JVI Accepts, published online ahead of print on 18 July...
Transcript of JVI Accepts, published online ahead of print on 18 July...
MOV10 inhibits IAP reverse transcription and retrotransposition 1
Running title: MOV10 inhibits IAP RTP 2
3
Chunye Lu1, Zeping Luo1, Stefanie Jaeger2, Nevan Krogan2, B. Matija Peterlin 1,3* 4
5
1Departments of Medicine, Microbiology and Immunology, Rosalind Russell Medical 6
Research Center, University of California at San Francisco (UCSF), San Francisco, CA 7
94143-0703, USA 8
2Department of Cellular and Molecular Pharmacology, University of California-San 9
Francisco, San Francisco, California 94158, USA 10
3Department of Virology, Haartman Institute, University of Helsinki, Helsinki, 00014 11
Finland 12
*Corresponding author: 13 B. Matija Peterlin 14 533 Parnassus Avenue 15 San Francisco,CA 94143-0703 16 Phone: 415-5021905 17 Fax: 415-5021901 18 E-mail: [email protected] 19
Abbreviations: Intracisternal A Particle (IAP); endogenous retroviruses (ERVs); Reverse 20 transcription (RT); virus-like particle (VLP); retrotransposition (RTP); Endogenous 21 reverse transcription (ERT) 22
23 24 Abstract: 160 words; text: 3649 words 25
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.00868-12 JVI Accepts, published online ahead of print on 18 July 2012
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ABSTRACT 29
Moloney leukemia virus 10 (MOV10) is an RNA helicase that is induced by type I 30
interferon. It inhibits HIV replication at several steps of its replicative cycle. Of interest, 31
MOV10 is a component of mRNA processing (P) bodies, which inhibit retrotransposition 32
(RTP) of Intracisternal A particles (IAP). In this report, we studied effects of MOV10 on 33
IAP RTP and its dependence on P bodies. Indeed, MOV10 inhibited IAP RTP. It 34
decreased significantly not only products of reverse transcriptase but also its 35
endogenous activity. MOV10 also associated with IAP RNA. Furthermore, although it 36
was found in IAP virus like particles, it did not affect their incorporation of IAP RNA, 37
primer tRNAPhe or IAP Gag. Concerning P bodies, the exogenously expressed MOV10 38
had no effect on their size and number, and the inhibition of IAP RTP persisted despite 39
the depletion of their RCK subunit. Thus, by interfering with reverse transcription, 40
MOV10 inhibits IAP RTP, and this inhibition is independent of P bodies. 41
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INTRODUCTION 44
Intracisternal A particles (IAPs) are LTR-retrotransposons (endogenous retroviruses, 45
ERVs) in the mammalian genome. About 100 out of 1000-2000 IAP ERVs in the mouse 46
genome are still active for autonomous intracellular retrotransposition (RTP). In humans, 47
no recent RTP events of IAPs were reported. However, there is evidence that IAPs are 48
transcriptionally active and can express proteins in human cell lines and tissues (10, 12). 49
Indeed, IAP virus like particles (VLPs) were found associated with monocytes and in 50
salivary tissues in patients with CD4+ T-cell deficiencies and Sjögren’s syndrome, 51
respectively (10, 12). These observations also suggested that active transcription and 52
production of IAP proteins does not necessarily lead to integration, the final step of RTP, 53
due to restrictions at different steps in its replicative cycle: (1) transcription, (2) 54
translation, (3) reverse transcription (RT) and (4) integration. 55
56
To prevent deleterious effects of RTP, host cells have developed multiple defense 57
mechanisms. Innate immunity conferred by cellular restriction factors is an important 58
anti-viral defense against endogenous and exogenous retroviruses. One of these is the 59
Moloney leukemia virus 10 (MOV10), an RNA helicase that belongs to the DExD box 60
superfamily (7). It consists of seven highly conserved helicase motifs. MOV10 is also a 61
component of mRNA processing (P) bodies, which harbor Argonaute proteins (Ago1 62
and Ago2) and are involved in miRNA-guided mRNA silencing (17). MOV10 is also 63
induced by type I interferon (20). Three groups have reported that MOV10 inhibits 64
human immunodeficiency virus type-1 (HIV) replication at a post-entry step (5, 9, 23). 65
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However, the molecular mechanism by which MOV10 functions to restrict HIV 66
replication is not clear. Previously we published that P bodies inhibit IAP RTP (14). 67
Therefore, we now wanted to determine if MOV10 also inhibits IAP RTP, and whether 68
its effects depend on P bodies. 69
70
In this study, we found that MOV10 is not only incorporated into IAP VLPs but inhibits 71
IAP RTP by blocking its RT. MOV10 also associated with IAP RNA, but did not affect 72
IAP RNA, primer tRNAPhe and IAP Gag incorporation into VLPs. Lastly, the 73
exogenous expression of MOV10 had no effect on the size and number of P bodies, 74
and it inhibited IAP RTP to a similar degree in the presence and absence of RCK. 75
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MATERIALS AND METHODS 78
Cells, plasmids and siRNAs. 79
Human embryonic kidney 293 cells were maintained at 37°C with 5% CO2 in Dulbecco 80
modified Eagle medium containing 10% fetal bovine serum (FBS), 100 mM l-glutamine. 81
The IAP reporter plasmid pFL was a kind gift from Kyoji Horie. pFlAG/HA-MOV10 was 82
purchased from Addgene (Addgene Plasmid #10976). To clone MOV10.YFP, primers 83
(F, 5’- CAG ATC TCG AGA TGC CCA GTA AGT TCA GC - 3’; R, 5’- CCG G TG GAT 84
CCA GGA GCT CAT TCC TCC ACT C -3’) were used to amplify MOV10 coding 85
sequences from pFlAG/HA-MOV10. The amplicon was ligated into the pEYFP-N1 86
(Clontech) after digesting with XhoI and BamHI. MOV10 siRNA (ON-TARGETplus 87
SMARTpool, L-014162-00)) and control siRNA (ON-TARGETplus non-targeting pool, D-88
001810-10-05)) were purchased from Dharmacon. Human RCK siRNA and its control 89
siRNA were described previously (14). 90
91
Transfections. 92
Lipofectamine™ 2000 (Invitrogen) was used to reverse transfect 293 cells with 1 µg of 93
pFL and 1 µg (unless otherwise indicated in the figure legend) of pFLAG/HA-MOV10 or 94
the empty plasmid vector, according to manufacturer's instructions. siRNAs were 95
transfected at a final concentration of 40 nM. 96
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Western blotting. 99
Western blotting was performed as described (13). Rabbit anti-MOV10 (10370-1-AP, 100
protein-tech group), mouse anti-actin (ab8227, Abcam), rabbit anti-IAP Gag (a kind gift 101
from Bryan Cullen), mouse anti-Ago2 (ab57113, Abcam), and goat anti-RCK (sc-51415, 102
Santa Cruz Biotechnology) were used as primary antibodies. 103
104
RNA immunoprecipitation (RNA-IP). 105
RNA-IP was performed as described (3). Briefly, 293 cells were collected and washed in 106
cold PBS. Cells were resuspended and lysed in 1.5 volumes of polysome lysis buffer 107
(10 mM HEPES [pH 7.0], 100 mM KCl, 5 mM MgCl2, 25 mM EDTA, 0.5% IGEPAL, 2 108
mM dithiothreitol [DTT], 0.2 mg/ml Heparin, 50 U/ml RNase OUT [Invitrogen], 50 U/ml 109
Superase IN [Ambion], 1 tablet of complete protease inhibitor [Roche]), followed by 110
centrifugation at 14,000 g at 4⁰C for 10 min. 50 µl protein A sepharose beads 111
(Amersham) were equilibrated in NT2 buffer containing 50 mM Tris-HCl [pH 7.5], 150 112
mM NaCl, 1 mM MgCl2, 0.05% IGEPAL, 5% BSA (Equitech Bio), 0.02% sodium azide 113
and 0.02 mg/ml heparin. 2.2 µg of rabbit anti-MOV10 or normal rabbit IgG were added 114
to the protein-A beads and were incubated for 12 hours at 4⁰C. The beads were 115
subsequently washed three times in NT2 buffer and resuspended in 10 ml NT2 buffer 116
supplemented with 30 mM EDTA (pH 8.0), 1 mM DTT, 50 U/ml RNase OUT and 50 117
U/ml Superase IN. 293 cell extracts were added to the antibody-coated beads, 118
incubated for 6 hours at 4⁰C. The beads were then thoroughly washed four times in ice-119
cold NT2 buffer and RNP complexes were eluted twice with 500 µl SDS-EDTA (50 mM 120
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Tris [pH 8.0], 100 mM NaCl, 10 mM EDTA, 1% SDS) containing 40 U/ml RNAase 121
inhibitor for 10 min at 65⁰C. To isolate RNA from the RNA-IP elution, the elution was 122
incubated in 200 mM NaCl and 20 µg of proteinase K for 1 hour at 42⁰C. 980 µl of acid-123
phenol:chloroform was added, followed by centrifuging at 10,000 x g at room 124
temperature for 3 min. The aqueous layer was transferred to a new microcentrifuge tube. 125
RNA was precipitated by adding 98 µl of 3M sodium acetate, 10 µg Yeast tRNA, 2450 µl 126
ice-cold ethanol to the aqueous layer and incubating for 2 hours at 80⁰C, followed by 127
centrifuging for 30 min at 4⁰C. The RNA pellet was washed with 70% ethanol and 128
dissolved in DEPC-water. 129
130
Immunoprecipitation (IP). 131
Cells were lysed in lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 132
mM EDTA, 1% NP-40, 10% glycerol and protease inhibitor (Sigma, P8340). Lysate was 133
precleared with A-Sepharose (GE Healthcare) and then incubated with rabbit anti-134
MOV10 antibody or normal rabbit IgG overnight, followed by 4 h incubation with the 135
protein A-Sepharose beads. Beads were washed three times in 0.5 mL lysis buffer, and 136
proteins were eluted from the beads with SDS sample buffer by incubating for 5 min at 137
95⁰C. Immunoprecipitates were then resolved in SDS-PAGE gel followed by western 138
blotting. 139
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Immunofluorescence staining. 143
Immunofluorescence was performed as described previously (14). Anti-eIF4E-T 144
antibody was used as primary antibody and donkey anti-goat IgGs conjugated to Alexa 145
Fluor dye 594 (Molecular Probes, Eugene, OR) was used as secondary antibody. 146
Samples were counterstained with DAPI and visualized with a Zeiss LSM 510 confocal 147
microscope, with a 63X1.4-numerical-aperture lens (Carl Zeiss Inc.). 148
149
RTP assay. 150
RTP assay was performed as described (14). Briefly, the RTP reporter plasmid pFL was 151
transfected with pFlAG/HA-MOV10, or MOV10 or RCK siRNAs into 293 cells. 3 days 152
later, cells were collected and subjected to FACS analysis. 153
154
Quantitative PCR (qPCR). 155
Total RNA or DNA was extracted and qPCR was performed using Stratagene Mx3500. 156
The primers used in this study included: primers for detecting IAP RNA levels, IAP Gag 157
(F, 5’- ACC CAG GAA GCA GTC AGA GA-3’, R, 5’- CCT TTA GGG CTT GAG CAC 158
AG-3’), two sets of primers for detecting IAP RT product, GFP (F1, 5’- TGA AGA AGT 159
CGC TGA TGT CCT-3’, R1, 5’-GAG CAA GCA GAT CCT GAA GAA-3’), GFP (CF1, 5’- 160
ACG CGG TAC ACG AAC ATC TC-3’, CR1, 5’-TCG CCT TCG ACA TCC TGA GC-3’), 161
Human β-actin (F, 5’-AAA GAC CTG TACGCC AAC AC-3’, R, 5’-GTC ATA CTC CTG 162
CTT GCT GAT-3’), tRNAPhe (F, 5’-GCC GAA ATA GCT CAG TTG GG-3’, R, 5’- TGG 163
TGC CGA AWY CCG GGA TC-3’). 164
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165
Endogenous reverse transcription assay. 166
Endogenous reverse transcription activity was assayed using 30 μg of purified IAPs in 167
50µl of reaction mixture: 50mM Tris.HCl (PH8.3), 10 mM MgCl2, 8 mM DTT, 400 µM 168
each of dNTPs, and 0.05% NP40. The reaction was incubated at 37⁰С for 3 hours and 169
was stopped by incubating in stop solution (10 mM Tris-HCl pH7.4, 10 mM EDTA, 20 170
µg/ml of salmon sperm DNA and 50 µg/ml protease K) at 37⁰С for 15 min, followed by 171
10 min at 95⁰С. 10 µl of the stopped reaction mixture was used as template for qPCR to 172
measure IAP cDNA from endogenous reverse transcription. 173
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VLP isolation and extraction of RNA from VLPs. 175
Endogenous A particles were isolated from 293 cells as described (15). Briefly, 293 176
cells were collected from 4 100-mm dishes and washed with cold PBS. Cells were lysed 177
in 2 ml of buffer A (0.25 M sucrose, 0.6% Triton X-100, 0.05 M Tris.HCl pH 7.6, 0.025 M 178
KCl, and 0.005 M MgCl2. Nuclei was removed by centrifugation at 700 x g for 10 min. 179
Cytoplasmic extract was collected in 0.01 M EDTA and was layered over a 17.5% (W/W) 180
sucrose cushion in 10 mm Tris.HCl (pH 7.4) and 1 mm EDTA before centrifugation in 181
SW 41.T1 rotor for 60 min at 40200 RPM. A particles was collected as a triton:EDTA-182
insoluble pellet and was resuspended in a small volume of sodium phosphate buffer 183
(pH7.1). All procedures were performed at 4⁰С. RNA was isolated from same amount 184
of purified IAPs using TRIzoL reagent according to manufacturer’s instruction. 185
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RESULTS 187
MOV10 inhibits IAP RTP in a dose-dependent manner. 188
To monitor IAP RTP, we used a previously described reporter IAP plasmid (pFL) (14). 189
Briefly, pFL contains a GFP reporter gene, which is disrupted by the γ-globin intron and 190
expresses GFP only if IAP had undergone RTP. Thus, RTP rate is determined by 191
counting GFP positive cells. To determine if MOV10 inhibits IAP RTP, we co-192
transfected 293 cells with pFL (1 µg) and different amounts of pFLAG/HA-MOV10 193
plasmid ranging from 0 to 1 µg. 3 days after the transfection, cells were collected. 194
Fluorescent activated cell sorting (FACS) was used to count GFP-positive cells and 195
estimate the IAP RTP rate. As presented in Fig. 1A and B, IAP RTP was reduced in 196
cells expressing MOV10 exogenously (Fig. 1A and B). Indeed, increasing amounts of 197
pFLAG/HA-MOV10 correlated positively with this inhibition (Fig. 1A). These dose-198
dependent effects suggest that the inhibition of IAP RTP is MOV10-specific. 199
200
MOV10 decreases IAP RT products but not levels of IAP RNA and protein. 201
To limit deleterious effects of ERVs, mammalian cells use multiple strategies that target 202
various stages of their replicative cycles. IAPs have an obligatory RNA-intermediate 203
step, similar to that of exogenous retroviruses like HIV, except that they lack an 204
extracellular phase. Thus, the host applies similar defense mechanisms that target: (1) 205
transcription, (2) translation, (3) RT and (4) integration of new IAP cDNA. To pinpoint 206
where MOV10 inhibits IAP, products from these different steps were examined. First, 207
levels of IAP RNA and IAP Gag were determined by real-time quantitative PCR (RT-208
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qPCR) and western blotting, respectively. As presented in Fig. 2A and B, the 209
exogenous expression of MOV10 resulted in little to no change in levels of IAP RNA 210
and IAP Gag. Next, we examined RT products (cDNA) of IAP by conventional qPCR 211
using the primer set (Fig. 2C, F1 and R1) that detects amplicons from the parental pFL 212
plasmid containing the γ-globin intron (Fig. 2D, top left panel) and the reverse 213
transcribed pFL cDNA, where this intron had been spliced out (Fig. 2D, bottom left 214
panel). In contrast to levels of IAP RNA and proteins, markedly decreased amounts of 215
IAP cDNA were detected in cells that expressed MOV10 exogenously (Fig. 2D, bottom 216
left panel). To better quantify decreased IAP RT products, we also performed RT-qPCR 217
using a primer set (Fig. 2C, CF1 and CR1), which only detects the amplicon from 218
reverse transcribed pFL. These IAP RT products were reduced 3-fold (Fig. 2D). Since 219
the GFP cassette in pFL was inserted into 3’ Env open reading frame of IAP (Fig. 2C) 220
and we detected these RT products using our GFP primer sets, we conclude that early 221
RT is inhibited by MOV10. 222
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MOV10 inhibits the endogenous IAP RT activity. 224
To confirm that MOV10 inhibited IAP RT, we examined the endogenous IAP RT activity 225
in VLPs. 293 cells were co-transfected with pFL (1 μg) and pFLAG/HA-MOV10 (1 μg) or 226
the empty plasmid vector (1 μg). Three days later, IAP VLPs were isolated from cells as 227
described (15). Endogenous RT (ERT) activity was assayed by incubating 30 µg of 228
purified IAP VLPs in 50 µl of reaction mixture, which included dNTPs, for 3 hours. RT 229
was stopped and 10 µl of this reaction mixture was analyzed by qPCR to measure 230
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levels of IAP cDNA. Consistent with decreased steady state cDNA levels in Fig. 2D, the 231
endogenous IAP RT activity was markedly decreased in cells expressing MOV10 232
exogenously (Fig. 3). 233
234
MOV10 is incorporated into IAP VLPs. 235
The exogenous expression of MOV10 inhibited IAP RT activity, which occurs in VLPs. 236
To determine is MOV10 is also incorporated into IAP VLPs, they were isolated from 237
cells. Next, we determined levels of MOV 10 and IAP Gag in these VLPs and lysates by 238
western blotting. Indeed, MOV10 was enriched in VLPs, compared to its levels in total 239
cell lysates (Fig. 4). Although the exogenous expression of MOV10 increased its 240
incorporation, it did not change levels of IAP Gag in VLPs (Fig.4), suggesting that 241
MOV10 does not interact with IAP Gag or that interactions between MOV10 and IAP 242
Gag are not required for the incorporation of MOV10 into VLPs. Intriguingly, Ago2, 243
another component of RNA-Induced Silencing Complex (RISC) that associates with 244
MOV10 (17), was also enriched in IAP VLPs (Fig. 4), which is consistent with findings 245
with HIV (4). However, the exogenous MOV10 expression did not increase Ago2 246
incorporation (Fig. 4), suggesting that its interaction with Ago2 is not required for its 247
encapsidation in VLPs. 248
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MOV10 associates with IAP RNA but not IAP Gag. 252
Next, we studied interactions between MOV10, IAP RNA and IAP Gag. 293 cells were 253
co-transfected with pFL, pFLAG/HA-MOV10 or the empty plasmid vector. Two days 254
post-transfection, cells were lysed and subjected to protein and RNA 255
immunoprecipitations (IP and RNA-IP). Immunoprecipitations with anti-MOV10 256
antibodies were probed for the presence of Ago2 and IAP Gag. As presented in Fig. 5, 257
Ago2, a known partner, but not IAP Gag co-precipitated with MOV10 (Fig.5A). This 258
observation suggests that MOV10 does not interact with IAP Gag. After expressing pFL 259
and FLAG/HA-MOV10 in 293 cells, we used anti-HA and anti-Flag antibodies to 260
precipitate proteins bound to MOV10. These immunoprecipitations were subjected to 261
LC-MS/MS. No IAP Gag was identified in either immunoprecipitation (data not shown), 262
confirming that MOV10 does not interact with IAP Gag. In contrast, RNA-IP revealed 263
that IAP RNA was enriched 400-fold with anti-MOV10 antibodies compared to the IgG 264
control (Fig. 5B and C), indicating that MOV10 interacts with IAP RNA. 265
266
MOV10 does not affect the incorporation of IAP RNA or primer tRNAPhe into 267
VLPs. 268
Decreased RT by MOV10 could also result from decreased incorporation of IAP RNA, 269
which serves as the template for RT, or tRNAPhe, the tRNA that primes IAP RT (18, 19). 270
To determine if levels of IAP RNA or tRNAPhe in VLPs were decreased by MOV10, 271
VLPs were isolated from cells expressing pFL with or without exogenous MOV10. Total 272
RNA was isolated from purified IAP VLPs and subjected to RT-qPCR. PCR 273
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amplifications revealed that MOV10 did not affect levels of IAP RNA or tRNAPhe in 274
VLPs (Fig. 6A and B). In addition, the ratio between tRNAPhe and IAP RNA remained 275
the same (Fig. 6C). Thus, MOV10 does not inhibit IAP RT by affecting the incorporation 276
of IAP RNA or primer tRNAPhe into new VLPs. 277
278
Knockdown of MOV10 inhibits IAP RTP. 279
To determine if the endogenous MOV10 protein also inhibits IAP RTP, we co-280
transfected 293 cells with pFL and previously validated MOV10 siRNAs (9). 3 days later, 281
cells were collected for FACS and VLPs were isolated for western blotting. Surprisingly, 282
depleting MOV10 also inhibited IAP RTP (Fig. 7A), albeit to a lesser degree than that by 283
its exogenous expression, suggesting that MOV10 also plays a positive role in IAP RTP. 284
This observation is consistent with findings with HIV (5, 9). Furthermore, this depletion 285
decreased MOV10 but not Ago2 incorporation into VLPs (Fig. 7B). These observations 286
again suggest that MOV10 and Ago2 are incorporated into VLPs independently, which 287
is consistent with data presented in Fig. 4. 288
289
The exogenous MOV10 expression does not affect the size and number of P 290
bodies. 291
Previously, we reported that the disruption of P bodies increases IAP RTP (14). MOV10 292
is a component of P bodies and the exogenous expression of some of their subunits 293
can affect their size and number (6, 8, 21). Thus, whereas the exogenous expression of 294
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P body component SMG7 increases their size (21), that of hDcp2, RCK/p54, or hEdc3 295
can reduce their number significantly (8). To determine if MOV10 could also affect P 296
bodies, we examined these RNA granules in cells, which expressed the MOV10.YFP 297
fusion protein exogenously, with anti-eIF4E-T antibodies by immunofluorescence. 298
eIF4E-T is a resident of P bodies (2). As presented in Fig.8, MOV10 did not affect the 299
size or number of P bodies (Fig. 8A and B). Further comparisons between cells 300
expressing or not MOV10.YFP exogenously from the same transfection confirmed this 301
finding (Fig. 8B, compare cells 1, 2 to cells 3, 4). To further address whether P bodies 302
are required for the MOV10 inhibition of IAP RTP, we also depleted RCK in these cells 303
with siRNA (14). Two days later, MOV10 inhibited IAP RTP to a similar extent with or 304
without RCK (Fig. 9). Taken together, these observations suggest that MOV10 inhibits 305
IAP RTP by a mechanism that does not involve P bodies. 306
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DISCUSSION 309
In this study, we demonstrated that the RNA helicase MOV10 inhibits IAP RTP. Indeed, 310
the exogenous expression of MOV10 did not affect levels of IAP mRNA and proteins, 311
but decreased those of IAP RT products. Consistent with decreased IAP RT products, 312
the endogenous RT activity from IAP VLPs was markedly decreased. Whereas MOV10 313
associated with IAP RNA and was incorporated into IAP VLPs, it did not bind to IAP 314
Gag. Importantly, MOV10 did not affect the size and number of P bodies. Depleting P 315
body component RCK did not affect the inhibition of IAP RTP by MOV10. These 316
findings suggest that MOV10 inhibits IAP RT by steric hindrance or structural changes 317
in IAP RNA. 318
319
MOV10 was packaged efficiently into IAP VLPs. Moreover, it did not affect levels of IAP 320
RNA or IAP Gag in cells or IAP VLPs, which is similar to findings with HIV (9, 23). 321
However, unlike HIV, we only detected interactions between MOV10 and IAP RNA but 322
not IAP Gag. Likewise, the binding between MOV10 and HIV RNA was important for its 323
incorporation into HIV (5). Consistent with our observations with IAP, Ago2 is also 324
incorporated into HIV (4). Of note, Ago2 and MOV10 are also components of RISC (17). 325
They warrant an investigation of how other subunits of RISC and/or translational 326
silencing are involved in the replicative cycle of endogenous and exogenous 327
retroviruses. 328
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MOV10 inhibits early and late HIV RT products (9, 23). Consistent with these findings, 330
our data indicate that early RT is inhibited by MOV10, suggesting that MOV10 could 331
have affected the incorporation of the primer tRNA. Phenylalanine tRNA (tRNAPhe) 332
was identified as the primer tRNA for IAP in mouse and hamster cells (18, 19). However 333
we found that MOV10 did not interfere with tRNAPhe incorporation. Thus MOV10 could 334
decrease RT in VLPs due to steric hindrance or changes in IAP RNA secondary 335
structure. The specific molecular mechanism by which MOV10 inhibits RT remains to 336
be investigated. 337
338
Of interest, instead of increasing, depletion of MOV10 also inhibited IAP RTP, which is 339
consistent with findings with HIV (5, 9). Thus, MOV10 could be involved in multiple 340
steps of IAP and HIV RNA metabolism, where its exogenous expression and depletion 341
affect distinct steps in viral replication. Alternatively, a complex containing MOV10 is 342
required and is disrupted by both processes leading to these inhibitory effects. It will be 343
of interest to determine the underlying mechanism by which depletion of MOV10 inhibits 344
IAP RTP. 345
346
MOV10 is concentrated in cytoplasmic P bodies (17). However, the exogenous MOV10 347
expression did not change the size and number of P bodies, confirming that MOV10 is 348
not an essential component of these RNA granules. In addition, the depletion of the 349
essential P body component RCK did not affect the inhibition of IAP RTP by MOV10. 350
These findings suggest that MOV10 inhibits IAP RTP independently of P bodies. In sum, 351
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MOV10 can restrict exogenous and endogenous retroviruses in mammals, where it acts 352
primarily at the level of RT. 353
354
ACKNOWLEDGMENTS 355
We thank members of the Peterlin laboratory for helpful advice and discussions, Kyoji 356
Horie for the pFL plasmid, the HARC Center for Flag- and HA-IP followed by LC-MS/MS 357
analysis. This work was supported by grants from the NIH (P50 GMO82250) and the 358
Campbell Foundation. 359
360 361
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FIGURE LEGENDS 362
Fig. 1. MOV10 decreases IAP RTP in a dose-dependent manner. 363
(A) Inhibition of IAP RTP increases with higher ratios of MOV10 over pFL plasmids 364
(0.2:1, 0.5:1, 1:1) in co-transfections. Data are presented relative to control (% 365
RTP set to 100) as averages ± standard deviations (SD) from two independent 366
experiments. 367
(B) MOV10 expression was detected by western blotting. β-actin was used as the 368
loading control. 369
Fig. 2. Exogenous MOV10 expression decreases IAP RT products. 370
(A) Exogenous MOV10 expression does not affect levels of IAP RNA. Relative levels 371
of RNA were set to 1 in the control. β-actin was used as the reference RNA. 372
(B) Exogenous MOV10 expression does not affect levels of IAP Gag. MOV10 and 373
IAP Gag proteins were detected by western blotting. β-actin was used as the 374
control. 375
(C) Schematic representation of the GFP cassette within pFL. GFP is in the anti-376
sense orientation, as indicated by large black arrows. The γ-globin intron is in the 377
sense orientation with marked donor (GT) and acceptor (AG) splice sites. Small 378
arrows indicate primer sets used in (D). 379
(D) Exogenous MOV10 expression inhibits IAP RT products in 293 cells. IAP RT 380
products were detected by qPCR (left panel, black arrows, F1-R1) and RT-qPCR 381
(right panel, gray arrows, CF1-CR1), using IAP genomic DNA as the template. 382
F1-R1 detects two amplicons: one containing the intron (top left panel) and the 383
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other where the intron was spliced out (bottom left panel, RT-pFL). CF1-CR1 384
only detects amplicons from spliced and reverse transcribed pFL. Relative levels 385
of RT products by RT-qPCR were set to 1 in the control. Data in (A) and (D) 386
represent averages ± standard deviations (SD) from three independent 387
experiments. 388
Fig. 3. Exogenous MOV10 expression decreases endogenous IAP RT activity in VLPs. 389
Levels of RT products synthesized by endogenous IAP RT were measured by 390
RT-qPCR. Relative levels of RT products were set to 1 in the control. Data 391
represent averages ± standard deviations (SD) from three independent 392
experiments. 393
Fig. 4. MOV10 and Ago2 are incorporated into IAP VLPs, and MOV10 does not affect 394
levels of IAP Gag. VLPs were isolated from 293 cells that expressed or not the 395
exogenous MOV10 protein. MOV10, IAP Gag, Ago2 and β-actin proteins in VLPs 396
(lane 1 and 2) and total cell lysates (TCL, lane 3 and 4) were detected with anti-397
MOV10, IAP Gag, Ago2 and β-actin antibodies by western blotting. β-actin was 398
used as the loading control. 399
Fig. 5. MOV10 associates with IAP RNA. 400
(A) MOV10 does not interact with IAP Gag. IPs were performed with IgG or anti-401
MOV10 antibodies on lysates from cells expressing pFL and MOV10. Levels of 402
MOV10, IAP Gag and Ago2 were determined by western blotting. 403
(B) IAP RNA is enriched about 400-fold in immunoprecipitations with anti-MOV10 404
compared to IgG antibodies. RNA-IPs were performed with cell lysates over-405
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expressing MOV10 and pFL. RNA was isolated from immunoprecipitations with 406
IgG or anti-MOV10 antibodies and quantified by RT-qPCR. Data are presented 407
as averages ± standard deviations (SD) from two independent experiments. 408
(C) MOV10 was immunoprecipitated with anti-MOV10 antibodies in RNA-IPs. 409
Western blotting was used to detect MOV10 in immunoprecipitations with IgG or 410
anti-MOV10 antibodies. 411
Fig. 6. MOV10 does not affect levels of tRNAPhe or IAP RNA in VLPs. RNA was 412
extracted from VLPs isolated from 293 cells expressing the exogenous MOV10 413
protein. RT-qPCR was performed to detect levels of tRNAPhe (A) and IAP RNA 414
(B). 415
(C) MOV10 does not affect the ratio between tRNAPhe and IAP RNA. Data are 416
presented as averages ± standard deviations (SD) from three independent 417
experiments. 418
Fig. 7. Depletion of MOV10 decreases IAP RTP, and MOV10 but not Ago2 419
incorporation into VLPs. 420
(A) Depleting MOV10 decreases IAP RTP to 38% of the control. Data are presented 421
as averages ± standard deviations (SD) from three independent experiments. 422
(B) Whereas depleting MOV10 decreases its incorporation into VLPs, Ago2 423
incorporation remains the same. VLPs (lanes 1 and 2) and total cell lysates (TCL, 424
lanes 3 and 4) were used to detect levels of MOV10 and Ago2 by western 425
blotting. 426
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Fig. 8. MOV10 co-localizes with eIF4E-T to P bodies (panels B-C, MOV10.YFP) and its 427
exogenous expression does not affect the number and size of these RNA 428
granules. Mov10.YFP marks transfected cells (panel C, MOV10, green 429
fluorescence), where it co-localizes with P bodies (panel B, red fluorescence and 430
panel D, merged, yellow fluorescence). The size and number of P bodies does 431
not change with the exogenous expression of MOV10.YFP (panel A, control, and 432
panel B, eIF4E, compare transfected cells 1 and 2 to non-transfected cells 3 and 433
4). Red fluorescent signals mark P bodies (panels A, B and D). Green florescent 434
signals indicate MOV10.YFP (panels C and D). In panel D, images B and C 435
were merged digitally. White arrows indicate P bodies in cells expressing the 436
exogenous MOV10.YFP protein. Data are representative of two independent 437
experiments. 438
Fig. 9. MOV10 inhibits IAP RTP to a similar extent in the presence or absence of RCK. 439
(A) MOV10 decreases IAP RTP to 23% and 30% of the control in the absence and 440
presence of RCK, respectively. Data are presented as averages ± standard 441
deviations (SD) from three independent experiments. 442
(B) Two days after the co-transfection of pFL with control or RCK siRNAs, levels of 443
MOV10 and RCK proteins were detected by western blotting. β-actin was used 444
as the loading control. 445
446
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