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Development of continuous cell culture of brown planthopper to trace the early infection 1
process of oryzaviruses in insect vector cells 2
3
Hongyan Chen, Limin Zheng, Qianzhuo Mao, Qifei Liu, Dongsheng Jia, and Taiyun Wei# 4 5
Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian 6
Agriculture and Forestry University, Fuzhou, Fujian 350002, PR China 7
8
Running title: The assembly of viroplasm of an oryzavirus 9
Key words: Nilaparvata lugens, Continuous cell culture, RRSV, Viroplasm, Viral early 10
replication 11
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The number of words in abstract: 239 words 13
The number of words in text: 5003 words 14
The number of figures: 9 15
Contents Category field: Plant Viruses 16
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18 #Author to whom all correspondence should be addressed, as follows: 19
Dr. Taiyun Wei, Institute of Plant Virology, Fujian Agriculture and Forestry University, 20
Fuzhou, Fujian 350002, PR China 21
Tel: +86-591-83789270. Fax: +86-591-83789439. 22
E-mail: [email protected] 23
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JVI Accepts, published online ahead of print on 29 January 2014J. Virol. doi:10.1128/JVI.03466-13Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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ABSTRACT: 31
Rice ragged stunt virus (RRSV), an oryzavirus in the family Reoviridae, is transmitted 32
by the brown planthopper, Nilaparvata lugens, in a persistent-propagative manner. Here, we 33
established a continuous cell line of brown planthopper to investigate the mechanism 34
underlying the formation of the viroplasm, the putative site for viral replication and assembly, 35
during infection of RRSV in its insect vector cells. Within 24 h of viral infection of cultured 36
cells, the viroplasm had formed and contained viral nonstructural proteins Pns6 and Pns10, 37
known to be constituents of viroplasm. Core capsid protein P3, core particles and newly 38
synthesized viral RNAs were accumulated inside the viroplasm, while outer capsid protein P8 39
and virions were accumulated at the periphery of the viroplasm, confirming that the viroplasm 40
induced by RRSV infection was the site for viral replication and assembly. Pns10 formed 41
viroplasm-like inclusions in the absence of viral infection, suggesting that the viroplasm 42
matrix was largely composed of Pns10. Pns6 was recruited in the viroplasm by direct 43
interaction with Pns10. Core capsid protein P3 was recruited to the viroplasm through specific 44
association with Pns6. Knockdown of Pns6 and Pns10 expression using RNA interference 45
inhibited viroplasm formation, virion assembly, viral proteins expression and viral dsRNAs 46
synthesis. Thus, the present study shows that both Pns6 and Pns10 of RRSV play important 47
roles in the early stages of viral life cycle in its insect vector cells, by recruiting or retaining 48
components necessary for viral replication and assembly. 49
The brown planthopper, a commonly distributed pest of rice in Asia, is the host of 50
numerous insect endosymbionts, and the major vector of two rice viruses (RRSV and rice 51
grassy stunt virus). For the first time, we successfully established the continuous cell line of 52
brown planthopper. The unique uniformity of brown planthopper cells in the monolayer can 53
support a consistent, synchronous infection by endosymbionts or viral pathogens, improving 54
our understanding of molecular insect-microbe interactions. 55
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INTRODUCTION: 56
Viruses in the Reoviridae family replicate and assemble within cytoplasmic viral 57
inclusions called viroplasms or viral factories (1). Plant reoviruses, which comprise the genera 58
Phytoreovirus, Fijivirus and Oryzavirus in the family Reoviridae, are transmitted 59
propagatively by cicadellid leafhoppers or planthoppers (1). Plant reoviruses are icosahedral, 60
double-layered particles with 10 or 12 dsRNA segments (1). The genesis and maturation of 61
viroplasms of phytoreoviruses and fijiviruses in their insect vectors are beginning to be 62
understood due to the development of culture systems for insect vector cells (2, 3, 4, 5, 6, 7). 63
Continuous cell cultures derived from rice green leafhopper, Nephotettix cincticeps 64
provide a good system for studying the replication cycles of rice dwarf virus (RDV), a 65
phytoreovirus, in its vector cells, including viral entry, replication and spread (3, 5, 6, 7). 66
Viroplasms, globular electron-dense inclusion bodies, are formed within 6 h post-infection 67
(hpi) of RDV in leafhopper cultured cells (7). Nonstructural protein Pns12 of RDV is essential 68
for the formation of viroplasm matrix and the nucleation of viral assembly complexes for the 69
production of viral progeny virions (7). Recently, we established the continuous cell cultures 70
derived from white-backed planthopper, Sogatella furcifera, to investigate the genesis and 71
maturation of viroplasms induced by southern rice black streaked dwarf virus (SRBSDV), a 72
fijivirus, in its insect vector cells (2, 4). The viroplasm of SRBSDV consists of a granular 73
region formed by viral nonstructural proteins P6 and P9-1 for the accumulation of viral RNAs 74
and a filamentous region formed by viral nonstructural proteins P5 and P6 for the assembly of 75
progeny virions (2, 4). Currently, the mechanisms responsible for the genesis and maturation 76
of the viroplasm induced by oryzaviruses remain largely unknown due to the lack of 77
continuous cultured cells derived from their insect vectors. 78
Rice ragged stunt virus (RRSV), an oryzavirus, is transmitted by the brown planthopper 79
(BPH), Nilaparvata lugens, in a persistent-propagative manner and has spread throughout 80
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southern China (8, 9, 10). RRSV virions are icosahedral, double-layered particles with 65 to 81
70 nm in diameter (11). The genome of RRSV encodes at least seven putative structural 82
proteins (P1, P2, P3, P4A, P5, P8B, and P9) and three nonstructural proteins (Pns6, Pns7 and 83
Pns10) (11, 12, 13, 14, 15, 16, 17). The core particles of RRSV contain core capsid protein P3, 84
RNA dependent RNA polymerase P4A protein and 10 segments of dsRNAs (11, 12, 15, 18). 85
The major outer capsid protein P8 and spike protein P9 are added onto the core particles to 86
assemble double-layered particles of RRSV (12, 19). A continuous cell culture derived from 87
BPH would enable us to define the process of progeny virion assembly during early infection 88
by RRSV. 89
The assembly of progeny core and virions of RRSV is believed to occur within the 90
viroplasms, globular electron-dense inclusion bodies, induced by RRSV infection in host 91
plants or insect vectors (9, 20). Our previous study indicates that the nonstructural protein 92
Pns10 of RRSV plays an essential role in viroplasm formation and viral replication (20). 93
Formation of the viroplasm for assembly of progeny virions is essential for the establishment 94
of a persistent infection of RRSV in its BPH vector (20). Whether other nonstructural proteins 95
are involved in the genesis and maturation of the viroplasm induced by RRSV infection, 96
however, is still unknown. The nonstructural protein Pns7 forms filament-like structures in the 97
absence of viral infection and has not been detected in RRSV-infected BPH vectors by 98
Western blot assay (20; unpublished data). Our previous study shows that the nonstructural 99
protein Pns10 can recruit Pns6 into the viroplasm-like structures formed by Pns10 during 100
co-expression of these two proteins in cells of the nonhost insect Spodoptera frugiperda (Sf9) 101
(20), suggesting that Pns6 might be one of the components of viroplasm matrix induced by 102
RRSV infection. 103
In the present study, we developed a continuous culture system derived from BPH to 104
trace the early infection process of RRSV. Our results indicated that the nonstructural proteins 105
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Pns6 and Pns10 of RRSV played important roles in the formation of the initial viroplasm 106
matrix, and Pns6 could recruit or retain components necessary for viral replication and 107
assembly. 108
109
MATERIALS AND METHODS: 110
Establishment of continuous cell culture derived from BPH for RRSV infection 111
The continuous cell culture derived from BPH was established by adapting the protocols 112
for similar systems for the white-backed planthopper and small brown planthopper, as 113
described by Ma et al (21) and Mao et al (4). The embryos at the blastokinetic stage, with red 114
eye spots on the BPH eggs on day 8 after oviposition, proved suitable for primary cell culture. 115
The eggs were sterilized with 70% ethanol for 5 min, and washed with Tyrode's solution. The 116
embryonic fragments were dissected from the eggs and then incubated with 0.25% trypsin in 117
Tyrode's solution (pH 6.7) for 30 min at room temperature. The embryonic tissues were 118
transferred to a centrifuge tube and centrifuged at 250×g for 3 min. The pellet was 119
resuspended in Kimura’s insect medium (3) and transferred to a culture flask. The culture was 120
incubated at 25°C and the medium was changed at intervals of 7-10 days. Epithelial-like cells 121
grew out from the explants of embryonic tissue to form a monolayer of primary culture cells 122
by 16 days. The primary culture reached almost confluence in the culture flask within 60 days. 123
The cells were then passed to culture flasks for further subculturing. The intervals of 124
subculturing were gradually shortened, from monthly intervals to 12–16-day intervals after 125
the 20th passage. After 27 passages, a continuous cell culture derived from BPH was 126
established and used for viral infection. 127
Fresh RRSV inoculum for infecting BPH cells was prepared from infected plants, 128
essentially as described previously (22). Synchronous infection of cultured cells of BPH by 129
RRSV was developed as described by Kimura (22). 130
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Baculovirus expression of P3, Pns6 and Pns10 of RRSV 131
Baculovirus expression of P3, Pns6 or Pns10 was performed according to the 132
manufacturer’s instructions (Invitrogen). Briefly, recombinant baculovirus vectors containing 133
P3, Pns6 or Pns10 were introduced into DH10Bac for transposition into the bacmid. The 134
recombinant bacmids were transfected into Sf9 cell via cellfectin reagent (Invitrogen). Sf9 135
cells infected with recombinant bacmids were processed for immunofluorescence microscopy. 136
Immunofluorescence microscopy 137
Rabbit polyclonal antisera against structural proteins P3 and P8 and nonstructural 138
proteins Pns6 and Pns10 of RRSV were prepared as described previously (2). IgG isolated 139
from polyclonal antisera was directly conjugated to fluorescein isothiocyanate (FITC), 140
rhodamine or Alexa Fluor 647 carboxylic acid according to the instructions of the user 141
manual (Invitrogen). BPH cells infected with RRSV or Sf9 cells infected with recombinant 142
bacmids on coverslips were fixed for 30 min in 4% paraformaldehyde, permeabilized for 5 143
min in 0.1% Triton X-100, and then processed for immunofluorescence microscopy, as 144
described previously (2, 23). Cells on coverslips were incubated with a 50-fold-diluted 145
solution of the directly conjugated IgG. Samples were then imaged by a Leica TCS SP5 146
confocal microscope, as described previously (2, 23). 147
Immunoelectron microscopy 148
BPH cells infected with RRSV or Sf9 cells on coverslips were fixed in 2% 149
paraformaldehyde plus 2% glutaraldehye, and then processed for immunoelectron microscopy, 150
as described previously (2, 4, 23). Cell sections were treated with antibodies against P3, P8, 151
Pns6 and Pns10 of RRSV, and then with anti-rabbit IgG conjugated to 15 nm gold particles 152
(Sigma). Samples were observed with a Hitachi H-7650 electron microscope as described 153
previously (2, 4, 23). 154
Immunofluorescence detection of newly synthesized viral RNAs 155
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Infection of BPH cells by RRSV was allowed to proceed for 28 or 56 h, at which time 156
cells were treated for 1 h with 10 µg/ml actinomycin D (Sigma) to inhibit cellular RNA 157
polymerase II (24). Cells were then transfected with 10 mM BrUTP (Sigma) via cellfectin 158
reagent (Invitrogen) and incubated for an additional 1 h before fixation and 159
immunofluorescence microscopy. BrUTP-labeled viral RNA was immunostained with 160
anti-BrdU from mouse (Sigma), followed by anti-mouse IgG conjugated to FITC (Sigma). 161
Yeast two-hybrid assay 162
The yeast two-hybrid assay for detecting interactions among P3, Pns6 and Pns10 of 163
RRSV was performed according to the instructions in the DUALmembrane starter kit user 164
manual (Dualsystems Biotech). The prey and bait vectors containing P3, Pns6 or Pns10 were 165
cotransformed into yeast strain NMY51 using the Yeast transformation kit (Dualsystems 166
Biotech). Independent positive transformants were selected and grown in 167
SD/−Leu/−Trp/−His/−Ade liquid medium at 30°C for 4 days. 168
Effect of synthesized dsRNAs from Pns6 and Pns10 genes of RRSV on viroplasm 169
formation and viral infection in the continuous cell cultures of BPH 170
DNA fragment spanning a 1000-bp segment of Pns6 gene or a 793-bp segment of Pns10 171
gene of RRSV was used for dsRNA synthesis according to the protocol of a T7 RiboMAX 172
Express RNAi System kit (Promega), as described previously (20). BPH cells on coverslips 173
were transfected with 0.5 µg/µL dsRNAs from Pns6 gene (dsPns6), from Pns10 gene 174
(dsPns10), or from green fluorescence protein (GFP)-encoding gene (dsGFP) via cellfectin 175
reagent (Invitrogen), as reported by Chen et al. (23). After 24 h, the cells were inoculated with 176
RRSV and grown further in culture medium. The cells were fixed 30 hpi or 84 hpi and 177
processed for immunofluorescence microscopy. Alternatively, at 84 hpi, total proteins were 178
extracted from infected cells and further analysed by immunoblotting with antibodies against 179
P3, P8, Pns6 and Pns10, respectively. Insect actin was detected with actin-specific antibodies 180
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(Sigma) as a control. Furthermore, viral dsRNAs were isolated from BPH cells, and then 181
separated in 10% polyacrylamide gels, as described previously (25). 182
183
RESULTS: 184
Establishment of continuous cell cultures derived from BPH 185
Primary cell cultures of BPH, originally established from the embryonic fragments 186
dissected from BPH eggs that had been oviposited 8 days earlier, were maintained at 25°C in 187
Kimura’s insect medium. After 27 passages of subculturing at 12–16-day intervals, the 188
monolayer of the epithelial-like cells, approximately 45–65 μm in diameter, was established 189
(Fig. 1). About 15% of frozen BPH cells after one year storage in liquid nitrogen were 190
recovered by the procedure described previously (data not shown; 3). This is the first report of 191
the establishment of a continuous BPH cell line. These vector cells in a monolayer (VCM) 192
were used to investigate early infection of RRSV in its insect vector. 193
Viral nonstructural proteins Pns6 and Pns10 colocalized in viroplasm matrix during 194
viral infection 195
The persistent-propagative infection of RRSV in the bodies of BPH initiates the 196
formation of the viroplasms for viral replication and the assembly of progeny virions (20). To 197
determine the roles of nonstructural proteins Pns6 and Pns10 in the formation of the 198
viroplasm matrix, we used immunoelectron microscopy to localize these two proteins in 199
infected VCMs at 84 hpi. In ultrathin sections of RRSV-infected VCMs, electron-dense 200
inclusions with core-like particles of ~50 nm in diameter interspersed within or with 201
double-layered particles of ~70 nm in diameter distributed at the periphery of the matrix, 202
namely, viroplasms (Fig. 2A). Our results showed that both Pns6 and Pns10 antibodies 203
reacted specifically with the matrix of viroplasm, but they were not associated with virions 204
(Fig. 2B, C). 205
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To confirm our observations, we immunostained RRSV-infected VCMs with 206
Pns10-specific IgG conjugated to FITC (Pns10-FITC) and Pns6-specific IgG conjugated to 207
rhodamine (Pns6-rhodamine). As early as 24 hpi, Pns6 and Pns10 were colocalized in 208
numerous, small viral inclusions throughout the cytoplasm of infected VCMs (Fig. 2D, 24 209
hpi). As infection proceeded, by 84 hpi, Pns6 and Pns10 continued to colocalize in viral 210
inclusions as they grew in size but decreased in number (Fig. 2D, 84 hpi). These viral 211
inclusions obviously corresponded to the viroplasm viewed with electron microscopy (Fig. 2B, 212
C). Taken together, our results indicated that viral nonstructural proteins Pns6 and Pns10 were 213
the major constituents of the matrix of viroplasm in virus-infected cells. 214
Progeny core particles assembled inside the viroplasm matrix, while progeny virions 215
assembled at the periphery of the matrix 216
To further confirm whether the viroplasm matrix was the site for assembly of progeny 217
virions during virus infection, we used immunoelectron microscopy to observe the 218
distribution of the core capsid protein P3 and outer capsid protein P8 within the viroplasm. P3 219
antibodies reacted strongly with the viroplasm matrix and virions at the periphery of the 220
viroplasm (Fig. 3A). Furthermore, P8 antibodies specifically reacted with virions at the 221
periphery of the viroplasm, but not with the viroplasm matrix (Fig. 3B). 222
To confirm our observations, we examined the distribution of structural proteins relative 223
to viral inclusions of Pns6 using immunofluorescence microscopy. Infected cells were 224
immunostained with core capsid protein P3-specific IgG conjugated to FITC (P3-FITC) and 225
Pns6-rhodamine, with outer capsid protein P8-specific IgG conjugated to FITC (P8-FITC) 226
and Pns6-rhodamine, or with P8-FITC and P3-specific IgG conjugated to rhodamine 227
(P3-rhodamine). As early as 24 hpi, numerous small viral inclusions of Pns6 were clearly 228
observed, but the structural proteins P3 and P8 still did not form clear fluorescent inclusions 229
(Fig. 4A, B, 24 hpi). By 30 hpi, the core capsid protein P3 was colocalized with viral 230
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inclusions of Pns6, while the outer capsid protein P8 was localized to the ring-like structures 231
at the margin of viral inclusions of Pns6 (Fig. 4A, B, 30 hpi). By 84 hpi, larger viral 232
inclusions of Pns6 were observed, with the core capsid protein P3 concentrated in the center 233
and the outer capsid protein P8 concentrated at the periphery (Fig. 4A, B, C, 84 hpi). All these 234
results indicated that progeny core particles assembled inside the viroplasm matrix, while 235
progeny virions assembled at the periphery of the matrix. 236
Accumulation of newly synthesized viral RNAs within the viroplasm matrix 237
Our electron and confocal microscopic observations indicated that core particles of 238
RRSV are embedded in the viroplasm matrix in infected cells. Because core particles 239
synthesize the plus-strand RNAs (1, 26), we hypothesized that the localization of core 240
particles of RRSV to the viroplasm may result in the production of viral RNAs within the 241
viroplasm during infection. To test this hypothesis, we investigated the distribution of newly 242
synthesized viral RNAs within the viroplasm matrix by colocalization of BrUTP and Pns6. 243
Our results showed that RRSV RNAs were accumulated within the viroplasm matrix at 30 244
and 58 hpi (Fig. 5), supporting our hypothesis that the newly synthesized viral RNAs 245
accumulated inside the viroplasm matrix during viral infection. 246
Core protein P3 was recruited to viroplasm through interaction with Pns6 247
Given our observations that viral nonstructural proteins Pns6 and Pns10, as well as the 248
core protein P3, are concentrated within the viroplasm matrix in infected cells, we 249
hypothesized that Pns6 or Pns10 may recruit and concentrate P3 to the viroplasm. To address 250
this hypothesis, we coinfected Sf9 cells with recombinant baculoviruses containing P3, Pns6, 251
or Pns10 and examined the localization by immunofluorescence microscopy. When expressed 252
alone, P3 or Pns10 aggregated to form punctate inclusions in the cytoplasm, and Pns6 was 253
associated with the plasma membrane (Fig. 6A). Furthermore, immunoelectron microscopy 254
showed that Pns10 antibodies reacted with the electron-dense inclusions, whose morphology 255
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was similar to viroplasm matrix (Fig. 6B). The coinfection led to the redistribution of Pns6 256
into the inclusions of Pns10 (Fig. 6C), as described previously (20). By contrast, the 257
coinfection showed that the inclusions of P3 did not overlap with the inclusions of Pns10 (Fig. 258
6D). However, Pns6 was clearly associated with P3 on the inclusions (Fig. 6E). As expected, 259
these three proteins could colocalize on the inclusions when we triply infected Sf9 cells with 260
recombinant baculoviruses containing P3, Pns6 or Pns10 (Fig. 6F). The yeast two-hybrid 261
assay further confirmed that Pns6 interacted directly with P3 or Pns10, while Pns10 did not 262
interact with P3 (Fig. 6G). These findings, combined with our observation that nonstructural 263
proteins Pns6 and Pns10 preceded the core capsid protein P3 in localization to viroplasms 264
(Fig. 4), provided compelling evidence that the core capsid protein P3 was recruited to the 265
viroplasm matrix via its direct interaction with Pns6 in virus-infected cells. 266
RNAi induced by dsRNAs from Pns6 and Pns10 genes inhibited the assembly of 267
viroplasms and viral infection in insect vector cell cultures 268
Our previous study showed that RNA interference (RNAi) induced by ingestion of 269
dsRNA from Pns10 gene of RRSV via membrane feeding knocked down Pns10 expression 270
and strongly inhibited viroplasm formation, preventing efficient viral infection in its insect 271
vectors (20). To determine whether Pns6 and Pns10 play a crucial role in viral replication or 272
assembly in cultured insect vector cells, we use RNAi strategy to knock down the expression 273
of Pns6 or Pns10 and then analysed the effects on the formation of viroplasm and viral 274
infection in virus-infected VCMs. At 30 hpi or 84 hpi, immunofluorescence microcopy 275
showed that the treatment of dsPns6 or dsPns10 significantly inhibited the formation of 276
viroplasm containing Pns6 and Pns10, and the accumulation of core or virions containing P3 277
or P8 (Figs. 7, 8; data not shown). As expected, the expression viral proteins (Pns6, Pns10, P3 278
and P8) and the synthesis of viral dsRNAs were significantly decreased in VCMs transfected 279
with dsPns6 or dsPns10 (Fig. 9). Taken together, all these results suggested that Pns6 and 280
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Pns10 of RRSV played critical roles in viral replication and assembly during viral infection in 281
insect vector cells. 282
283
DISCUSSION： 284
The brown planthopper (BPH, Nilaparvata lugens), a typical large-scale migratory rice 285
pest in Asia, is a major vector of RRSV. In this study, for the first time, we successfully 286
established the continuous cell cultures derived from BPH. The unique uniformity of BPH 287
cells in the monolayer (VCM) supports a consistent, synchronous infection of its insect vector 288
cells by RRSV, allowing us to define the early process of viral replication and assembly 289
during viral infection. Following the entry of the virus into the cytoplasm of the VCMs, the 290
RRSV core particles transcribe viral plus-strand RNAs, as in the case of other viruses in the 291
family Reoviridae (1, 26). The nonstructural proteins Pns6 and Pns10 of RRSV were 292
expressed first by the host’s cellular translational machinery, then associated together to form 293
the initial viroplasm matrix, the site for viral replication and assembly (Fig. 2). Pns10 formed 294
viroplasm-like inclusions in the absence of viral infection, suggesting that the viroplasm 295
matrix induced by RRSV infection was formed primarily by Pns10 (20). Pns6 was recruited in 296
the viroplasm matrix by direct interaction with Pns10 (Fig. 6). Thus, both Pns6 and Pns10 297
seem to play important roles in the early stages of the viral life cycle, by recruiting or 298
retaining components necessary for viral replication and assembly. 299
To determine whether Pns6 and Pns10 of RRSV play a crucial role in viral replication 300
and assembly, we used the RNAi strategy to knock down the expression of Pns6 and Pns10 in 301
virus-infected VCMs. Our results indicated that the knockdown of Pns6 and Pns10 expression 302
due to RNAi induced by dsPns6 and dsPns10 led to the significant inhibition of viroplasm 303
formation, virion assembly, viral proteins expression and viral dsRNAs synthesis in VCMs 304
(Figs. 7, 8, 9). This finding, together with the observation that Pns6 and Pns10 were the 305
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components of the viroplasm matrix, suggested that Pns6 and Pns10 were essential for viral 306
replication and assembly in its insect vector cells. 307
How is Pns6 of RRSV involved in viral replication and assembly in virus-infected insect 308
vector cells? Previous data showed that Pns6 is a viral movement protein and a viral 309
RNA-silencing suppressor in plant hosts (16, 17). Furthermore, Pns6 of RRSV binds to both 310
dsRNA and ssRNA in a sequence-independent manner, showing a preference for RRSV 311
ssRNA over the rice ssRNA (27). Here, we showed that Pns6 rather than Pns10 specifically 312
interacted with core capsid P3 (Fig. 6). Thus, Pns6 may be involved in the recruitment or 313
retention of viral RNAs and core proteins to viroplasm matrix during viral replication or 314
assembly. The direct interaction of Pns6 with core capsid protein P3 indicated that Pns6 has 315
the ability to bind core particles within the interior regions of viroplasm. In RRSV-infected 316
VCMs, core particles localized to the interior regions of viroplasms, while intact virions 317
accumulated in peripheral regions of the viroplasms (Figs. 2, 3, 4). It seems that the peripheral 318
zone of the viroplasms, which contains both viral core and outer capsid proteins (Figs. 2, 3, 4), 319
is the site of assembly of progeny virions where outer capsid proteins are attached to core 320
particles. For viruses in the family Reoviridae, the core particles, rather than intact virions, 321
serve as the transcriptase particles for the production of viral plus-strand RNAs (1, 26). Based 322
on these analyses, we deduced that the binding of Pns6 with core capsid protein P3 within the 323
interior regions of viroplasm might delay the attachment of outer capsid proteins to core 324
particles, allowing core particles to continue synthesizing more viral plus-strand RNAs. This 325
hypothesis was supported by our finding that the newly synthesized viral RNAs accumulated 326
abundantly in viroplasm matrix induced by RRSV infection (Fig. 5). In the case of viruses in 327
the family Reoviridae, due to the lack of ribosomes within the viroplasm, viral RNAs 328
produced by core particles could be released into the cytoplasm surrounding the viroplasm for 329
translation of viral proteins (1). Thus, our results suggested that the binding of Pns6 with core 330
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capsid protein P3 might benefit the replication and assembly of RRSV in its insect vector cells, 331
by enhancing the synthesis of viral RNAs and the expression of viral proteins. The 332
RNA-binding activity of Pns6 suggested that this protein might also function during the 333
assembly of core particles, possibly in the packaging of the 10 different viral genome 334
segments of RRSV in the early steps of progeny core particle assembly. All these analyses 335
support our hypothesis that Pns6 of RRSV plays important roles in the formation of viroplasm 336
matrix, and in the recruitment or or retention viral RNAs and proteins to viroplasms for viral 337
replication and assembly. 338
Viruses in the family Reoviridae encode specific nonstructural proteins that function in 339
forming the organizational viroplasm matrix. For example, µNS of reoviruses, NS2 of 340
orbiviruses, NSP2/NSP5 of rotaviruses, Pns12 of the phytoreovirus RDV, P9-1/P5 of the 341
fijivirus SRBSDV, and Pns10 of RRSV, the oryzavirus under study here, have been shown to 342
be the viral proteins whose expression is required for forming viroplasm-like structures in the 343
absence of viral infection ( 2, 4, 7, 20, 28, 29, 30, 31, 32, 33, 34), suggesting that these 344
proteins might play similar roles in formation of the viroplasm matrix during virus replication 345
cycles. Furthermore, these viroplasm matrix proteins, such as reovirus µNS, orbivirus NS2, 346
rotavirus NSP5, are involved in recruiting or retaining core capsid proteins to their respective 347
viroplasm matrices (32, 33, 34). However, in the case of RRSV, an oryzavirus, the core capsid 348
protein is recruited or retained by the nonstructural protein Pns6, rather than the viroplasm 349
matrix protein Pns10, to the viroplasm. Thus, Pns6 and Pns10 of RRSV appeared to “split” 350
the functional duties of reovirus µNS, orbivirus NS2 or rotavirus NSP5. 351
Based on this discussion, we propose a model for the genesis and maturation of 352
viroplasm induced by RRSV in its insect vector cells. Early in the infection process, Pns6 and 353
Pns10 of RRSV associated together to form the initial viroplasm m atrix. During infection, 354
Pns6 might act as a scaffold for the recruitment of core capsid protein P3, whereas other 355
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structural proteins are recruited to the viroplasms through interactions with as-yet-unknown 356
factors. RRSV RNA was bound by Pns6 or other viral proteins to form replication and 357
assembly complexes for the production of progeny core particles within the interior regions of 358
viroplasm. Pns6 might bind to core particles to enhance the production of viral RNA by 359
blocking or delaying outer capsid assembly on these particles. The intact virions were 360
assembled at the periphery of the viroplasm. Thus, the development of the continuous BPH 361
cell culture can further advance our understanding of the early infection process of 362
oryzaviruses and related pathogens in their insect vector cells. 363
By development continuous insect vector cell cultures, we already revealed the 364
mechanisms for the genesis and maturation of the viroplasms induced by plant reoviruses 365
including phytoreovirus RDV, oryzavirus RRSV and fijivirus SRBSDV (4, 7, this study). The 366
viroplasm matrices of RDV and RRSV consist of a granular region for viral replication and 367
the assembly of progeny virions (7, Fig. 2, 3, 4). However, in the case of SRBSDV, the 368
viroplasm matrices consist of a granular region for the accumulation of viral RNAs and a 369
filamentous region for the assembly of progeny virions (2, 4). It is interesting to find that the 370
granular region of SRBSDV viroplasm was not suitable for the assembly of progeny virions 371
(4). We determined that the filamentous region of SRBSDV viroplasm would provide a larger 372
surface area for producing many more progeny virions than RDV or RRSV can in their 373
respective insect vector cells (4). These analyses suggest that SRBSDV, a new identified plant 374
reovirus, has evolved to be well adapted for persistent transmission by its WBPH vector, 375
consistent with the fact that the minimum latent periods for RDV, RRSV and SRBSDV in 376
their insect vectors are 6 days, 9 days and 14 days, respectively (1, 9, 35, 36). 377
378
ACKNOWLEDGMENTS: 379
This work was supported by grants from the programs for the National Basic Research 380
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Program of China (2014CB138400), the Specialized Research Fund for the Ministry of 381
Agriculture (201003031, 201303021), the National Natural Science Foundation of China 382
(31130044, 31200118, 31171821), and the Natural Science Foundation of Fujian Province 383
(2012J01087). 384
385
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491
FIGURE LEGENDS: 492
Fig. 1. Monolayer of established continuous cell culture derived from BPH after 27 passages 493
of subculturing. Bar, 30 μm. 494
495
Fig. 2. Viral nonstructural proteins Pns6 and Pns10 are the constituents of viroplasm matrix 496
induced by RRSV infection in VCMs. (A) Morphogenesis of viroplasm. Inset is enlarged 497
image of the boxed area. Black arrows: intact virions, red arrows: core-like 498
particles. Bars, 100 nm. (B, C) Immunogold labeling of Pns6 (B) and Pns10 (C) in the 499
viroplasm matrix at 84 hpi. VCMs were labelled for Pns6 and Pns10 with Pns6- and 500
Pns10-specific antibodies in panels B and C, respectively, as primary antibodies, followed by 501
anti-rabbit IgG conjugated to 15 nm gold particles as secondary antibodies. Insets are 502
enlarged images of the boxed areas. Black arrows: virions, red arrows: gold particles. Bars, 503
100 nm. (D) Confocal immunofluorescence micrographs of the colocalization of Pns6 and 504
Pns10 within the viroplasm. RRSV-infected VCMs were immunostained with Pns10-FITC 505
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and Pns6-rhodamine at 24 and 84 hpi. Bars, 5 μm. 506
507
Fig. 3. The distribution of core capsid protein P3 and outer capsid protein P8 of RRSV within 508
the viroplasm matrix in virus-infected VCMs 84 hpi. VCMs were labelled for P3 (A) and P8 509
(B) with P3-and P8-specific antibodies, respectively, as primary antibodies, followed by 510
anti-rabbit IgG conjugated to 15 nm gold particles as secondary antibodies. Panels II: 511
enlarged images of boxed areas in panels I. Black arrows: virions, red arrows: gold particles. 512
Bars, 100 nm. 513
514
Fig. 4. Confocal immunofluorescence micrographs of the colocalization of RRSV core protein 515
P3 and outer capsid protein P8 with nonstructural protein Pns6 within the viroplasm matrix 516
induced by RRSV infection in VCMs. RRSV-infected VCMs were immunostained with 517
P3-FITC and Pns6-rhodamine (A), with P8-FITC and Pns6-rhodamine (B), or with P8-FITC 518
and P3-rhodamine (C). Hours post-inoculation (hpi) are indicated. Bars, 5 μm. 519
520
Fig. 5. Intracellular sites of viral RNAs synthesis in mock- or RRSV-infected VCMs. 521
BrUTP-labeled viral RNA was immunostained with anti-BrdU from mouse, followed by 522
anti-mouse IgG conjugated to FITC. Viroplasm matrix is immunostained with 523
Pns6-rhodamine. Hours post-inoculation (hpi) are indicated. Bar, 5 μm. 524
525
Fig. 6. Core protein P3 was recruited to the viroplasm matrix through interaction with Pns6. 526
(A–D) The localization of Pns6, Pns10 and P3 in the absence of viral infection. Sf9 cells 527
infected with recombinant baculoviruses containing Pns6, Pns10 or P3 were fixed 3 days after 528
infection and processed for immunofluorescence and immunoelectron microscopy. (A) Pns6, 529
Pns10 and P3 were expressed alone. Sf9 cells were immunostained with P3-rhodamine, 530
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Pns6-specific IgG conjugated to FITC (Pns6-FITC) or Pns10-specific IgG conjugated to 531
rhodamine (Pns10-rhodamine). Bars, 5 μm. (B) Immunogold labeling of Pns10 associated 532
with electron-dense inclusion in Sf9 cells infected with recombinant baculovirus containing 533
Pns10. Cells were immunostained with Pns10 antibodies and goat antibodies against rabbit 534
IgG that had been conjugated with 15-nm-diameter gold particles as secondary antibodies. 535
Bar, 100 nm. (C) Pns6 and Pns10 were coexpressed. Sf9 cells were immunostained with 536
Pns6-FITC and Pns10-rhodamine. Bar, 5 μm. (D) P3 and Pns10 were coexpressed. Cells were 537
immunostained with P3-FITC and Pns10-rhodamine. Bar, 5 μm. (E) P3 and Pns6 were 538
coexpressed. Sf9 cells were immunostained with Pns6-FITC and P3-rhodamine. Bar, 5 μm. (F) 539
Pns6, Pns10 and P3 were coexpressed. Sf9 cells were immunostained with Pns6-FITC, 540
Pns10-rhodamine and P3-specific IgG conjugated to Alexa Fluor 647 carboxylic acid. Bar, 5 541
μm. (G) Yeast two-hybrid assay of protein–protein interactions among the RRSV P3, Pns6 542
and Pns10 proteins. Transformants were plated on SD/−Leu/−Trp/−His/−Ade medium for 4 543
days. +, positive control, i.e., pBT3-STE+pOst1-NubI; −, negative control, i.e., 544
pBT3-STE+pPR3-N; P3+Pns10, pBT3-STE-P3+pPR3-N-Pns10; Pns6+Pns10, 545
pBT3-STE-Pns6+pPR3-N-Pns10; P3+Pns6, pBT3-STE-P3+pPR3-N-Pns6. 546
547
Fig. 7. RNAi induced by dsPns6 inhibited the assembly of viroplasm and the infection of 548
RRSV in VCMs. At 24 h after transfection with dsGFP or dsPns6, VCMs were inoculated 549
with RRSV. At 84 hpi, VCMs were immunostained with Pns10-FITC and Pns6-rhodamine 550
(A), with P3-FITC and Pns6-rhodamine (B), or with P8-FITC and Pns6-rhodamine (C) and 551
then examined with confocal microscopy. Images are representative results of multiple 552
experiments with multiple preparations. Bars, 10 μm. 553
554
Fig. 8. RNAi induced by dsPns10 inhibited the assembly of viroplasm and the infection of 555
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RRSV in VCMs. At 24 h after transfection with dsGFP or dsPns10, VCMs were inoculated 556
with RRSV. At 84 hpi, VCMs were immunostained with Pns6-FITC and Pns10-rhodamine 557
(A), with P3-FITC and Pns10-rhodamine (B), or with P8-FITC and Pns10-rhodamine (C) and 558
then examined with confocal microscopy. Images are representative results of multiple 559
experiments with multiple preparations. Bars, 10 μm. 560
561
Fig. 9. Inhibition of viral protein expression and dsRNA synthesis in VCMs by RNAi induced 562
by dsPns6 or dsPns10. At 24 h after transfection with dsGFP, dsPns6 or dsPns10, VCMs were 563
inoculated with RRSV and kept in cultured medium for 84 h. (A, B) Lysates prepared from 564
infected cells were analyzed by immunoblotting using polyclonal antisera against P3, P8, 565
Pns6 and Pns10 of RRSV. An actin-specific antibody was used as control. (C, D) Viral 566
dsRNAs from infected VCMs was resolved by electrophoresis in 10% polyacrylamide gels 567
and analyzed by ethidium bromide staining. Size classes of viral dsRNA segments are 568
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B C
MergedPns10 Pns6D
24 hpi
84 hpi
Figure 2
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B
I II
I II
Figure 3
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MergedP3 Pns6
24 hpi
A
30 hpi
24 hpi
84 hpi
MergedP8 Pns6
24 hpi
B
30 hpi
84 hpi
MergedP8 P3
84 hpi
C
Figure 4
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MergedBrUTP Pns6
Mock
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58 hpi
Figure 5
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Pns10P3 Pns6 Pns10A B
MergedPns6 Pns10C
II IIII
MergedP3 Pns10D
MergedPns6 P3E
MergedPns6 Pns10 P3FF
+ P3+P 6 P 6+P 10 P3+P 10
G
Figure 6
+ P3+Pns6 Pns6+Pns10 P3+Pns10
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dsGFP treatment
AMergedPns10 Pns6
dsPns6 treatmenttreatment
BMergedP3 Pns6
dsGFP treatment
dsPns6 treatment
dsGFP treatment
CMergedP8 Pns6
dsPns6 t t ttreatment
Figure 7
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AMergedPns6 Pns10
dsPns10 t t ttreatment
BMergedP3 Pns10
dsGFP treatment
dsPns10 treatment
dsGFP treatment
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dsPns10 treatment
Figure 8
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dsGFP dsPns6 S1S2S3S4
dsGFP dsPns6
Pns6
CA
S5
S6S7S8
Pns10
P3
DB
S9S10
P8
Actin
dsGFP dsPns10
Pns10
dsGFP dsPns10
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P3
S1S2S3S4
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Figure 9
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