JVI Accepted Manuscript Posted Online 21 January 2015 J. Virol. doi:10.1128/JVI.03328-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
1 2
Identification of PB2 mutations responsible for the efficient replication
3
of H5N1 influenza viruses in human lung epithelial cells
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Reina Yamaji1, Shinya Yamada1##, Mai Q. Le2, Chengjun Li3, Hualan Chen3, Ema Qurnianingsih4, Chairul A. Nidom4, Mutsumi Ito1, Yuko Sakai-Tagawa1, Yoshihiro Kawaoka1, 5, 6 #
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1
Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
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2
National Institute of Hygiene and Epidemiology, Hanoi, Vietnam
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State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Nangang District, Harbin, China 150001.
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4
Avian Influenza Research Center, Airlangga University, Surabaya, Indonesia
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Wisconsin-Madison, WI 53711
Department of Pathobiological Sciences, School of Veterinary Medicine, University of
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Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
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Running title: Adaptive mutations of H5N1 virus
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Document totals: Word count for abstract: 190 Word count for main text: 3622
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Address for correspondence: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. E-mail: [email protected]
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Corresponding author. Tel.: +81-3-5449-5504; fax: +81-3-5449-5408 Co-corresponding author. Tel.: +81-3-5449-5504; fax: +81-3-5449-5408
2 35 36 37 38
Address for co-correspondence: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. E-mail: [email protected]
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Abstract
40
Highly pathogenic H5N1 avian influenza viruses have caused outbreaks among poultry
41
worldwide, resulting in sporadic infections in humans, approximately 60% mortality of which
42
have been fatal. However, efficient transmission of H5N1 viruses among humans has yet to
43
occur, suggesting that further adaptation of H5N1 viruses to humans is required for their
44
efficient transmission among humans. Viral determinants for efficient replication in humans
45
are currently poorly understood. Here, we report that the polymerase PB2 protein of an
46
H5N1 influenza virus isolated from a human in Vietnam (A/Vietnam/UT36285/2010, 36285)
47
increased the growth ability of an avian H5N1 virus (A/wild bird/Anhui/82/2005, Wb/AH82) in
48
human lung epithelial A549 cells (although, the reassortant virus did not replicate more
49
efficiently than human 36285 virus). Furthermore, we demonstrate that amino acid residues
50
at positions 249, 309, and 339 of the PB2 protein from this human isolate were responsible
51
for its efficient replication in A549 cells. PB2-249G and 339M, which are found in the human
52
H5N1 virus, are rare in H5N1 viruses from both human and avian sources. Interestingly,
53
PB2-249G is found in over 30% of human seasonal H3N2 viruses, which suggests that
54
H5N1 viruses may replicate well in human cells when they acquire this mutation. Our data
55
are of value to H5N1 virus surveillance.
56
4 57
Importance
58
Highly pathogenic H5N1 avian influenza viruses must acquire mutations to overcome the
59
species barrier between avian species and humans. When H5N1 viruses replicate in human
60
respiratory cells, they can acquire amino acid mutations that allow them to adapt to humans
61
through continuous selective pressure. Several amino acid mutations have been shown to
62
be advantageous for virus adaptation to mammalian hosts. Here, we found that amino acid
63
changes at positions 249, 309, and 339 of PB2 contribute to efficient replication of avian
64
H5N1 viruses in human lung cells. These findings are beneficial for evaluating the pandemic
65
risk of circulating avian viruses and for further functional analysis of PB2.
66 67
5 68
Introduction
69
Highly pathogenic H5N1 influenza A viruses continue to circulate among avian species. As a
70
result, sporadic avian-to-human transmission has occurred and the threat of a pandemic has
71
persisted. The first human case caused by a highly pathogenic H5N1 influenza A virus was
72
reported in Hong Kong in 1997 (1, 2). Although the mass culling ordered by the Hong Kong
73
government temporarily ended the outbreak, a second outbreak of H5N1 viruses occurred in
74
2003. With the spread of H5N1 viruses among migrating birds and poultry, these viruses
75
quickly spread beyond East Asia (3). This spread of H5N1 viruses has been accompanied
76
by increasing reports of cases of avian-to-human transmission. As of January 2015, the total
77
number of confirmed human cases of highly pathogenic H5N1 influenza A virus infection had
78
reached 694, with 402 deaths ( http://www.who.int/, 4, 5). However, the human cases of
79
H5N1 infection have been limited mainly to individuals in close contact with infected poultry,
80
and reports of human-to-human transmission have been extremely rare (6, 7). For an H5N1
81
virus to overcome the host barrier, the following conditions must be met: efficient
82
transmission via droplets, efficient replication in the host, and immune vulnerability of the
83
infected populace. Consequently, viruses harboring mutations that promote efficient
84
transmission and replication in humans could lead to a pandemic.
85
A number of viral determinants have been identified that contribute to adaptation of avian
86
influenza viruses to mammalian hosts (8-28), and several large-scale comparative analyses
87
of proteins from avian and human viruses have been performed to catalog the amino acids
6
88
that are conserved in each host species (29-31). The PB2, PB1, and PA subunits of the
89
polymerase complex play a major role in virus pathogenicity and efficient viral growth in
90
mammals. PB2 is particularly important in overcoming species barriers. For example, lysine
91
(K) at residue 627 of PB2 enhances the polymerase activity, replication efficiency, and
92
virulence of H5N1 viruses in mammals (8-10). In addition, arginine at residue 591, which is
93
located close to the amino acid at position 627 of PB2 in the three-dimensional structure of
94
the protein, compensates for the lack of lysine at residue 627 and confers efficient viral
95
replication to pandemic H1N1 viruses in mammals (11). Other amino acid substitutions, such
96
as 701N, 271A, and 158G, in PB2 have also been identified as important markers that
97
confer a replicative advantage and high pathogenicity to influenza viruses in mammals
98
(12-15). Recently, we showed that the combination of amino acid substitutions 147T, 339T,
99
and 588T in PB2 confer a high polymerase activity to avian viruses in human cells (28). It is
100
not clear, however, whether we have identified all of the possible amino acid changes that
101
are needed for efficient growth of H5N1 viruses in mammals.
102
In this study, we attempted to identify amino acids that support the efficient growth of H5N1
103
viruses in human lung cells. Accumulation of information on adaptive mutations to humans
104
helps us to prepare for an unpredictable but potential H5N1 pandemic and to assess the risk
105
of future isolates with pandemic potential. Here, we attempted to identify new molecular
106
determinants associated with the efficient growth of a human-isolated H5N1 virus in human
7
107
lung cells by generating reassortant and mutant viruses in the genetic background of an
108
avian H5N1 virus (with low replicative ability in human lung cells) and a human H5N1 virus
109
(with high replicative ability in human lung cells).
110
8 111
Materials and Methods
112
Cells
113
Madin-Darby canine kidney (MDCK) cells were grown in minimal essential medium (MEM)
114
containing 5% newborn calf serum, vitamins, essential amino acids, and antibiotics. Human
115
embryonic kidney 293T cells, A549 human lung adenocarcinoma epithelial cells, and DF-1
116
chicken embryo fibroblast cells were grown in Dulbecco modified Eagle medium (DMEM)
117
containing 10% fetal bovine serum (FBS) and antibiotics. MDCK, 293T, and A549 cells were
118
cultured at 37℃ with 5% CO2. DF-1 cells were cultured at 39℃ with 5% CO2 unless
119
otherwise stated.
120 121
Viruses
122
In this study, we used the following ten H5N1 viruses: A/wild bird/Anhui/82/2005 (Wb/AH82)
123
(accession numbers: PB2 KP638500, PB1 KP638559, PA KP638509, HA KP638516, NP
124
KP638524, NA KP638533, M KP638542, NS KP638551), A/chicken/Vietnam/TY31/2005
125
(Ck/TY31) (accession numbers: PB2 KP638493, PB1 KP638558, PA KP638501, HA
126
EU118136.1, NP KP638525, NA EU118127.1, M KP638534, NS KP638550), A/chicken/Central
127
Java/UT3091/2005
128
GQ122415, and GQ122395),
129
PB2 KP638492, PB1 KP638552, PA KP638507, HA KP638560, NP KP638523, NA
130
KP638530, M KP638539, NS KP638547), A/Vietnam/UT36282/2010 (36282) (accession
(Ck/UT3091)
(accession
numbers:
GQ122490
to
GQ122495,
A/Vietnam/UT36285/2010 (36285) (accession numbers:
9
131
numbers: PB2 KP638494, PB1 KP638557, PA KP638506, HA KP638515, NP KP638522,
132
NA KP638532, M KP638541, NS KP638549), A/Vietnam/UT36236/2010 (36236) (accession
133
numbers: PB2 KP638495, PB1 KP638556, PA KP638505, HA KP638514, NP KP638520,
134
NA KP638531, M KP638540, NS KP638548), A/Vietnam/HN31676DH/2009 (31676)
135
(accession numbers: PB2 KP638496, PB1 KP638553, PA KP638504, HA KP638513, NP
136
KP638519, NA KP638526, M KP638535, NS KP638543), A/Vietnam/UT31641 Ⅱ /2008
137
(31641) (accession numbers: PB2 KP638497, PB1 KP638554, PA KP638503, HA
138
KP638512,
139
A/Vietnam/UT31604Ⅰ/2009 (31604) (accession numbers: PB2 KP638498, PB1 KP638561,
140
PA KP638502, HA KP638511, NP KP638517, NA KP638527, M KP638536, NS KP638544),
141
and A/Vietnam/UT36250 Ⅰ /2010 (36250) (accession numbers: PB2 KP638499, PB1
142
KP638555, PA KP638508, HA KP638510, NP KP638521, NA KP638529, M KP638538, NS
143
KP638546). Throat swabs were collected from H5N1 influenza virus-infected patients in
144
northern Vietnam and were sent to the National Institute of Hygiene and Epidemiology in
145
Hanoi, Vietnam. To isolate H5N1 virus, clinical specimens were inoculated to MDCK cells in
146
MEM containing 0.3% BSA and incubated at 37℃ for 48 hours. Stock viruses were
147
propagated in MDCK cells at 37℃ and stored at -80℃. 36285 was isolated from a patient
148
who exhibited influenza symptoms and recovered (a two-year-old female, onset on April 2,
149
2010, hospitalized on April 4, 2010). All experiments with H5N1 viruses were performed in a
NP
KP638518,
NA
KP638528,
M
KP638537,
NS
KP638545),
10
150
biosafety level 3 containment laboratory approved for such use by the Ministry of Agriculture,
151
Forestry, and Fisheries, Japan.
152 153
Isolation of viral RNA, RT-PCR, and generation of viruses by use of reverse genetics.
154
Viral RNA was extracted from the supernatants of virus-infected MDCK cells by using a
155
QIAamp viral RNA minikit (Qiagen, Hilden, Germany). Extracted RNA was reverse
156
transcribed with Superscript Ⅲ (Invitrogen, Carlsbad, CA) and the universal primers specific
157
for influenza A virus genes to generate cDNA. The resulting products were PCR-amplified by
158
using KOD Fx DNA polymerase (Toyobo, Osaka, Japan) with specific primers for each virus
159
gene and cloned into the RNA polymerase I plasmid pHH21 (ref). Mutations in the PB2 gene
160
of Wb/AH82 were generated by PCR amplification of the respective PB2 construct with
161
primers possessing the desired mutations. Primer sequences are available upon request. All
162
constructs were sequenced to ensure the absence of unwanted mutations. All avian,
163
reassortant, and mutant viruses were generated by use of plasmid-based reverse genetics,
164
as described previously (32). The culture supernatant derived from transfected cells was
165
amplified in DF-1 cells grown in DMEM containing 0.3% bovine serum albumin. At 48 h
166
post-infection, the culture supernatant was harvested, clarified, divided into aliquots, and
167
stored at -80℃. The virus titers of all human and avian viruses were determined by using
168
plaque assays in MDCK cells.
11
169 170
Mouse experiments
171
Six-week-old female BALB/c mice (Japan SLC) were used for these experiments. To
172
determine the dose lethal to 50% of mice (MLD50), 5 mice/group were anesthetized with
173
isoflurane and inoculated intranasally with 101 to 105 plaque-forming units (PFU) in a 50-µl
174
volume. The mice were monitored daily for clinical signs of infection and checked for
175
changes in body weight and mortality for 14 days post-infection. MLD50 values were
176
calculated by using the method of Reed & Muench (1938). All experiments with mice were
177
performed in accordance with the University of Tokyo's Regulations for Animal Care and Use
178
and were approved by the Animal Experiment Committee of the Institute of Medical Science,
179
the University of Tokyo.
180 181
Viral replication assay
182
Triplicate wells of confluent A549 cells or DF-1 cells were infected with viruses at a
183
multiplicity of infection (MOI) of 0.0002, and incubated for 1 h at 37℃ or 39℃, respectively.
184
After the 1-h incubation, A549 cells were further incubated in MEM containing 0.3% bovine
185
serum albumin (BSA) at 33℃ and 37℃. DF-1 cells were further incubated in DMEM
186
containing 0.3% bovine serum albumin (BSA) at 39℃. Aliquots of supernatants were
187
harvested at 1, 24, 48, 72, and 96 h post-infection, and frozen at -80℃. Virus titers in the
12
188
culture supernatants at each time point were determined by plaque assays in MDCK cells.
189 190
Statistical analysis
191
Differences in the virus titers of the supernatants were statistically analyzed by using the
192
Student’s t-test. Differences in mean maximum body weight loss in mice were also
193
statistically analyzed by using the Student’s t- test.
194 195
13 196
Results
197
Comparison of the growth ability of H5N1 viruses in A549 and DF-1 cells
198
To identify human adaptive mutations in H5N1 influenza viruses, we first compared the
199
growth properties at a multiplicity of infection (MOI) of 0.0002 in carcinomic human alveolar
200
basal
201
A/Vietnam/UT36285/2010
202
A/Vietnam/UT36236/2010
203
A/Vietnam/UT31641 Ⅱ /2008
204
A/Vietnam/UT36250Ⅰ/2010 (36250). All seven human H5N1 viruses grew well in A549 cells
205
at 37˚C with maximum titers of over 106 PFU/ml, except for 31676 which grew to slightly
206
lower titers (Fig. 1A). Sequence analysis revealed that 36250, 36236, 31604, and 31641
207
possess lysine at position 627 of the PB2 protein (PB2-627K), whereas 36282 possesses
208
PB2-591R. However, viruses 31676 and 36285 do not have any known PB2 markers that
209
could account for the efficient replication in mammals (Table 1). These findings suggest that
210
the human H5N1 viruses 31676 and 36285 may have unreported amino acids that enhance
211
their viral growth ability in A549 cells. Therefore, in this study we focused on the replicative
212
efficiency of 36285. First, we generated the 36285 virus by use of reverse genetics
213
(36285-RG) and confirmed that 36285-RG grew as well as wild-type 36285 in A549 cells
214
(Fig.1B). We next compared the growth property of 36285-RG in A549 cells at 37˚C with that
215
of three avian H5N1 viruses generated by use of reverse genetics: A/wild bird/Anhui/82/2005
epithelial
A549
cells
of
seven
(36285), (36236), (31641),
H5N1
viruses
isolated
from
humans:
A/Vietnam/UT36282/2010
(36282),
A/Vietnam/HN31676DH/2009
(31676),
A/Vietnam/UT31604 Ⅰ /2009
(31604),
and
14
216
(Wb/AH82-RG), A/chicken/Vietnam/TY31/2005 (Ck/TY31-RG), and A/chicken/Central
217
Java/UT3091/2005 (Ck/UT3091-RG) (Fig. 1C). Ck/TY31-RG and Wb/AH82-RG grew poorly
218
with maximum titers of less than 104.5 PFU/ml (Fig. 1C). Of note, both 36285 and Wb/AH82
219
belong to the same H5 HA sub-clade 2.3.4 and are genetically closely related (Table 2).
220
Therefore, we compared 36285 and Wb/AH82 to elucidate the mechanism of H5N1
221
adaptation to humans. In chicken embryo fibroblast DF-1 cells, 36285-RG and Wb/AH82-RG
222
both grew well at 39˚C (Fig. 2A), demonstrating their potential to replicate in avian cells; in
223
contrast, 36285-RG grew much better than Wb/AH82-RG in A549 cells at 33˚C (Fig. 2B),
224
demonstrating its potential to replicate in mammalian cells. We compared the growth
225
capability of H5N1 viruses in A549 cells at both 37℃ and 33℃ because in humans, the
226
temperature in the lungs is 37℃, and that in the upper airway is 33℃. However, the body
227
temperature of birds is 39℃; therefore, we compared the growth capability of H5N1 viruses
228
in chicken DF-1 cells only at 39℃.
229 230
Comparison of the pathogenicity of Wb/AH82-RG and 36285-RG in mice
231
We next examined the pathogenicity of Wb/AH82-RG and 36285-RG in mice. Inoculation of
232
103, 104, and 105 PFU of 36285-RG virus resulted in 100% mortality, and a 40% fatality rate
233
was observed in the mice that received 101 and 102 PFU of virus. In contrast, inoculation of
234
104 and 105 PFU of Wb/AH82-RG resulted in 100% mortality and a 60% fatality rate was
15
235
observed in mice that received 102 and 103 PFU of virus. The MLD50 values for 36285-RG
236
and Wb/AH82-RG were 101.5 PFU and 102.7 PFU, respectively (Fig. 3C, 3D). Thus,
237
36285-RG and Wb/AH82-RG differed in their mouse virulence by only one log MLD50 unit.
238 239
The PB2 gene segment from 36285 is responsible for its efficient growth in human
240
cells.
241
To elucidate the molecular basis for the replicative difference between Wb/AH82 and 36285
242
in A549 cells, we generated a series of reassortant viruses (as illustrated in Fig. 4A) and
243
compared their growth properties in A549 cells. The reassortant viruses were named
244
according to the origin of their Wb/AH82 or 36285 genes. For instance, “36285(PB2)”
245
indicates a virus possessing PB2 from 36285 and the rest of its gene segments from
246
Wb/AH82. Three reassortant viruses possessing the PB2, PB1, PA, and NP of 36285 [i.e.,
247
36285(3P+NP), 36285(3P+NP, HA, NA), and 36285(3P+NP, M, NS)] were comparable in
248
their growth to 36285-RG; none of these viruses replicated more efficiently than 36285-RG
249
(Fig. 4B). The replicative ability of 36285(M, NS) was similar to that of Wb/AH82-RG,
250
indicating that the M and NS proteins of 36285 do not have a large impact on the difference
251
in the growth capabilities of Wb/AH82-RG and 36285-RG. Of note, the HA and NA of 36285
252
also contributed, to some extent, to the difference in the growth properties of Wb/AH82-RG
253
and 36285-RG; however, they enhanced viral replication to a much lesser extent than did
16
254
the polymerase subunits and NP of 36285. These results suggest that the PB2, PB1, PA,
255
and/or NP gene segments of 36285 were the most responsible for the difference in growth
256
capability between Wb/AH82-RG and 36285-RG in A549 cells.
257
To determine which viral segment(s) among PB2, PB1, PA, and NP contributed to the high
258
replication of 36285-RG, we generated a range of reassortants (Fig. 5A) and compared their
259
growth properties in A549 cells. Only viruses possessing 36285(PB2) grew well (Fig. 5B). By
260
contrast, 36285(PB1, PA, NP) grew poorly, similarly to Wb/AH82-RG. These results
261
demonstrate that the PB2 of 36285 makes an important contribution to the difference in
262
growth capability between Wb/AH82-RG and 36285-RG in A549 cells.
263 264
A single G-to-D mutation at 309 and the double mutations E249G and T339M enhance
265
the replication of Wb/AH82-RG.
266
Sequence comparison of the PB2 proteins of Wb/AH82 and 36285 revealed twenty-one
267
amino acid differences (Table 3). To identify the specific changes that give rise to efficient
268
viral replication, we generated mutant viruses possessing a chimeric PB2 protein (Fig. 6A);
269
the remaining gene segments were derived from Wb/AH82. In A549 cells, the mutant
270
viruses possessing chimera 1, 5, or 6 grew poorly, whereas those with chimera 2 or 3 grew
271
as well as that with 36285 PB2. These results suggest that residues 221 to 460 contribute
272
most to the difference between 36285-RG and Wb/AH82-RG in terms of growth in A549
17
273
cells.
274
To further define which amino acid residues enhance the viral replication, we generated
275
viruses with additional chimeric PB2 proteins (Fig.7A). The mutant viruses possessing
276
chimera 2, 7, 8, and 11 grew well, whereas the growth of the viruses with chimera 1, 9, and
277
10 was lower compared with that of the viruses possessing residues 221 to 345 from 36285.
278
These results indicate that residues 221 to 345 from 36285 provide the replicative
279
advantage to 36285-RG (Fig. 7B).
280
We then tried to determine which amino acids among residues 221 to 345 of PB2 enhanced
281
the growth properties of 36285-RG. There are three amino acid differences in this region
282
between Wb/AH82 and 36285: 249E/G, 309G/D, and 339T/M. We singly or in combination
283
introduced the three residues of 249G, 309D, and 339M into the PB2 of Wb/AH82 (Fig. 8A)
284
and generated viruses possessing the mutant Wb/AH82 PB2 gene in the background of the
285
remaining
286
changes found among circulating influenza viruses). Indeed, the combination of PB2-249G,
287
-309D, and -339M markedly increased the growth capability of Wb/AH82-RG by around 102
288
fold compared with Wb/AH82-RG (Fig. 8B, 8C). Although the single introduction of
289
PB2-249G, -PB2-339M, or -PB2-309D enhanced the growth capability of Wb/AH82-RG,
290
PB2-339M was least effective (Fig. 8B). All mutants that possessed combinations of two of
291
the PB2 mutations tested grew better than Wb/AH82-RG by more than 101.5-fold (Fig. 8C).
Wb/AH82 genes (note that all mutant Wb/AH82 viruses possess amino acid
18
292
These results indicate that PB2-249G, PB2-339M, and PB2-309D support efficient viral
293
growth in A549 cells.
294 295
The amino acid mutations E249G and T339M may be associated with the adaptation of
296
H5N1 virus to humans.
297
To further evaluate the potential role of the PB2 amino acid residues 249, 309, and 339 in
298
adaptation to humans, we collected full-length PB2 sequences of influenza viruses of
299
various subtypes from the Influenza sequence database (ISD) (Table 4). Almost all of H5N1
300
viruses contained PB2 amino acid 309D, which is highly conserved among influenza viruses
301
of various subtypes regardless of the host species, implying that this residue does not need
302
to change for a virus to break through the species barrier. In contrast, PB2 amino acids
303
249G and 339M were rare among the avian H5N1 viruses. Intriguingly, no less than 49.2%
304
of the human seasonal H3N2 viruses had PB2 amino acid 249G. This finding supports the
305
hypothesis that PB2 amino acid 249G is the amino acid mutation that the virus gains during
306
replication in human respiratory cells and increases its fitness for human adaptation.
307 308
19 309
Discussion
310
Several amino acid mutations associated with the adaptation of H5N1 virus to humans have
311
been identified, including 627K (8-10), 591R, 591K (11), 701N (9, 13), 271A (14), and 158G
312
(15) of PB2. We thought that further analysis of H5N1 viruses isolated from humans could
313
identify new mutations needed for their efficient replication in human cells. Even though the
314
36285 H5N1 virus did not have any amino acid markers that have been previously identified
315
to provide a replicative advantage in mammalian cells, it grew well in human lung cells. By
316
using a viral replication assay, we identified three amino acids in PB2 that are responsible for
317
the replicative efficiency of H5N1 virus in A549 cells. Viruses with these amino acids in PB2
318
circulate in nature; we did not generate viruses possessing ‘novel’ amino acid changes.
319
These data will provide information of value for pandemic preparedness and for use in
320
evaluating the pandemic risk potential of future isolates.
321
Pflug et al. (33) recently determined the complete crystal structure of the heterotrimeric
322
influenza A polymerase bound to the vRNA. This structure revealed that all three amino acid
323
substitutions identified in this study (i.e., PB2-E249G, G309D, and T339M) are located on
324
the surface of the PB2 protein. This finding supports the concept that these residues likely
325
interact with viral or host proteins, leading to the replicative advantage of the 36285 virus.
326
PB2-E249G and T339M have an interesting common feature in that they belong to areas
327
identified as ‘cap-binding' sites (34-37). Influenza virus has developed a mechanism called
328
‘cap-snatching,’ to steal the cap from the host cellular RNA in order to synthesize viral
20
329
protein. The polymerase PB2 subunit binds to the 5’ cap of host pre-mRNAs, which are
330
cleaved after 10–13 nucleotides by the PA subunit (38-40). An X-ray structure of the
331
cap-binding domain shows that the amino acid at position 339 of PB2 is located at the edge
332
of the cap-binding pocket (37). The precise cap-binding site, however, remains controversial;
333
one study identified the PB2 amino acid residues responsible for RNA cap-binding as
334
PB2-363F and PB2-404F (35), but another study identified the PB2 amino acid residues
335
242–282 and 538–577 as important (36), and yet another study suggested that amino acid
336
residues 318–483 were involved (37). The effect of the amino acid 339T in vivo and in vitro
337
on biologic events is also inconsistent. We recently found that 339T has a major impact on
338
the polymerase activity of H5N1 virus jointly with 147T and 588T (28). However, another
339
paper reported that K339T reduced PB2 cap binding activity and influenza polymerase
340
activity in vitro, and also attenuated virulence in mice (41). This latter paper suggested that
341
the K339T substitution in PB2 reduced the cap binding affinity because the side chain of
342
threonine is shorter than that of lysine and threonine is uncharged (41). In the current study,
343
we found that PB2 amino acid T339M helped to facilitate virus growth in A549 cells. Both
344
threonine and methionine are uncharged but the side chain of methionine is longer than that
345
of threonine. Although the function of the three amino acid mutations identified in this study
346
remains unclear, our findings may help improve our understanding of PB2 functions.
347
Human influenza A virus acquired the PB2 amino acid mutation E249G upon mouse
21
348
adaptation (42), suggesting that the amino acid at position 249 probably is involved in some
349
mechanism for adaptation to mammals. Sequence analysis showed that the prevalence of
350
PB2 amino acid 249G in human seasonal H3N2 viruses was almost 50%. Almost all of the
351
seasonal human H3N2 viruses isolated since 2006 have glycine at position 249, implying
352
that some positive pressure for adaptation to humans led to glycine selection over aspartic
353
acid. These data reinforce the notion that PB2 amino acid 249G plays a role in adaptation to
354
humans or mammals.
355
The MLD50 values of 36285-RG virus and Wb/AH82-RG virus in mice differed by one log unit
356
(Fig. 4), whereas the titers of the two viruses in A549 cells differed by more than 3 log units
357
(Fig. 3). The HA and NA genes are involved, to some extent, in the difference in the growth
358
ability of Wb/AH82-RG and 36285-RG in A549 cells. The pathogenicity in mice could have
359
been influenced by HA, because the dominant types of receptor to influenza viruses that are
360
distributed on bronchi and lungs differ with the host species (43-46). Humans primarily have
361
sialic acid linked to galactose by an α2,6-linkage (Sia-α2,6Gal) (43-45), whereas mice
362
primarily have Sia-α2,3Gal-type linkages (46). One study showed that on the surface of
363
A549 cells there are large amounts of Sia-α2,6Gal and a small amount of Sia-α2,3Gal (47).
364
Perhaps, the difference between the receptor specificities of the viruses and the receptor
365
types displayed on the lung cells of mice may have influenced the pathogenicity in mice.
366
In conclusion, here we found that the PB2 amino acid substitutions E249G, G309D, and
22
367
T339M enhance the replicative ability of H5N1 virus in A549 cells. Our study suggests that
368
these PB2 substitutions could assist H5N1 viruses in adapting to human lung cells. Although
369
the contribution of the PB2 C-terminal domain to virus host range is now well established,
370
that of the middle portion of the PB2 segment remains largely unknown. The full structure of
371
PB2 is needed to better understand its functions and role in host adaptation.
372 373
23 374
Acknowledgements
375
We are grateful to Susan Watson for editing the manuscript. We thank Kiyoko
376
Iwatsuki-Horimoto and Maki Kiso for technical assistance with mouse experiments. We also
377
thank Shufang Fan, Masato Hatta, Gabriele Neumann, Amie J. Eisfeld, and Takeo Gorai for
378
fruitful discussion. This work was supported by the Japan Initiative for Global Research
379
Network on Infectious Diseases from the Ministry of Education, Culture, Sports, Science and
380
Technology, Japan; by grants-in-aid from the Ministry of Health, Labour and Welfare, Japan;
381
and by the National Institute of Allergy and Infectious Diseases (NIAID), the National
382
Institutes of Health (NIH).
383 384 385 386
The authors do not have any competing interests.
24 387
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33 542
Figure legends
543
Fig. 1: Comparison of the growth properties of H5N1 viruses isolated from humans
544
and birds.
545
(A) A549 cells were infected with H5N1 human viruses isolated in Vietnam in 2010 at a
546
multiplicity of infection (m.o.i.) of 0.0002 and cultured at 37℃. Virus release into cell culture
547
supernatant was titrated by plaque assays with MDCK cells at the indicated time points. The
548
standard deviation represents 3 independent experiments.
549
(B) A549 cells were infected with the H5N1 human viruses 36285-wild type and 36285-RG
550
at an m.o.i. of 0.0002 and cultured at 37℃. Error bars represent the standard deviation of
551
three independent experiments.
552
(C) A549 cells were infected with H5N1 human virus 36285-RG and avian viruses at an m.o.i.
553
of 0.0002 and cultured at 37℃. Supernatants were titrated by plaque assays with MDCK
554
cells at the indicated time points. The standard deviation represents 3 independent
555
experiments. *The titers of the 36285-RG virus are significantly different from those of the
556
Wb/AH82-RG virus (p
1
1 2
Identification of PB2 mutations responsible for the efficient replication
3
of H5N1 influenza viruses in human lung epithelial cells
4 5 6 7
Reina Yamaji1, Shinya Yamada1##, Mai Q. Le2, Chengjun Li3, Hualan Chen3, Ema Qurnianingsih4, Chairul A. Nidom4, Mutsumi Ito1, Yuko Sakai-Tagawa1, Yoshihiro Kawaoka1, 5, 6 #
8 9 10
1
Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
11 12
2
National Institute of Hygiene and Epidemiology, Hanoi, Vietnam
13 14 15 16
3
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Nangang District, Harbin, China 150001.
17 18
4
Avian Influenza Research Center, Airlangga University, Surabaya, Indonesia
19 20
5
21
Wisconsin-Madison, WI 53711
Department of Pathobiological Sciences, School of Veterinary Medicine, University of
22 23 24
6
Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
25 26
Running title: Adaptive mutations of H5N1 virus
27
Document totals: Word count for abstract: 190 Word count for main text: 3622
28 29 30
#
31
##
32
Address for correspondence: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. E-mail: [email protected]
33 34
Corresponding author. Tel.: +81-3-5449-5504; fax: +81-3-5449-5408 Co-corresponding author. Tel.: +81-3-5449-5504; fax: +81-3-5449-5408
2 35 36 37 38
Address for co-correspondence: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. E-mail: [email protected]
3
39
Abstract
40
Highly pathogenic H5N1 avian influenza viruses have caused outbreaks among poultry
41
worldwide, resulting in sporadic infections in humans, approximately 60% mortality of which
42
have been fatal. However, efficient transmission of H5N1 viruses among humans has yet to
43
occur, suggesting that further adaptation of H5N1 viruses to humans is required for their
44
efficient transmission among humans. Viral determinants for efficient replication in humans
45
are currently poorly understood. Here, we report that the polymerase PB2 protein of an
46
H5N1 influenza virus isolated from a human in Vietnam (A/Vietnam/UT36285/2010, 36285)
47
increased the growth ability of an avian H5N1 virus (A/wild bird/Anhui/82/2005, Wb/AH82) in
48
human lung epithelial A549 cells (although, the reassortant virus did not replicate more
49
efficiently than human 36285 virus). Furthermore, we demonstrate that amino acid residues
50
at positions 249, 309, and 339 of the PB2 protein from this human isolate were responsible
51
for its efficient replication in A549 cells. PB2-249G and 339M, which are found in the human
52
H5N1 virus, are rare in H5N1 viruses from both human and avian sources. Interestingly,
53
PB2-249G is found in over 30% of human seasonal H3N2 viruses, which suggests that
54
H5N1 viruses may replicate well in human cells when they acquire this mutation. Our data
55
are of value to H5N1 virus surveillance.
56
4 57
Importance
58
Highly pathogenic H5N1 avian influenza viruses must acquire mutations to overcome the
59
species barrier between avian species and humans. When H5N1 viruses replicate in human
60
respiratory cells, they can acquire amino acid mutations that allow them to adapt to humans
61
through continuous selective pressure. Several amino acid mutations have been shown to
62
be advantageous for virus adaptation to mammalian hosts. Here, we found that amino acid
63
changes at positions 249, 309, and 339 of PB2 contribute to efficient replication of avian
64
H5N1 viruses in human lung cells. These findings are beneficial for evaluating the pandemic
65
risk of circulating avian viruses and for further functional analysis of PB2.
66 67
5 68
Introduction
69
Highly pathogenic H5N1 influenza A viruses continue to circulate among avian species. As a
70
result, sporadic avian-to-human transmission has occurred and the threat of a pandemic has
71
persisted. The first human case caused by a highly pathogenic H5N1 influenza A virus was
72
reported in Hong Kong in 1997 (1, 2). Although the mass culling ordered by the Hong Kong
73
government temporarily ended the outbreak, a second outbreak of H5N1 viruses occurred in
74
2003. With the spread of H5N1 viruses among migrating birds and poultry, these viruses
75
quickly spread beyond East Asia (3). This spread of H5N1 viruses has been accompanied
76
by increasing reports of cases of avian-to-human transmission. As of January 2015, the total
77
number of confirmed human cases of highly pathogenic H5N1 influenza A virus infection had
78
reached 694, with 402 deaths ( http://www.who.int/, 4, 5). However, the human cases of
79
H5N1 infection have been limited mainly to individuals in close contact with infected poultry,
80
and reports of human-to-human transmission have been extremely rare (6, 7). For an H5N1
81
virus to overcome the host barrier, the following conditions must be met: efficient
82
transmission via droplets, efficient replication in the host, and immune vulnerability of the
83
infected populace. Consequently, viruses harboring mutations that promote efficient
84
transmission and replication in humans could lead to a pandemic.
85
A number of viral determinants have been identified that contribute to adaptation of avian
86
influenza viruses to mammalian hosts (8-28), and several large-scale comparative analyses
87
of proteins from avian and human viruses have been performed to catalog the amino acids
6
88
that are conserved in each host species (29-31). The PB2, PB1, and PA subunits of the
89
polymerase complex play a major role in virus pathogenicity and efficient viral growth in
90
mammals. PB2 is particularly important in overcoming species barriers. For example, lysine
91
(K) at residue 627 of PB2 enhances the polymerase activity, replication efficiency, and
92
virulence of H5N1 viruses in mammals (8-10). In addition, arginine at residue 591, which is
93
located close to the amino acid at position 627 of PB2 in the three-dimensional structure of
94
the protein, compensates for the lack of lysine at residue 627 and confers efficient viral
95
replication to pandemic H1N1 viruses in mammals (11). Other amino acid substitutions, such
96
as 701N, 271A, and 158G, in PB2 have also been identified as important markers that
97
confer a replicative advantage and high pathogenicity to influenza viruses in mammals
98
(12-15). Recently, we showed that the combination of amino acid substitutions 147T, 339T,
99
and 588T in PB2 confer a high polymerase activity to avian viruses in human cells (28). It is
100
not clear, however, whether we have identified all of the possible amino acid changes that
101
are needed for efficient growth of H5N1 viruses in mammals.
102
In this study, we attempted to identify amino acids that support the efficient growth of H5N1
103
viruses in human lung cells. Accumulation of information on adaptive mutations to humans
104
helps us to prepare for an unpredictable but potential H5N1 pandemic and to assess the risk
105
of future isolates with pandemic potential. Here, we attempted to identify new molecular
106
determinants associated with the efficient growth of a human-isolated H5N1 virus in human
7
107
lung cells by generating reassortant and mutant viruses in the genetic background of an
108
avian H5N1 virus (with low replicative ability in human lung cells) and a human H5N1 virus
109
(with high replicative ability in human lung cells).
110
8 111
Materials and Methods
112
Cells
113
Madin-Darby canine kidney (MDCK) cells were grown in minimal essential medium (MEM)
114
containing 5% newborn calf serum, vitamins, essential amino acids, and antibiotics. Human
115
embryonic kidney 293T cells, A549 human lung adenocarcinoma epithelial cells, and DF-1
116
chicken embryo fibroblast cells were grown in Dulbecco modified Eagle medium (DMEM)
117
containing 10% fetal bovine serum (FBS) and antibiotics. MDCK, 293T, and A549 cells were
118
cultured at 37℃ with 5% CO2. DF-1 cells were cultured at 39℃ with 5% CO2 unless
119
otherwise stated.
120 121
Viruses
122
In this study, we used the following ten H5N1 viruses: A/wild bird/Anhui/82/2005 (Wb/AH82)
123
(accession numbers: PB2 KP638500, PB1 KP638559, PA KP638509, HA KP638516, NP
124
KP638524, NA KP638533, M KP638542, NS KP638551), A/chicken/Vietnam/TY31/2005
125
(Ck/TY31) (accession numbers: PB2 KP638493, PB1 KP638558, PA KP638501, HA
126
EU118136.1, NP KP638525, NA EU118127.1, M KP638534, NS KP638550), A/chicken/Central
127
Java/UT3091/2005
128
GQ122415, and GQ122395),
129
PB2 KP638492, PB1 KP638552, PA KP638507, HA KP638560, NP KP638523, NA
130
KP638530, M KP638539, NS KP638547), A/Vietnam/UT36282/2010 (36282) (accession
(Ck/UT3091)
(accession
numbers:
GQ122490
to
GQ122495,
A/Vietnam/UT36285/2010 (36285) (accession numbers:
9
131
numbers: PB2 KP638494, PB1 KP638557, PA KP638506, HA KP638515, NP KP638522,
132
NA KP638532, M KP638541, NS KP638549), A/Vietnam/UT36236/2010 (36236) (accession
133
numbers: PB2 KP638495, PB1 KP638556, PA KP638505, HA KP638514, NP KP638520,
134
NA KP638531, M KP638540, NS KP638548), A/Vietnam/HN31676DH/2009 (31676)
135
(accession numbers: PB2 KP638496, PB1 KP638553, PA KP638504, HA KP638513, NP
136
KP638519, NA KP638526, M KP638535, NS KP638543), A/Vietnam/UT31641 Ⅱ /2008
137
(31641) (accession numbers: PB2 KP638497, PB1 KP638554, PA KP638503, HA
138
KP638512,
139
A/Vietnam/UT31604Ⅰ/2009 (31604) (accession numbers: PB2 KP638498, PB1 KP638561,
140
PA KP638502, HA KP638511, NP KP638517, NA KP638527, M KP638536, NS KP638544),
141
and A/Vietnam/UT36250 Ⅰ /2010 (36250) (accession numbers: PB2 KP638499, PB1
142
KP638555, PA KP638508, HA KP638510, NP KP638521, NA KP638529, M KP638538, NS
143
KP638546). Throat swabs were collected from H5N1 influenza virus-infected patients in
144
northern Vietnam and were sent to the National Institute of Hygiene and Epidemiology in
145
Hanoi, Vietnam. To isolate H5N1 virus, clinical specimens were inoculated to MDCK cells in
146
MEM containing 0.3% BSA and incubated at 37℃ for 48 hours. Stock viruses were
147
propagated in MDCK cells at 37℃ and stored at -80℃. 36285 was isolated from a patient
148
who exhibited influenza symptoms and recovered (a two-year-old female, onset on April 2,
149
2010, hospitalized on April 4, 2010). All experiments with H5N1 viruses were performed in a
NP
KP638518,
NA
KP638528,
M
KP638537,
NS
KP638545),
10
150
biosafety level 3 containment laboratory approved for such use by the Ministry of Agriculture,
151
Forestry, and Fisheries, Japan.
152 153
Isolation of viral RNA, RT-PCR, and generation of viruses by use of reverse genetics.
154
Viral RNA was extracted from the supernatants of virus-infected MDCK cells by using a
155
QIAamp viral RNA minikit (Qiagen, Hilden, Germany). Extracted RNA was reverse
156
transcribed with Superscript Ⅲ (Invitrogen, Carlsbad, CA) and the universal primers specific
157
for influenza A virus genes to generate cDNA. The resulting products were PCR-amplified by
158
using KOD Fx DNA polymerase (Toyobo, Osaka, Japan) with specific primers for each virus
159
gene and cloned into the RNA polymerase I plasmid pHH21 (ref). Mutations in the PB2 gene
160
of Wb/AH82 were generated by PCR amplification of the respective PB2 construct with
161
primers possessing the desired mutations. Primer sequences are available upon request. All
162
constructs were sequenced to ensure the absence of unwanted mutations. All avian,
163
reassortant, and mutant viruses were generated by use of plasmid-based reverse genetics,
164
as described previously (32). The culture supernatant derived from transfected cells was
165
amplified in DF-1 cells grown in DMEM containing 0.3% bovine serum albumin. At 48 h
166
post-infection, the culture supernatant was harvested, clarified, divided into aliquots, and
167
stored at -80℃. The virus titers of all human and avian viruses were determined by using
168
plaque assays in MDCK cells.
11
169 170
Mouse experiments
171
Six-week-old female BALB/c mice (Japan SLC) were used for these experiments. To
172
determine the dose lethal to 50% of mice (MLD50), 5 mice/group were anesthetized with
173
isoflurane and inoculated intranasally with 101 to 105 plaque-forming units (PFU) in a 50-µl
174
volume. The mice were monitored daily for clinical signs of infection and checked for
175
changes in body weight and mortality for 14 days post-infection. MLD50 values were
176
calculated by using the method of Reed & Muench (1938). All experiments with mice were
177
performed in accordance with the University of Tokyo's Regulations for Animal Care and Use
178
and were approved by the Animal Experiment Committee of the Institute of Medical Science,
179
the University of Tokyo.
180 181
Viral replication assay
182
Triplicate wells of confluent A549 cells or DF-1 cells were infected with viruses at a
183
multiplicity of infection (MOI) of 0.0002, and incubated for 1 h at 37℃ or 39℃, respectively.
184
After the 1-h incubation, A549 cells were further incubated in MEM containing 0.3% bovine
185
serum albumin (BSA) at 33℃ and 37℃. DF-1 cells were further incubated in DMEM
186
containing 0.3% bovine serum albumin (BSA) at 39℃. Aliquots of supernatants were
187
harvested at 1, 24, 48, 72, and 96 h post-infection, and frozen at -80℃. Virus titers in the
12
188
culture supernatants at each time point were determined by plaque assays in MDCK cells.
189 190
Statistical analysis
191
Differences in the virus titers of the supernatants were statistically analyzed by using the
192
Student’s t-test. Differences in mean maximum body weight loss in mice were also
193
statistically analyzed by using the Student’s t- test.
194 195
13 196
Results
197
Comparison of the growth ability of H5N1 viruses in A549 and DF-1 cells
198
To identify human adaptive mutations in H5N1 influenza viruses, we first compared the
199
growth properties at a multiplicity of infection (MOI) of 0.0002 in carcinomic human alveolar
200
basal
201
A/Vietnam/UT36285/2010
202
A/Vietnam/UT36236/2010
203
A/Vietnam/UT31641 Ⅱ /2008
204
A/Vietnam/UT36250Ⅰ/2010 (36250). All seven human H5N1 viruses grew well in A549 cells
205
at 37˚C with maximum titers of over 106 PFU/ml, except for 31676 which grew to slightly
206
lower titers (Fig. 1A). Sequence analysis revealed that 36250, 36236, 31604, and 31641
207
possess lysine at position 627 of the PB2 protein (PB2-627K), whereas 36282 possesses
208
PB2-591R. However, viruses 31676 and 36285 do not have any known PB2 markers that
209
could account for the efficient replication in mammals (Table 1). These findings suggest that
210
the human H5N1 viruses 31676 and 36285 may have unreported amino acids that enhance
211
their viral growth ability in A549 cells. Therefore, in this study we focused on the replicative
212
efficiency of 36285. First, we generated the 36285 virus by use of reverse genetics
213
(36285-RG) and confirmed that 36285-RG grew as well as wild-type 36285 in A549 cells
214
(Fig.1B). We next compared the growth property of 36285-RG in A549 cells at 37˚C with that
215
of three avian H5N1 viruses generated by use of reverse genetics: A/wild bird/Anhui/82/2005
epithelial
A549
cells
of
seven
(36285), (36236), (31641),
H5N1
viruses
isolated
from
humans:
A/Vietnam/UT36282/2010
(36282),
A/Vietnam/HN31676DH/2009
(31676),
A/Vietnam/UT31604 Ⅰ /2009
(31604),
and
14
216
(Wb/AH82-RG), A/chicken/Vietnam/TY31/2005 (Ck/TY31-RG), and A/chicken/Central
217
Java/UT3091/2005 (Ck/UT3091-RG) (Fig. 1C). Ck/TY31-RG and Wb/AH82-RG grew poorly
218
with maximum titers of less than 104.5 PFU/ml (Fig. 1C). Of note, both 36285 and Wb/AH82
219
belong to the same H5 HA sub-clade 2.3.4 and are genetically closely related (Table 2).
220
Therefore, we compared 36285 and Wb/AH82 to elucidate the mechanism of H5N1
221
adaptation to humans. In chicken embryo fibroblast DF-1 cells, 36285-RG and Wb/AH82-RG
222
both grew well at 39˚C (Fig. 2A), demonstrating their potential to replicate in avian cells; in
223
contrast, 36285-RG grew much better than Wb/AH82-RG in A549 cells at 33˚C (Fig. 2B),
224
demonstrating its potential to replicate in mammalian cells. We compared the growth
225
capability of H5N1 viruses in A549 cells at both 37℃ and 33℃ because in humans, the
226
temperature in the lungs is 37℃, and that in the upper airway is 33℃. However, the body
227
temperature of birds is 39℃; therefore, we compared the growth capability of H5N1 viruses
228
in chicken DF-1 cells only at 39℃.
229 230
Comparison of the pathogenicity of Wb/AH82-RG and 36285-RG in mice
231
We next examined the pathogenicity of Wb/AH82-RG and 36285-RG in mice. Inoculation of
232
103, 104, and 105 PFU of 36285-RG virus resulted in 100% mortality, and a 40% fatality rate
233
was observed in the mice that received 101 and 102 PFU of virus. In contrast, inoculation of
234
104 and 105 PFU of Wb/AH82-RG resulted in 100% mortality and a 60% fatality rate was
15
235
observed in mice that received 102 and 103 PFU of virus. The MLD50 values for 36285-RG
236
and Wb/AH82-RG were 101.5 PFU and 102.7 PFU, respectively (Fig. 3C, 3D). Thus,
237
36285-RG and Wb/AH82-RG differed in their mouse virulence by only one log MLD50 unit.
238 239
The PB2 gene segment from 36285 is responsible for its efficient growth in human
240
cells.
241
To elucidate the molecular basis for the replicative difference between Wb/AH82 and 36285
242
in A549 cells, we generated a series of reassortant viruses (as illustrated in Fig. 4A) and
243
compared their growth properties in A549 cells. The reassortant viruses were named
244
according to the origin of their Wb/AH82 or 36285 genes. For instance, “36285(PB2)”
245
indicates a virus possessing PB2 from 36285 and the rest of its gene segments from
246
Wb/AH82. Three reassortant viruses possessing the PB2, PB1, PA, and NP of 36285 [i.e.,
247
36285(3P+NP), 36285(3P+NP, HA, NA), and 36285(3P+NP, M, NS)] were comparable in
248
their growth to 36285-RG; none of these viruses replicated more efficiently than 36285-RG
249
(Fig. 4B). The replicative ability of 36285(M, NS) was similar to that of Wb/AH82-RG,
250
indicating that the M and NS proteins of 36285 do not have a large impact on the difference
251
in the growth capabilities of Wb/AH82-RG and 36285-RG. Of note, the HA and NA of 36285
252
also contributed, to some extent, to the difference in the growth properties of Wb/AH82-RG
253
and 36285-RG; however, they enhanced viral replication to a much lesser extent than did
16
254
the polymerase subunits and NP of 36285. These results suggest that the PB2, PB1, PA,
255
and/or NP gene segments of 36285 were the most responsible for the difference in growth
256
capability between Wb/AH82-RG and 36285-RG in A549 cells.
257
To determine which viral segment(s) among PB2, PB1, PA, and NP contributed to the high
258
replication of 36285-RG, we generated a range of reassortants (Fig. 5A) and compared their
259
growth properties in A549 cells. Only viruses possessing 36285(PB2) grew well (Fig. 5B). By
260
contrast, 36285(PB1, PA, NP) grew poorly, similarly to Wb/AH82-RG. These results
261
demonstrate that the PB2 of 36285 makes an important contribution to the difference in
262
growth capability between Wb/AH82-RG and 36285-RG in A549 cells.
263 264
A single G-to-D mutation at 309 and the double mutations E249G and T339M enhance
265
the replication of Wb/AH82-RG.
266
Sequence comparison of the PB2 proteins of Wb/AH82 and 36285 revealed twenty-one
267
amino acid differences (Table 3). To identify the specific changes that give rise to efficient
268
viral replication, we generated mutant viruses possessing a chimeric PB2 protein (Fig. 6A);
269
the remaining gene segments were derived from Wb/AH82. In A549 cells, the mutant
270
viruses possessing chimera 1, 5, or 6 grew poorly, whereas those with chimera 2 or 3 grew
271
as well as that with 36285 PB2. These results suggest that residues 221 to 460 contribute
272
most to the difference between 36285-RG and Wb/AH82-RG in terms of growth in A549
17
273
cells.
274
To further define which amino acid residues enhance the viral replication, we generated
275
viruses with additional chimeric PB2 proteins (Fig.7A). The mutant viruses possessing
276
chimera 2, 7, 8, and 11 grew well, whereas the growth of the viruses with chimera 1, 9, and
277
10 was lower compared with that of the viruses possessing residues 221 to 345 from 36285.
278
These results indicate that residues 221 to 345 from 36285 provide the replicative
279
advantage to 36285-RG (Fig. 7B).
280
We then tried to determine which amino acids among residues 221 to 345 of PB2 enhanced
281
the growth properties of 36285-RG. There are three amino acid differences in this region
282
between Wb/AH82 and 36285: 249E/G, 309G/D, and 339T/M. We singly or in combination
283
introduced the three residues of 249G, 309D, and 339M into the PB2 of Wb/AH82 (Fig. 8A)
284
and generated viruses possessing the mutant Wb/AH82 PB2 gene in the background of the
285
remaining
286
changes found among circulating influenza viruses). Indeed, the combination of PB2-249G,
287
-309D, and -339M markedly increased the growth capability of Wb/AH82-RG by around 102
288
fold compared with Wb/AH82-RG (Fig. 8B, 8C). Although the single introduction of
289
PB2-249G, -PB2-339M, or -PB2-309D enhanced the growth capability of Wb/AH82-RG,
290
PB2-339M was least effective (Fig. 8B). All mutants that possessed combinations of two of
291
the PB2 mutations tested grew better than Wb/AH82-RG by more than 101.5-fold (Fig. 8C).
Wb/AH82 genes (note that all mutant Wb/AH82 viruses possess amino acid
18
292
These results indicate that PB2-249G, PB2-339M, and PB2-309D support efficient viral
293
growth in A549 cells.
294 295
The amino acid mutations E249G and T339M may be associated with the adaptation of
296
H5N1 virus to humans.
297
To further evaluate the potential role of the PB2 amino acid residues 249, 309, and 339 in
298
adaptation to humans, we collected full-length PB2 sequences of influenza viruses of
299
various subtypes from the Influenza sequence database (ISD) (Table 4). Almost all of H5N1
300
viruses contained PB2 amino acid 309D, which is highly conserved among influenza viruses
301
of various subtypes regardless of the host species, implying that this residue does not need
302
to change for a virus to break through the species barrier. In contrast, PB2 amino acids
303
249G and 339M were rare among the avian H5N1 viruses. Intriguingly, no less than 49.2%
304
of the human seasonal H3N2 viruses had PB2 amino acid 249G. This finding supports the
305
hypothesis that PB2 amino acid 249G is the amino acid mutation that the virus gains during
306
replication in human respiratory cells and increases its fitness for human adaptation.
307 308
19 309
Discussion
310
Several amino acid mutations associated with the adaptation of H5N1 virus to humans have
311
been identified, including 627K (8-10), 591R, 591K (11), 701N (9, 13), 271A (14), and 158G
312
(15) of PB2. We thought that further analysis of H5N1 viruses isolated from humans could
313
identify new mutations needed for their efficient replication in human cells. Even though the
314
36285 H5N1 virus did not have any amino acid markers that have been previously identified
315
to provide a replicative advantage in mammalian cells, it grew well in human lung cells. By
316
using a viral replication assay, we identified three amino acids in PB2 that are responsible for
317
the replicative efficiency of H5N1 virus in A549 cells. Viruses with these amino acids in PB2
318
circulate in nature; we did not generate viruses possessing ‘novel’ amino acid changes.
319
These data will provide information of value for pandemic preparedness and for use in
320
evaluating the pandemic risk potential of future isolates.
321
Pflug et al. (33) recently determined the complete crystal structure of the heterotrimeric
322
influenza A polymerase bound to the vRNA. This structure revealed that all three amino acid
323
substitutions identified in this study (i.e., PB2-E249G, G309D, and T339M) are located on
324
the surface of the PB2 protein. This finding supports the concept that these residues likely
325
interact with viral or host proteins, leading to the replicative advantage of the 36285 virus.
326
PB2-E249G and T339M have an interesting common feature in that they belong to areas
327
identified as ‘cap-binding' sites (34-37). Influenza virus has developed a mechanism called
328
‘cap-snatching,’ to steal the cap from the host cellular RNA in order to synthesize viral
20
329
protein. The polymerase PB2 subunit binds to the 5’ cap of host pre-mRNAs, which are
330
cleaved after 10–13 nucleotides by the PA subunit (38-40). An X-ray structure of the
331
cap-binding domain shows that the amino acid at position 339 of PB2 is located at the edge
332
of the cap-binding pocket (37). The precise cap-binding site, however, remains controversial;
333
one study identified the PB2 amino acid residues responsible for RNA cap-binding as
334
PB2-363F and PB2-404F (35), but another study identified the PB2 amino acid residues
335
242–282 and 538–577 as important (36), and yet another study suggested that amino acid
336
residues 318–483 were involved (37). The effect of the amino acid 339T in vivo and in vitro
337
on biologic events is also inconsistent. We recently found that 339T has a major impact on
338
the polymerase activity of H5N1 virus jointly with 147T and 588T (28). However, another
339
paper reported that K339T reduced PB2 cap binding activity and influenza polymerase
340
activity in vitro, and also attenuated virulence in mice (41). This latter paper suggested that
341
the K339T substitution in PB2 reduced the cap binding affinity because the side chain of
342
threonine is shorter than that of lysine and threonine is uncharged (41). In the current study,
343
we found that PB2 amino acid T339M helped to facilitate virus growth in A549 cells. Both
344
threonine and methionine are uncharged but the side chain of methionine is longer than that
345
of threonine. Although the function of the three amino acid mutations identified in this study
346
remains unclear, our findings may help improve our understanding of PB2 functions.
347
Human influenza A virus acquired the PB2 amino acid mutation E249G upon mouse
21
348
adaptation (42), suggesting that the amino acid at position 249 probably is involved in some
349
mechanism for adaptation to mammals. Sequence analysis showed that the prevalence of
350
PB2 amino acid 249G in human seasonal H3N2 viruses was almost 50%. Almost all of the
351
seasonal human H3N2 viruses isolated since 2006 have glycine at position 249, implying
352
that some positive pressure for adaptation to humans led to glycine selection over aspartic
353
acid. These data reinforce the notion that PB2 amino acid 249G plays a role in adaptation to
354
humans or mammals.
355
The MLD50 values of 36285-RG virus and Wb/AH82-RG virus in mice differed by one log unit
356
(Fig. 4), whereas the titers of the two viruses in A549 cells differed by more than 3 log units
357
(Fig. 3). The HA and NA genes are involved, to some extent, in the difference in the growth
358
ability of Wb/AH82-RG and 36285-RG in A549 cells. The pathogenicity in mice could have
359
been influenced by HA, because the dominant types of receptor to influenza viruses that are
360
distributed on bronchi and lungs differ with the host species (43-46). Humans primarily have
361
sialic acid linked to galactose by an α2,6-linkage (Sia-α2,6Gal) (43-45), whereas mice
362
primarily have Sia-α2,3Gal-type linkages (46). One study showed that on the surface of
363
A549 cells there are large amounts of Sia-α2,6Gal and a small amount of Sia-α2,3Gal (47).
364
Perhaps, the difference between the receptor specificities of the viruses and the receptor
365
types displayed on the lung cells of mice may have influenced the pathogenicity in mice.
366
In conclusion, here we found that the PB2 amino acid substitutions E249G, G309D, and
22
367
T339M enhance the replicative ability of H5N1 virus in A549 cells. Our study suggests that
368
these PB2 substitutions could assist H5N1 viruses in adapting to human lung cells. Although
369
the contribution of the PB2 C-terminal domain to virus host range is now well established,
370
that of the middle portion of the PB2 segment remains largely unknown. The full structure of
371
PB2 is needed to better understand its functions and role in host adaptation.
372 373
23 374
Acknowledgements
375
We are grateful to Susan Watson for editing the manuscript. We thank Kiyoko
376
Iwatsuki-Horimoto and Maki Kiso for technical assistance with mouse experiments. We also
377
thank Shufang Fan, Masato Hatta, Gabriele Neumann, Amie J. Eisfeld, and Takeo Gorai for
378
fruitful discussion. This work was supported by the Japan Initiative for Global Research
379
Network on Infectious Diseases from the Ministry of Education, Culture, Sports, Science and
380
Technology, Japan; by grants-in-aid from the Ministry of Health, Labour and Welfare, Japan;
381
and by the National Institute of Allergy and Infectious Diseases (NIAID), the National
382
Institutes of Health (NIH).
383 384 385 386
The authors do not have any competing interests.
24 387
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Figure legends
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Fig. 1: Comparison of the growth properties of H5N1 viruses isolated from humans
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and birds.
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(A) A549 cells were infected with H5N1 human viruses isolated in Vietnam in 2010 at a
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multiplicity of infection (m.o.i.) of 0.0002 and cultured at 37℃. Virus release into cell culture
547
supernatant was titrated by plaque assays with MDCK cells at the indicated time points. The
548
standard deviation represents 3 independent experiments.
549
(B) A549 cells were infected with the H5N1 human viruses 36285-wild type and 36285-RG
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at an m.o.i. of 0.0002 and cultured at 37℃. Error bars represent the standard deviation of
551
three independent experiments.
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(C) A549 cells were infected with H5N1 human virus 36285-RG and avian viruses at an m.o.i.
553
of 0.0002 and cultured at 37℃. Supernatants were titrated by plaque assays with MDCK
554
cells at the indicated time points. The standard deviation represents 3 independent
555
experiments. *The titers of the 36285-RG virus are significantly different from those of the
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Wb/AH82-RG virus (p
