There are three conserved N-linked glycosites, namely, Asn10, Asn23, and

19

Asn286, at the stem region of hemagglutinin (HA) in H5N1 avian influenza viruses

20

(AIVs). To understand the effect of glycosylation in the stem domain of HA on the

21

biological characteristics of H5N1 AIVs, we used site-directed mutagenesis to

22

generate different patterns of stem glycans on the HA of A/Mallard/Huadong/S/2005.

23

The results indicated that these three N-glycans were dispensable for the generation of

24

replication-competent influenza viruses. However, when N-glycans at Asn10 plus

25

either Asn23 or Asn268 were removed, the cleavability of HA was almost completely

26

blocked, leading to a significant decrease of the growth rates of the mutant viruses in

27

MDCK and CEF in comparison with that of the wild-type (WT) virus. Moreover, the

28

mutant viruses lacking these oligosaccharides, particularly the N-glycan at Asn10,

29

revealed a significant decrease in thermostability and pH stability compared with the

30

WT virus. Interestingly, the mutant viruses induced a lower level of neutralizing

31

antibodies against the WT virus, and most of the mutant viruses were more sensitive

32

to neutralizing antibodies than the WT virus. Taken together, these data strongly

33

suggest that the HA stem glycans play a critical role on HA cleavage, replication,

34

thermostability, pH stability, and antigenicity of H5N1 AIVs.

35

Keywords: H5N1 avian influenza virus, Hemagglutinin, Stem glycans, Stability

36

INTRODUCTION

37

The H5N1 subtype avian influenza virus (AIV) infects wild birds and poultry in

38

several places worldwide, and H5N1 AIV infection causes a serious economic loss in

39

many countries (Chen et al., 2004; Gu et al., 2013; Li et al., 2010; Zhao et al., 2013).

40

The virus also causes a serious and frequently fatal illness in humans who were in

41

contact with infected birds, but the transmission between humans is limited (Claas et

42

al., 1998; Subbarao et al., 1998; Yen et al., 2007; Yuen et al., 1998). Several lines of

43

evidence indicate that the threat of an H5N1 pandemic is real, and that careful

44

planning is necessary to reduce the public health impact of H5N1 AIV infection (Gao

45

et al., 2009; Li & Chen, 2014; Song et al., 2011). Recent human isolates resistant to

46

adamantanes and neuraminidase inhibitors and the difficulty associated with vaccine

47

production against H5N1 AIV mutants illustrate the need for additional research on

48

the epidemiology and biology of H5N1 influenza virus (Proenca-Modena et al.,

49

2007).

50

Hemagglutinin (HA) is a major surface glycoprotein responsible for viral

51

attachment and penetration during infection and often undergoes N-linked

52

glycosylation at several sites (Kim & Park, 2012; Rossman & Lamb, 2011). Amino

53

acid sequence analysis has revealed that there is considerable variation in both the

54

number and location of potential glycosylation sites among different HA subtypes and

55

even among variants from a single subtype (Chen et al., 2012; Wang et al., 2009). In

56

contrast, the oligosaccharides attached to the upper globular domain at variable sites

57

of the HA molecule have been shown to modulate antigenic properties (Skehel et al.,

58

1984; Wang et al., 2009; Wang et al., 2010b; Zhang et al., 2015), receptor binding

59

(Gao et al., 2009; Liao et al., 2010; Matrosovich et al., 1999; Ohuchi et al., 1997a;

60

Wang et al., 2010b), and resistance to collectins (Hartshorn et al., 2008; Reading et al.,

61

2009; Tate et al., 2011). The conserved glycosylation sites are located within the stem

62

region of the HA molecule, which has been assumed to provide the main forces that

63

stabilize the HA trimer (Liao et al., 2010; Wiley & Skehel, 1987). In a previous study,

64

a vaccinia virus expression system was used to study the role of stem glycosylation

65

sites (12, 28 and 478) in the HA of a H7N1 influenza virus on the transport and

66

stability of the HA protein. The loss of all three oligosaccharides in the stem region

67

resulted in temperature sensitivity and complete transport blockage at the

68

nonpermissive temperature, whereas the loss of two oligosaccharides reduced the

69

transport rate of HA (Roberts et al., 1993). It has been further demonstrated that stem

70

glycans are important for the maintenance of the metastable conformation of the HA

71

protein required for fusion activity by expressing mutants of the HA protein lacking

72

stem glycans in CV1 cells (Ohuchi et al., 1997b). Moreover, it has also been

73

demonstrated that the N-glycan at Asn12 is crucial for virus replication using an RNA

74

polymerase I-based reverse genetics system (Wagner et al., 2002).

75

For H5 subtype influenza viruses, there are three highly conserved potential

76

glycosylation sites in the stem of the HA protein: Asn10/11, Asn23 and Asn286 (H5

77

numbering) (Chen et al., 2012). It has been previously reported that a single point

78

mutation in the HA of the Ck/Penn (H5N2) virus resulted in the loss of N-linked

79

glycans in the stalk region at residue 11, leading to virulence (Deshpande et al., 1987).

80

Moreover, it has been demonstrated that glycans in the stalk and the length of the

81

connecting peptide determine the cleavability of the H5 influenza virus HA (Kawaoka

82

& Webster, 1989). However, all of the above-described results are based on HA

83

sequence analysis or only involve a single potential glycosylation site, and the

84

functional importance of stem glycans for H5 viruses has yet to be fully defined. In

85

this study, we generated recombinant influenza viruses lacking the N-glycans in the

86

HA stem through site-specific mutagenesis

87

characteristics and antigenicity of the resulting mutants.

88

RESULTS

89

Generation of recombinant viruses differing in HA stem

90

glycosylation

and examined the biological

91

There were three potential glycosylation sites at Asn10/11, Asn23, and Asn286

92

(Fig. 1) within the HA stem, in which Asn10/11 (10NNST) was an overlapping

93

glycosylation sequence. To remove the glycosylation site of Asn10/11, three mutants

94

were constructed by substitution of 10NNST for 10NPST, 10NNSA, or 10-NNAA,

95

and

96

respectively. To remove the glycosylation sites Asn23 or Asn286, 23NVT and

97

286NSS were replaced by 23NVA and 286NSG, respectively. Accordingly, the

98

recombinant AIVs with the single, double, or triple deletion of potential glycosylation

99

sites were generated by combination of 10NPST, 23NVA, and 286NSG, and named

100

rS△10, rS△23, rS△286, rS△10/23, rS△10/286, rS△23/286, and rS△10/23/286. The

101

rescued WT virus (rS) was served as control. All recombinant viruses were confirmed

designated

as

rS△10/11NPST,

rS△10/11NNSA

and

rS△10/11NNAA,

102

by RT-PCR amplification of the viral RNA and sequence analysis. No spontaneous

103

mutation occurred during the rescue and amplification procedure.

104

Western blot analysis and indirect immunofluorescence assay

105

To verify whether N-glycans were missing from the HA1 proteins of the mutant

106

viruses, the protein profile of the recombinant viruses was determined by western blot.

107

As shown in Fig. 2A, HA1 of the rS△10/11NPST mutant virus showed a reduced

108

molecular weight, when compared with that of the WT virus, HA1 of the

109

rS△10/11NNSA mutant virus exhibited a similar molecular weight as that of the WT

110

virus, while both HA0 and HA1 of the rS△10/11NNAA were not detectable. However,

111

M1 proteins were all detectable in cells infected with the three mutant viruses as well

112

as the WT virus. The data suggested that 10Asn was served as a potential

113

glycosylation site and substitution of 10NNST for 10NPST could eliminate this

114

overlapping glycosylation without affecting the synthesis of the HA protein.

115

Since stem glycosylations are thought to play a role in appropriate folding and

116

HA conformation, the HA expression of the three mutant viruses was further

117

evaluated by indirect immunofluorescence assay. As shown in Fig. 3, no marked

118

difference in the intracellular distribution of the HA protein between the WT virus and

119

the three mutant viruses was observed. Thus, it appeared that the loss of the

120

glycosylation site at 10/11Asn by substitution of 10NNST for 10NPST dose not

121

resulted in accumulation of the HA protein in the ER. Therefore, combined with

122

western blot data, the rS△10/11NPST (abbreviation for rS△10) mutant virus was

123

chosen for subsequent study.

124

Compared with the WT HA1 protein, the HA1 proteins from the single, double,

125

or triple deletion mutant viruses showed reduced molecular masses corresponding to

126

the absence of one, two, or three oligosaccharide chains at the conserved glycosites

127

(Fig. 2B). However, due to the absence of two oligosaccharide chains, HA1 protein

128

from the rS△10/23 or rS△10/286 mutant virus was not detectable (Fig. 2B),

129

indicating that the removal of these oligosaccharide chains significantly affected the

130

cleavage of the H5N1 AVI HA protein. These data confirmed that the HA proteins of

131

the mutant viruses were glycosylated as expected and that the cleavability was

132

affected by deletion of stem glycosylation.

133

To evaluate the amount of total HA protein in the infected cells, equal loading of

134

protein samples was calibrated by M1 protein probed with an anti-M1 antibody (Fig.

135

2B). The HA expression level of the rS△10 and rS△23 mutant viruses was higher

136

than that of the WT virus, while slight decrease was observed in the HA expression

137

level of the rS△10/286 and rS△10/23/286 mutant viruses.

138

Virus growth

139

To assess the effects of the loss of N-linked glycosylation on the growth rates of

140

the recombinant viruses, CEF and MDCK cells were infected with the recombinant

141

viruses at an MOI of 0.001. In CEF cells, the loss of glycans had marginal effects on

142

the virus yields compared with the WT virus with the exception of the rS△10/23 and

143

rS△10/286 viruses, which showed retarded growth until 72 h p.i. (Fig. 4A). In MDCK

144

cells, the rS△10/23 and rS△10/286 mutant viruses exhibited markedly diminished

145

growth at 72 h p.i. and rS△10/23/286 showed a 10-fold difference in the TCID50 value

146

compared with the WT virus (Fig. 4B), while the other viruses replicated in a similar

147

manner to the WT virus. All rescued viruses replicated well in 10-day-old SPF eggs

148

(>108 EID50/mL) or in CEF cells (≥108 TCID50/mL), except for the rS△10/23 and

149

rS△10/286 mutant viruses, which showed a decreased virus titer compared with the

150

WT virus. Moreover, the rS△10/23, rS△10/286 and rS△10/23/286 mutant viruses

151

exhibited significant attenuation in embryonic eggs according to MDTs of the

152

recombinant viruses. All of the recombinant viruses produced similar plaque sizes in

153

MDCK cells (Fig. 5). However, the mutant viruses with losing of 10N glycosylations

154

(especially for rS△10 and rS△10/23) exhibited a decrease in the clearness of the

155

plaques compared with the WT virus.

156

Thermal stability of the recombinant viruses

157

To analyse the thermostabilities, the WT and seven mutant viruses with different

158

stem glycosylation patterns were exposed to 37°C for five days. All of the mutant

159

viruses exhibited a significant decrease in titre compared with the WT virus, and the

160

rS△10, rS△10/23 and rS△10/286 mutant viruses were completely inactivated within

161

three days when incubated at 37°C (P < 0.05, Fig. 6A). When incubated at 42°C, all

162

of the mutant viruses showed a similar pattern, and the rS△10, rS△10/23 and

163

rS△10/286 mutant viruses were completely inactivated within one day (Fig. 6B),

164

indicating that deletion of 10N glycosylation in the stem of HA exhibits a significant

165

effect on the thermostability of the H5N1 virus in vitro.

166

Low-pH stability of the recombinant viruses

167

To evaluate the low-pH stability of the recombinant viruses, their HA titres were

168

measured after pre-incubation for 10 min under the indicated acidic conditions (pH

169

4.0, 5.0 and 6.0). The results showed that all of the viruses were inactivated at pH 4.0.

170

The rS△286 mutant virus maintained the HA titre at pH 5.0 and 6.0, as did the WT

171

virus. The rS△10/23/286 mutant virus exhibited enhanced susceptibility at pH 5.0,

172

and the other mutant viruses showed a significant decrease in HA titre at pH 5.0 and

173

6.0 compared with the WT virus (P < 0.05, Fig. 7), indicating that the loss of 10N and

174

23N glycosylation in the stem of HA plays a part in the low-pH stability of the H5N1

175

virus in vitro.

176

Antigenicity

177

To evaluate the effect of stem glycosylation on the antigenic properties of the H5

178

viruses, we tested the Nt activities of sera obtained from chickens immunized with

179

each of the glycosylation mutant viruses against each other. As shown in Table 1, the

180

mutant viruses induced lower Nt antibody against the WT virus than the WT virus.

181

However, the mutant viruses were more susceptible to antisera induced by the WT or

182

mutant viruses than the WT virus, suggesting that the stem glycosylations of the HA

183

protein influence the antigenicity and stability of the H5N1 AVI virus.

184

DISCUSSION

185

It has been shown that HA stem glycans are potent regulators of influenza virus

186

(H7N1) replication (Wagner et al., 2002). For example, the N-glycan at Asn28 (H7

187

numbering) is indispensable for the formation of replication-competent influenza

188

viruses in the RNA polymerase I-based reverse genetics system. In this study, we were

189

able to rescue all of the mutant viruses lacking any of the stem glycans using an

190

eight-plasmid DNA transfection system (Hoffmann et al., 2000). Thus, it appears that

191

stem glycans are dispensable for the generation of replication-competent influenza

192

viruses. Previous studies in MDCK cells and embryonated chicken eggs have revealed

193

that the N-glycan at Asn12 is crucial for virus replication (Wagner et al., 2002). It has

194

proven that the HA sequence at 10-12, namely Asn-Asn-Ser, in either avirulent or

195

virulent strains of H5N2 AIV is not a glycosylation site and that the HA protein in an

196

avirulent H5N2 AIV is glycosylated at residue Asn11 (Deshpande et al., 1987). In this

197

study, the H5N1 AIV A/Mallard/Huadong/S/2005 strain possesses the same sequence

198

Asn-Asn-Ser-Thr at 10-13. We used the 10NPST, 10NNSA, or 10NNAA changes to

199

remove the potential glycosylation site and confirmed that 10Asn was served as a

200

potential glycosylation site, and that 10NPST change could eliminate this overlapping

201

glycosylation without affecting the synthesis of the HA protein. The mutant viruses

202

with a single N-glycan deletion at Asn10, Asn23, or Asn286 show a similar growth

203

rate as the WT virus in both CEF and MDCK cells; however, the rS△10/23 and

204

rS△10/286 mutant viruses exhibited a strong decrease in replication. We conclude that

205

glycans at Asn10 are involved in H5N1 virus replication in vitro, and there is a

206

synergistic effect between stem glycans on virus replication. This finding corresponds

207

closely with our other results, which show that the absence of the N-glycan at Asn10

208

has the most severe effect on thermal stability and low-pH stability.

209

Hemagglutinin is synthesized as a precursor (HA0) in the endoplasmic reticulum

210

and then exported to the cell surface via the Golgi network. On the cell surface, HA0

211

is cleaved by specific host proteases into HA1 and HA2 (Skehel & Wiley, 2000). It

212

has been previously reported that site-specific glycosylation affects the cleavage of

213

the H5N2 AIV HA and thus virulence (Deshpande et al., 1987). Presumably, the

214

presence of glycosylation at Asn10/11 may mask a recognition signal required by the

215

cleavage enzyme or perturb a structural feature such that it no longer serves as a

216

substrate for the processing enzyme. Further study confirmed that the relationship

217

between carbohydrates in the stalk and the length of the connecting peptide is a

218

critical determinant of cleavability (Kawaoka & Webster, 1989). HAs lacking an

219

oligosaccharide side chain at residue Asn10/11 were cleaved with four basic amino

220

acids at the cleavage site, whereas those with an oligosaccharide side chain resisted

221

cleavage unless additional basic amino acids were inserted. In this study, the HA of

222

the WT virus with five basic amino acids at the cleavage site was cleaved into HA1

223

and HA2 efficiently even if the strain possessed a potential glycosylation site at

224

residue Asn10. However, HAs that lacked glycosylation at Asn10 resisted cleavage,

225

and the cleavage of the HAs were almost completely blocked by the deletion of

226

glycosylation at Asn10 in combination with either Asn23 or Asn286. It is most likely

227

that the absence of carbohydrates may perturb a structural feature such that it no

228

longer serves as a substrate for the processing enzyme.

229

The deletion of glycans in the HA stem region leads to a change in the HA

230

structure (Liao et al., 2010), thereby possibly affecting the stability of the resulting

231

mutant viruses. It has been shown that temperature is negatively correlated with the

232

persistence time (Brown et al., 2007; Brown et al., 2009). In this study, we found that

233

the deletion of any N-glycan in the stem of HA exhibits a significant effect on the

234

thermostability, and this effect was particularly observed with the deletion of an

235

N-glycan at Asn10. In fact, the mutant viruses lacking an oligosaccharide side chain at

236

residue Asn10 are much more thermosensitive than the WT virus (Negovetich &

237

Webster, 2010). In addition to temperature, the persistence of virus in the environment

238

is also affected by pH (Brown et al., 2009). It has been reported that the stem glycan

239

can be regarded as a potent stabilizer of the metastable conformation of the HA

240

protein preventing premature denaturation (Wagner et al., 2002). Consistently, our

241

results showed that the deletion of glycans at Asn10 and Asn23 had a significant

242

effect on the hemagglutination activity of the mutant viruses under acidic conditions

243

(pH 5.0 and pH 6.0) compared with the WT virus. These data indicate that the glycan

244

at Asn10 is important for the maintenance of the thermostability and pH stability of

245

H5N1 AIVs.

246

The acquisition of N-linked glycosylation sites on the HA protein is thought to

247

play a beneficial role in virus variation by shielding antigenic sites from interaction

248

with antibodies (Kobayashi & Suzuki, 2012; Wanzeck et al., 2011). The removal of

249

glycosylation sites on the HA protein can expose conserved epitopes hidden by large

250

glycans to elicit an immune response that recognizes HA variants at a higher titre

251

(Wang et al., 2009). Broadly neutralizing HA2 stem-specific monoclonal antibodies

252

with neutralizing activity for H1 clade viruses (Ekiert et al., 2009; Sui et al., 2009)

253

and H3 subtype viruses (Bommakanti et al., 2010; Wang et al., 2010a) have been

254

developed. These studies indicate that epitopes in the stem of HA are conserved and

255

accessible to antibodies. In this study, the mutant viruses lacking stem glycosylation

256

of the HA are susceptible to antibodies induced by the WT virus; however, the mutant

257

viruses failed to induce higher neutralizing antibodies against the WT virus. Although

258

some mutant viruses showed higher HA protein expression when compared with the

259

WT virus, they still induced lower neutralizing antibodies against the WT virus.

260

Presumably, the deletion of stem glycans may expose more epitopes, resulting in virus

261

inactivation, and decrease the stability of the HA trimer, resulting in the induction of a

262

poor antibody response.

263

In

summary,

stem

glycans

are

dispensable

for

the

generation

of

264

replication-competent H5N1 influenza viruses, but stem glycans contribute to HA

265

cleavability and replication in tissue culture. Moreover, the deletion of stem glycans

266

decreases the thermostability and low-pH stability, which could result in antibody

267

susceptibility of the mutant viruses and induction of a lower antibody response. This

268

study underscores the importance of N-glycans attached to the stem domain of the HA

269

protein for the replication, thermostability and pH stability, and antigenicity of

270

influenza viruses and may be used as a method for the production of

271

temperature-sensitive, live attenuated influenza virus vaccines.

272

MATERIALS AND METHODS

273

Viruses and cells. A/Mallard/Huadong/S/2005 (S, H5N1) (Tang et al., 2009)

274

was isolated in eastern China by our laboratory and identified as a highly pathogenic

275

H5N1 AIV. All wild-type (WT) AIV and mutant strains were propagated in

276

10-day-old specific-pathogen-free (SPF) embryonated chicken eggs. Madin-Darby

277

canine kidney (MDCK) cells, human embryonic kidney (293T) cells (obtained from

278

the American Type Culture Collection, Manassas, VA, USA) and chicken embryo

279

fibroblast (CEF) cells were maintained in Dulbecco’s modified Eagle’s medium

280

(DMEM, Gibco, New York, USA) supplemented with 10% foetal bovine serum (FBS,

281

PAA, Pasching, Australia) at 37°C under 5% CO2.

282

Mutagenesis and virus rescue. Site-directed mutagenesis of the HA gene

283

of the H5N1 AIV S strain was performed by overlap-PCR with following primers. To

284

remove N-glycosylation sites at 10/11N, Asn11 was exchanged for Pro using the

285

primers

286

TCTGTCGAGGGGTTTGCATG -3’, Thr13 was exchanged for Ala using the primers

287

5’-

288

CAACCTGCTCTGCCGAGTTGTTTG -3’, or Ser12 and Thr13 were exchanged for

289

Ala using the primers 5’- CATGCAAACAACGCGGCAGAGCAGGTTG -3’ and 5’-

290

CAACCTGCTCTGCCGCGTTGTTTGCATG

291

glycosylation site at Asn23, Thr25 was exchanged for Ala using the primers 5’-

292

GGAAAAGAATGTTGCTGTTACAC

293

GTGTAACAGCAACATTCTTTTCC -3’. For remove of poteintial glycosylation site

5’-

CATGCAAACCCCTCGACAGA

CAAACAACTCGGCAGAGCAGGTTG

-3’.

-3’

-3’

and

-3’

For

and

remove

and

of

5’-

5’-

poteintial

5’-

294

at Asn286, Ser288 was exchanged for Gly with the primer pair

5’-

295

GATAAACTCTGGTATGCCATTCCAC

5’-

296

GTGGAATGGCATACCAGAGTTTATC -3’. The modified HA genes were cloned

297

into the PHW2000 vector (Hoffmann et al., 2000) and confirmed by sequence

298

analysis, and the positive plasmid was named pHW254-mHA. Eight rescue plasmids

299

(pHW251-PB2,

300

pHW256-NA, pHW257-M, and pHW258-NS) (Tang et al., 2008) with or without the

301

pHW254-mHA substitution plasmid were cotransfected into a mixture of 293T and

302

MDCK cells using the FuGENE HD Transfection Reagent (Promega, Madison, WI,

303

USA). After 48 h, the culture mixtures were inoculated into 10-day-old SPF eggs to

304

amplify the rescued viruses at 35°C. The allantoic fluids were tested individually for

305

the presence of infectious virus through a standard hemagglutination assay using

306

chicken red blood cells (CRBCs) (Killian, 2008). The RNAs of the propagated rescue

307

viruses were extracted and amplified, and each viral gene segment was sequenced to

308

ensure the absence of unwanted mutations.

309

Western blot analysis. To analyse the virus HA protein, CEF cells were

310

infected with the recombinant viruses at an MOI of 2 for 1 h at 37°C under 5% CO2.

311

The infected cells were washed three times with PBS, and fresh DMEM was then

312

added. At 12 h post-infection (p.i.), the cells were lysed with 200 μL of whole-cell

313

lysis buffer (protease inhibitor mixture, 150 mM NaCl, 1% Triton X-100, 20 mM

314

Tris-HCl, pH7.5) (Beyotime, Shanghai, China) on ice for 30 min. The cell lysates

315

were collected with a cell scraper, and the whole-cell extract was harvested by

pHW252-PB1,

pHW253-PA,

-3’

and

pHW254-HA,

pHW255-NP,

316

centrifugation at 13,000 rpm and 4°C for 10 min. The proteins were separated by

317

electrophoresis on a 12% tris-glycine gel and transferred to a nitrocellulose membrane.

318

The membrane was blocked in 5% skim milk, incubated with monoclonal antibody

319

3A9 (anti-HA1 of H5N1 AIV) and mouse serum (anti-M1 of H5N1 AIV), and then

320

followed by incubation with horseradish peroxidase (HRP)-conjugated goat

321

anti-mouse antibodies (EMD Chemicals Inc., San Diego, CA, USA). The protein

322

bands were visualized using enhanced chemiluminescence (Thermo Fisher Scientific

323

Inc., Rockford, IL, USA) on radiographic film.

324

Indirect immunofluorescence assay. CEF cells were infected with the

325

recombinant viruses at an MOI of 2 for 1 h at 37°C under 5% CO2, and then 2 mL of

326

DMEM was added. After incubation for 12 h, the cells were fixed with 4%

327

paraformaldehyde in PBS for 30 min, saturated with PBS containing 0.5% Triton

328

X-100 for 10 min and then blocked with 10% bovine serum albumin in PBS for 30

329

min. Cells were incubated with monoclonal antibody 3A9 (anti-HA) at 37°C for 1 h.

330

Cells were then washed three times with PBS and incubated at 37°C for 1 h with

331

fluorescein isothiocyanate (FITC)-coupled goat anti-mouse secondary antibodies

332

(Santa Cruz, Texas, USA). After incubation with secondary antibodies, cells were

333

washed three times with PBS and incubated with 4’,6-diamidino-2-phenylindole

334

(DAPI) (Beyotime, Shanghai, China) for 5 min and endoplasmic reticulum (ER)

335

Tracker Red (Beyotime, Shanghai, China) for 15 min. Cells were observed using a

336

Zeiss LSM 710 META/AxioVert 200 Confocal Microscope with a 40×lens.

337

Analysis of virus growth. For the growth curves, monolayer cells (CEF and

338

MDCK) were infected with the recombinant viruses at an MOI of 0.001 in DMEM for

339

1 h. The unbound viruses were washed with PBS, and serum-free DMEM was added.

340

The cells were incubated at 37°C under 5% CO2, and the virus titres in the supernatant

341

were monitored periodically by the determination of the TCID50 on CEF cells

342

(Wagner et al., 2000).

343

For the plaque assays, MDCK cells were seeded into 12-well plates and

344

incubated at 37°C in a humidified atmosphere with 5% CO2 for 24 h. The monolayer

345

cells were infected with 10-fold dilutions of the recombinant viruses in a total volume

346

of 0.5 ml of serum-free DMEM. After incubation for 1 h, the cells were washed three

347

times with PBS and then overlaid with DMEM containing 0.8% agar (Sigma, St.

348

Louis, MO, USA) and 2% FBS. The infected cells were incubated for an additional 72

349

h, and the plaques were visualized by staining with 0.1% crystal violet in a 10%

350

formaldehyde solution (Wagner et al., 2000). For each virus, 10 well-spaced plaques

351

were measured using the GNU image manipulation program (version 2.8,

352

www.gimp.org), as described previously (Negovetich & Webster, 2010).

353

Thermostability. The thermostability of the viruses was determined as

354

described previously (Negovetich & Webster, 2010; Yamanouchi & Barrett, 1994).

355

Prior to the incubations, the TCID50 values on CEF cells of the recombinant viruses in

356

allantoic fluid were determined. The starting titres (108 TCID50/mL) were

357

standardized by diluting the virus samples in DMEM containing 0.5% BSA and

358

aliquoted the samples into vials at volumes of 0.1 mL. The viruses in the sealed vials

359

were incubated at either 37°C or 42°C for up to five days. After incubation, the

360

aliquots were quickly cooled in ice water and stored at -80°C until the titre

361

determination on CEF cells with TCID50.

362

Low-pH stability. Fifty microliters of each virus was incubated with 50 μL of

363

100 mM acetate buffer (pH 4.0 and pH 5.0), 100 mM phosphate buffer (pH 6.0) or 1×

364

PBS (pH 7.2) at 37°C for 10 min, and the samples were then neutralized with PBS

365

(pH 7.4) immediately and maintained on ice (Takahashi et al., 2010; Wagner et al.,

366

2002). The virus titres were determined by hemagglutination assay.

367

Antigenicity. All of the animal studies were approved by the Jiangsu

368

Administrative

369

SYXKSU-2007-0005), and complied with the guidelines of laboratory animal welfare

370

and ethical of Jiangsu Administrative Committee for Laboratory Animals.

371

Four-week-old SPF White Leghorn chickens (four chickens in each group) were

372

inoculated intramuscularly with 0.2 mL of inactivated vaccine preparations containing

373

108 EID50 of each virus mixed with Freund’s adjuvant (Sigma, USA) and boosted

374

after two weeks. Sera were collected on day 21 after the second immunization, and

375

the serum antibody levels against the homologous and heterologous viruses were

376

determined using a neutralization (Nt) assay (Wang et al., 2010b). Briefly, two-fold

377

serial dilutions of chicken serum starting from the 1:2 dilution were incubated with an

378

equal volume of the indicated viruses at a concentration of 100 TCID50/50 μL in a

379

96-well plate for 1 h at 37°C. The virus-serum mixture was transferred to monolayers

380

of CEF cells and incubated at 37°C for four days. The neutralizing antibody titres

381

were defined as the reciprocal of the highest serum dilution that completely

Committee

for

Laboratory

Animals

(Permission

number:

382

neutralized the appropriate virus as defined by the absence of a cell pathology effect

383

(CPE) on day 4 p.i.

384

Statistical analysis. The viral titres and antibody titres are expressed as the

385

means ± standard deviation (SD). The statistical analyses were performed using an

386

independent-sample t test. Differences with a P value of less than 0.05 were regarded

387

to be statistically significant.

388

ACKNOWLEDGMENTS

389

This work was supported by the National Natural Science Foundation of China (No.

390

31372450, 31402229), the Special Fund for Agro-Scientific Research in the Public

391

Interest (201003012), the Major State Basic Research Development Program of China

392

(973 Program) (2011CB505003), the National High-Tech Research and Development

393

Program of China (2011AA10A209), and a Project Funded by the Priority Academic

394

Program Development of Jiangsu Higher Education Institutions. We are grateful to

395

Professor Xiulong Xu for his Language Editing on our manuscript.

396

REFERENCES

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influenza viruses in China. Vet Microbiol 163, 351-357.

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Figure Legends

532

Fig. 1. Location of N-glycosylation sites on the HA of the H5N1 AIV

533

A/Mallard/Huadong/S/2005 strain. Ribbon diagrams of monomeric H5 HA structures

534

were generated by the SWISS-MODEL automated protein structure homology

535

modelling server, and the images were obtained using the RasTop (Version 2.2)

536

program. The carbohydrates observed in the electron-density maps are coloured green,

537

and all of the asparagines sites in the HA1 protein are labelled. The location of the

538

polybasic cleavage site is highlighted in red with a dotted circle.

539

Fig. 2. Western blot analyses of the H5N1 avian influenza HA1 protein. Lysates of

540

H5N1 viruses were incubated with mAb3A9 (anti-HA1 of H5N1) and mouse serum

541

(anti-M1 of H5N1 AIV). The binding was visualized with 3-aminophthalate after

542

incubation with peroxidase-conjugated secondary antibodies. (A) Detection of HA

543

protein from the 10/11N glycosylation site deletion mutant viruses. (B) Detection of

544

HA protein from the single, double, and triple glycosylation deletion mutant viruses.

545

Equal loading of protein samples was demonstrated with anti-M1 antibodies. The

546

density of the protein band was determined by using Image J software. Numbers in

547

parentheses are density values to that of M1 protein.

548

Fig. 3. Indirect immunofluorescence assay. CEF cells were infected with rS(WT),

549

rS△10/11NPST, rS△10/11NNSA and rS△10/11NNAA at an MOI of 2 and fixed at 12

550

h p.i. Fixed cells were treated with HA monoclonal antibody (3A9), DAPI for nuclei

551

staining and ER Tracker Red for endoplasmic reticulum staining. A merged image of

552

all channels is also shown. Bar, 10 μm.

553

Fig. 4. Growth curves of mutants in CEF (A) and MDCK (B) cells. The cell

554

monolayers were infected with recombinant viruses bearing WT or glycan mutant

555

HAs from S virus at an MOI of 0.001, and the supernatants were monitored for virus

556

titres using TCID50 at the time points indicated. The error bars represent the standard

557

deviations (SD) of the means from three independent experiments. The statistical

558

significance of the differences in the growth properties between the WT virus and

559

mutant viruses was assessed through a Mann-Whitney test (* P < 0.05).

560

Fig. 5. Plaque assay in MDCK cells. MDCK cells were infected with recombinant

561

viruses. The monolayers were covered with an agarose-containing overlay for 72 h

562

and stained with crystal violet. The dilutions were chosen individually for each virus

563

stock to obtain optimal plaque pictures. No significant difference in plaque size was

564

found between the mutant viruses and wild-type virus.

565

Fig. 6. Effect on the activity of the mutant viruses at 37°C (A) or 42°C (B). The WT

566

virus and mutant viruses were incubated at 37°C or 42°C for 5 days. The titres of the

567

aliquots were determined on CEF cells with TCID50. The error bars denote the SD of

568

the mean of four independent titres at each time point. An asterisk indicates that the

569

titres of the mutant viruses were significantly different from those of the WT virus at

570

the indicated time points, as determined by the Mann-Whitney test (* P < 0.05).

571

Fig. 7. Low-pH stability of the mutant viruses. The recombinant viruses were

572

incubated in each buffer at 37°C for 10 min, and the viral titres were then determined

573

by hemagglutination assay. The results are presented as the log2 of the HA titres at the

574

indicated pH conditions. The error bars denote the SDs of the means of three

575

independent titres at each time point. The statistical significance of the differences in

576

low-pH stability between the WT virus and mutant viruses was assessed through the

577

Mann-Whitney test (* P < 0.05).

Figure Click here to download Figure: Fig. 1.doc

Fig. 1

Figure Click here to download Figure: Fig. 2.doc

Fig. 2

Figure Click here to download Figure: Fig. 3.doc

Fig. 3

Figure Click here to download Figure: Fig. 4.doc

A

10

CEF

*

*

*

*

*

60

*

*

48

6

*

36

Log1 0 TCID5 0/mL

8

*

4

* 2

*

*

72

24

12

0 Hours post-infection

B

10

MDCK

*

6

*

*

*

* 4 * 2

Hours post-infection

Fig. 4

72

60

48

36

24

0 12

Log1 0 TCID5 0/mL

8

rS rS△ 10 rS△ 23 rS△ 286 rS△ 10/23 rS△ 10/286 rS△ 23/286 rS△ 10/23/286

Figure Click here to download Figure: Fig. 5.doc

Fig. 5

Fig. 6 6

* * *

/2 8

B rS △

10

6

6

/2 8

/2 8 /2 3

23

6

*

/2 3

6

*

10

/2 8

** *

23

/2 8

* *

rS △

Viruses rS △

10

*

rS △

6

*

/2 8

rS △

/2 3

6

* *

10

10

28

23

*

rS △

/2 3

* *

10

rS △

rS △

rS △

10

*

rS △

6

* **

28

6

rS △

23

rS △

6

rS △

10

10

rS △

rS

Log 1 0 TCID5 0/mL 10

rS

Log 1 0 TCID5 0/mL

Figure Click here to download Figure: Fig. 6.doc

A

37℃

8

* *

4 *

4 *

0 **

Mock 1d 3d 5d

*

2

0 * * *

8

42℃ * * *

2 * *

Figure Click here to download Figure: Fig. 7.doc

6 * *

* *

* *

* *

*

* *

4 2

Fig. 7

6 /2 8

6 rS △

10

23

/2 3

/2 8

6 10 rS △

Viruses

rS △

/2 8

/2 3 rS △

10

28 6 rS △

23 rS △

rS △

10

0

rS

Hemagglutination (Log 2)

8 pH4.0 pH5.0 pH6.0 PBS

Table Click here to download Table: Table 1.doc

Table 1. Neutralization titres of chicken sera induced by the wild-type virus and the mutant viruses. Sera a Viruses rS (WT) rS△10 rS△23 rS△286 rS△10/23 rS△10/286 rS△23/286 rS△10/23/286 rS (WT) 80 160 40 20 160 80 80 1280 b rS△10 2560 320 160 40 320 160 160 160 rS△23 5120 640 320 160 320 320 160 160 rS△286 10240 320 320 160 640 160 640 320 rS△10/23 10240 1280 1280 640 5120 320 1280 640 rS△10/286 10240 10240 2560 10240 320 2560 640 10240 rS△23/286 320 5120 640 160 640 160 2560 1280 rS△10/23/286 2560 320 640 160 40 2560 160 320 a

The chickens were immunized with the indicated viruses with different HA glycosylations, and sera samples were collected for the neutralization assay on CEF cells. The values represent reciprocal geometric mean antibody titres (GMT) from three animals. b The neutralization titres against homologous viruses are shown in bold.