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

174

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

223

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

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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

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JVI Accepts, published online ahead of print on 18 July...

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