JVI Accepted Manuscript Posted Online 29 March 2017 J. Virol. doi:10.1128/JVI.00260-17 Copyright © 2017 American Society for Microbiology. All Rights Reserved.

The minute virus of canines (MVC) NP1 protein

2

governs the expression of a subset of essential NS proteins

3

via its role in RNA processing

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Olufemi O. Fasina, Stephanie Stupps, Wanda Figueroa-Cuilan, and David J. Pintel* Department of Molecular Microbiology and Immunology University of Missouri-Columbia, School of Medicine Bond Life Sciences Center, Columbia MO USA 65211

Running: MVC NP1 governs viral NS-gene expression

*Corresponding Author: 471f Bond Life Sciences Center 1201 Rollins Rd. Columbia, MO 65211 [email protected] 573-882-3920

1

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

1

32 33

ABSTRACT Parvoviruses use a variety of means to control the expression of their compact genomes. The Bocaparvovirus Minute Virus of Canines (MVC) encodes a small, genus-specific

35

protein, NP1, which governs access to the viral capsid gene via its role in alternative

36

polyadenylation and alternative splicing of the single MVC pre-mRNA. In addition to NP1, MVC

37

encodes five additional non-structural proteins (NS) that share an initiation codon at the left end

38

of the genome and which are individually encoded by alternative multiply-spliced mRNAs. We

39

found that three of these proteins were encoded by mRNAs that excise the NP1-regulated MVC

40

intron immediately upstream of the internal polyadenylation site (pA)p, and that generation of

41

these proteins was thus regulated by NP1. Splicing of their progenitor mRNAs joined the amino

42

terminus of these proteins into the NP1 open reading frame, and splice-site mutations that

43

prevented their expression inhibited virus replication in a host-cell dependent manner. Thus, in

44

addition to controlling capsid gene access, NP1 also controls expression of three of the five

45

identified NS proteins via its role in governing MVC pre-mRNA splicing.

46

IMPORTANCE

47

The Parvovirinae are small non-enveloped icosahedral viruses that are important

48

pathogens in many animal species including humans. Minute virus of canine (MVC) is an

49

autonomous parvovirus in the Bocaparvovirus genus. It has a single promoter that generates a

50

single pre-mRNA. NP1, a small genus-specific MVC protein, participates in the processing of

51

this pre-mRNA and so controls capsid gene access via its role in alternative internal

52

polyadenylation and splicing. We show here that NP1 also controls expression of three of the

53

five identified NS proteins via its role in governing MVC pre-mRNA splicing. These NS proteins

54

are together required for virus replication in a host-cell dependent manner.

55 56

2

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

34

57

INTRODUCTION

58

Parvoviruses use multiple mechanisms to maximize the coding potential from their compact genomes, including alternative transcription initiation, alternative splicing, alternative

60

polyadenylation and alternative translation initiation mechanisms (1-3).

61

Infection with minute virus of canines (MVC), a member of the Bocaparvovirus genus (4), can

62

cause abortion and stillbirth in pregnant dogs, as well as mild gastroenteritis and respiratory

63

disease in puppies (5-8). MVC generates a single pre-mRNA from a promoter at the left-hand

64

end of the genome (P6) that is processed via alternative splicing and alternative polyadenylation

65

into multiple non-structural- and capsid-encoding transcripts (9, 10). As with other parvoviruses,

66

an open reading frame (ORF) in the left half of the genome encodes nonstructural (NS)

67

proteins, while an ORF in the right half encodes the capsid proteins VP1 and VP2 (2). The

68

bocaparvoviruses also encode a genus-specific protein, NP1, from a small ORF spanning the

69

center of the genome (9, 11). As seen for other parvoviruses, the NS proteins of MVC were

70

predicted to play multifunctional roles during infection; inspection of the MVC NS ORF identifies

71

a DNA binding/endonuclease domain within their shared amino-terminus, a helicase domain in

72

the relative center of the ORF, and a zinc-finger motif at its carboxyl-terminus. MVC NS proteins

73

have been shown to help initiate and sustain the replication of the viral genome, are required for

74

virus packaging, and mediate a number of important viral/host-cell interactions (12-18). NP1 is a

75

distinct Bocaparvovirus non-structural protein that modulates RNA processing via the

76

suppression of the internal polyadenylation signal (pA)p located in the middle of the genome. It

77

is also required for the splicing of the 3D/3A intron that lies immediately upstream of (pA)p (19).

78

Both of these processes are necessary to gain proper access to the capsid gene ORF (19, 20) .

79

We have found that that MVC generates a greater diversity of NS proteins than has

80

been previously identified; alternative splicing of RNAs from the MVC NS region led to the

81

generation of five NS proteins. Three of these proteins were generated from spliced mRNAs 3

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

59

that fused their carboxyl termini in-frame with the carboxyl terminus of NP1. The mRNAs

83

encoding these proteins utilize the 3D/3A intron and, consistent with our previous

84

characterization of the role of NP1 in splicing of this intron, we found that generation of these

85

mRNAs was dependent upon NP1. All NS isoforms share the same initiating AUG and the origin

86

binding/endonuclease domain that is conserved among Parvoviridae NS1 and Rep proteins.

87

Splice-site mutations which prevented expression of all three of the proteins derived from RNAs

88

using the 3D/3A intron inhibited virus replication in a host cell-dependent manner. Thus, in

89

addition to its role in accessing the viral capsid genes, NP1 is also necessary for efficient

90

expression of a subset of essential NS proteins, by virtue of its role in RNA processing.

91 MATERIALS AND METHODS

92 93

Cells and viruses/infections and transfections. All experiments were carried out as

94

specified in Walter Reed - 3873D (WRD) canine cells, Madin-Darby canine kidney (MDCK) cells

95

or human 293T, which were propagated as previously described (17, 21) in Dulbecco’s modified

96

Eagle medium (DMEM) with 10% or 5% fetal calf serum. The minute virus of canines (MVC)

97

used in this study was the original strain (GA3), obtained from Colin Parrish at Cornell

98

University. The infectious molecular clone pIMVC, GenBank accession number FJ214110.1,

99

used for transfection and generation of mutants was constructed by J. Qiu and described

100

previously (10). Transfections of WRD, MDCK and 293T cells were performed with either

101

Lipofectamine Plus (Life technologies, CA) or LipoD293 transfection reagent (SignaGen

102

Laboratories, MD).

103

Immunoblot analyses. Cells grown and transfected in 60mm dishes with various

104

constructs as described in the text were harvested 48hrs post-transfection, and lysed in either

105

Laemmli buffer with 2%SDS, RIPA buffer (10mM Tris pH7.5, 150mM NaCl, 1 mM EDTA,

4

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

82

1%Triton X100,0.5% Sodium Deoxycholate and 0.1%SDS) or WL lysis buffer (50mM Tris

107

pH8.0, 400mM NaCl, 1mM EDTA, 0.5%NP40, and 10% glycerol) which contained protease and

108

phosphatase inhibitors. Bradford assay of cell lysates was carried out to determine total protein

109

concentration in each lysate. SDS-PAGE and Western blotting were performed as previously

110

described (20).

111

Ribonuclease protection assays. Total RNA was isolated using the TRIzol reagent

112

(Invitrogen) and RNase protections were performed as previously described (22).The 2A\3D

113

probe (nts 2344-2550) which spanned the MVC second acceptor at nt 2386 and third donor at

114

nt 2490 was used to analyze the splicing across the MVC second and third introns.

115

Antibodies. Polyclonal antibodies directed against MVC NS epitope (NS ORF amino

116

acids 687-700; PKKQRKTEHKVLID) and MVC NP1 epitope (NP1 amino acids 1-13;

117

MSTRHMSKRSKAR) were utilized for MVC NS protein immunoblotting. Although 4 of 13 amino

118

acids of the NS protein antibody are missing in NS84 and NS50, this antibody detects NS100,

119

NS84, NS66 and NS50 at similar ratios to the proteins tagged with FLAG or HA and detected

120

with commercial anti-tag antibodies (see Figures 2B and 3B). Monoclonal antibodies against

121

FLAG, HA and tubulin epitopes (Sigma, St. Louis) were used for immunoblotting as previously

122

described (20).

123

Immunofluorescence analyses. Cells grown in 6-well plates on cover slips and

124

transfected constructs as described in the text were harvested 48hrs post-transfection, and fixed

125

in 4% paraformaldehyde in phosphate buffered saline (PBS) for 15 minutes. Cells were washed

126

twice with PBS and then permeabilized with 0.5% Triton-X in PBS for 10 minutes. Primary and

127

secondary antibodies were diluted in 3% bovine serum albumin (BSA) in phosphate buffered

128

saline (PBS). Cells were incubated with primary antibodies for 1hr followed by a wash with 3%

129

BSA in PBS. This was followed by incubation with appropriate Alexa-488 tagged secondary

130

antibodies. Nuclei were visualized with 4’, 6-diamidino-2-phenylindole (DAPI). The slips were 5

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

106

131

mounted on slides in Fluoromount-G (Southern Biotech, AL). Image analysis was carried out

132

with a Leica TCP SP8 MP confocal microscope. All images were taken with 63X objective. Southern blotting. WRD and 293T cells were transfected with the pIMVC and pIMVC-

134

derived mutant constructs as described in the text. DNA was extracted 48hrs post-transfection

135

and DNA replication was assayed by Southern blotting as described previously (17, 23) using a

136

random primed radio-labelled Not I digested genomic fragment of the pIMVC as probe. Loading

137

of samples was standardized using a Nanodrop spectrophotometer.

138

Plasmid constructs. The generation of the multiple plasmid constructs used in this

139

study are described below.

140

pIMVC (WT), pIMVC 3Am, : the wild type infectious clone (WT) (10) and the third intron

141

acceptor mutant (3Am) (20) were generated as previously described.

142

pIMVC 1Am: mutation of the splice acceptor site of MVC first intron (nt 2199) was generated as

143

an in-frame G-to-A substitution.

144

pIMVC 1AmΔ3DA: this mutant was generated by site directed mutagenesis by insertion of a

145

gene-block synthesized oligonucleotide (IDT, IA) that lacked nts 2490-3037 between the EcoRI

146

site (nt 2345) and PvuII site (nt 3060). This generated a construct with deletion of MVC third

147

intron in pIMVC1Am background.

148

pIMVC NS-66, pIMVC NS-50, and pIMVC NS-40: these constructs were generated by cloning

149

the respective cDNAs encoding each NS isoform into pIMVC WT with primers flanking the XbaI

150

(nt 868) and PvuII (nt 3060) sites.

151

3XF NS-66, 3XF NS-50 and 3XF NS-40: the cDNAs encoding NS-66, NS-50 and NS-40 were

152

cloned into the CMV 3XFLAG 7.1 expression vector (Sigma, St. Louis).

153

pIMVC NSNP1fus: a single nucleotide deletion at nt 2536 fused the NS ORF to NP1 ORF.

154

TCGGCCGAGCAATATGTC to TCGGCCGAGCAAATG

6

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

133

pIMVC NP1m: this functionally defective mutant of NP1 was generated by introducing five

156

consecutive proline substitutions into the NP1 ORF at nt 2780 [the previously described 5X-pro

157

mutant,(20) ].

158

pIMVC 3DAm was generated by debilitating the third intron splice donor at nt 2491 via an in-

159

frame G-to-A substitution in the NS ORF in combination with the third nucleotide substitutions

160

between nts 3026-3040: GTTCTCACACAGGAT to GTGCTGACACAAGAC which surround the

161

third intron acceptor (indicated in bold and underlined).

162

pIMVC 3097HA (WT, NP1m): this mutant, created both in the wild-type and NP1-mutant

163

infectious molecular clone backgrounds, was generated by site directed mutagenesis which

164

introduced an HA epitope upstream of NP1 termination codon.

165 166

RESULTS

167

MVC encodes a larger spectrum of nonstructural proteins than previously determined. In

168

previous studies we identified two proteins encoded by the left-end nonstructural gene of MVC

169

in addition to the centrally encoded NP1 protein (20). These NS proteins migrated at ~84 and

170

~66 kDa, respectively. We demonstrated that the ~66 kDa protein was encoded by a doubly-

171

spliced RNA (R3; as diagrammed in Fig. 1C). While the derivation of the ~84 kDa protein was

172

not determined, we speculated (in retrospect, incorrectly), based on predicted molecular weight,

173

that it was encoded by the uninterrupted ORF which spanned the entire NS region present

174

within the unspliced RNA that had been described (indicated as R1 in Fig 1C).

175

A more detailed examination using an antibody specific to an epitope within the

176

carboxyl-terminal region of the MVC NS ORF (nts 403-2727), indicated by the star in Fig. 1C),

177

allowed us to identify 4 proteins that utilize the NS gene region during virus infection (Fig. 1A,

178

lane 2). (A fifth NS protein will be discussed below.) In addition to the previously identified

179

abundant ~84 kDa and ~66kDa proteins, this antibody revealed NS proteins of ~100 kDa and

7

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

155

~50 kDa that appeared at lower abundance. Expression of these four proteins was also

181

detected following the transfection of the infectious clone of MVC in 293T cells (Fig. 1A, lane 4).

182

Insertion of a terminating TAA codon at nt 427 in the infectious clone of MVC confirmed that,

183

following transfection of 293T cells, all four NS proteins (but not NP1) utilize the previously

184

identified NS gene AUG at nt 403 (Fig. 1A, lane 5).

185

Three of the newly identified NS proteins are generated from RNAs which excise

186

the 3D/3A intron and join the NS ORF to the NP1 ORF. To identify the coding regions for

187

these newly identified NS proteins we first performed RT-PCR assays to identify spliced RNAs

188

from the NS region that could potentially encode these proteins. Using probes spanning nts 868

189

to 3097 (diagrammed in Fig.1C), RNA generated by the infectious clone pIMVC generated

190

distinct RT-PCR species of 1683 nts, 1224 nts, 678 nts, and 464 nts (Fig. 1B) following

191

transfection of 293T cells. These products were individually cloned into bacterial plasmids, and

192

sequencing identified these as corresponding to the RNA species R2, R3, R4, and R5,

193

respectively, as diagrammed in Fig. 1C. Of note, RNAs R2, R4 and R5 utilize the viral 3A

194

acceptor, and thus putatively fused the carboxyl-terminal region of NP1 in-frame with upstream

195

NS ORF.

196

The individual cDNAs representing the various splicing patterns were then cloned in

197

place into the full-length clone of MVC. These constructs were transfected into 293T cells and

198

extracts were analyzed by immunoblot using the same antibody to the C-terminal region of NS

199

as described above. The 1224 nt cDNA, representing the R3 transcripts (see Fig. 1B),

200

generated a single protein with a mobility of ~66k Da as expected (Fig. 2A, lane 5). Similarly,

201

the 678 nt cDNA, representing the R4 transcripts (see Fig. 1B), generated a single protein with

202

a mobility of approximately 50 kDa (Fig 2A, lane 6). As expected, expression of the 464 nt

203

cDNA, which represents the R5 transcripts, did not generate a protein detected by this antibody

204

(data not shown). This will be addressed further below. 8

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

180

205

When we expressed the largest 1683 nt cDNA, representative of the R2 transcripts, we were surprised to see the generation of a protein of ~50 kDa (data not shown). Upon further

207

examination, we found that as expressed, this RNA had been further spliced at both upstream

208

introns, yielding an R4-like transcript. In a separate set of experiments, we fortuitously found

209

that silent mutation of the 1A donor site, constructed so as to not change the NS amino acid

210

sequence, prevented all splicing of the MVC pre-mRNAs. This mutant generated only a single

211

protein with a mobility of approximately 100 kDa (Fig. 2A, lane 3). The reason that this mutant

212

did not undergo additional splicing has not yet been determined. However, the result confirmed

213

that the unspliced R1 transcripts encodes a protein of 100 kDa - rather than the largest of the

214

proteins we had identified in our previous study (i.e., the 84 kDa species) - which had been our

215

previous assumption based solely on predicted molecular weight. Cloning the 3D/3A cDNA into

216

the 1A mutant background to prevent further splicing (generating, in essence, the R2 transcript

217

diagrammed in Figure 1C), produced a single protein of 84 kDA (Fig. 2A, lane 4). These results

218

confirmed that the 100kDa, 84 kDa, 66 kdA, and 50 kDa proteins detected in Fig. 1, were

219

generated by the R1, R2, R3, and R4 classes of transcripts, respectively.

220

As mentioned, sequencing of the 464 nt cDNA indicated that it lacked the epitope used

221

for detection in the experiment shown in Fig. 1A and Fig 2A. Thus, we cloned the 464 nt cDNA -

222

and in addition the 1224 nt and 678 nt cDNAs (encoding the 66 and 50 kDa proteins,

223

respectively) as controls - into a CMV-driven expression vector containing a FLAG tag at the

224

amino terminus of the NS open reading frame. As seen previously, immunoblotting of extracts

225

generated following transfection of these constructs using the anti-NS antibody detected only

226

the 66 and 50 kDa proteins (Fig. 2B, lanes 3-5), while the anti-FLAG antibody identified the 40

227

kDa protein as the product of the 464 cDNA (Fig. 2B, lanes 6-8), which represented the R5

228

transcripts diagrammed in Figure 1C.

9

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

206

229

To confirm that the 84 kDa, 50 kDa and 40 kDa NS proteins utilize a different carboxylterminal exon than the 66kDa and 100 kDa proteins, we explored the prediction that deletion of

231

a single nucleotide which fuses the NS ORF to the NP1 ORF (within the 3D/3A intron, nt 2536),

232

would increase the size of only the 66 kDa and 100 kDa NS proteins (mutation diagrammed in

233

Fig. 2C; c.f. Fig. 1C). The results following transfection of this mutant (NSNP1fus; Fig. 2D, lane

234

3) demonstrate this to be the case. The levels of the 84 and 50 kDa proteins generated by the

235

NS/NP1 fusion shown in Fig. 2D, lane 3 are relatively low, likely because as a fusion protein, a

236

significant proportion of the NP1 present is mutant and poorly functional. NP1 is required for

237

splicing of the 3A/3D intron (19) and the potential role for NP1 in this observation can be

238

explained as discussed below.

239

NP1 governs expression of three NS proteins via its role in splicing the 3D/3A

240

intron. Since NP1 is required for splicing of the 3A/3D intron (19), it seemed likely that NP1 may

241

govern expression of the 84 kDa, 50 kDA, and 44 kDa NS proteins. To investigate this

242

possibility, we analyzed expression following transfection of 293T cells of the wild-type and

243

NP1-mutant MVC infectious clones bearing an HA epitope inserted at nt 3097 at the carboxyl

244

termini of the NP1 ORF (triangle in Fig. 1C). For these experiments we assayed both RNA

245

production using RNase protection assays, and protein production using antibodies directed

246

either to HA or to the previously described epitope at the carboxyl-terminus of the NS ORF (star

247

in Fig. 1C). The HA insertion itself had only a slight effect on splicing of the 3D/3A intron in the

248

presence of otherwise wild-type NP1 (Fig. 3A, compare lane 5 to lane 2). Mutation of NP1 [the

249

previously described mutant bearing an insertion of 5 proline residues which abrogated NP1’s

250

roles in 3D/3A splicing and suppression of (pA)p (19) ], led to a decrease in splicing of the

251

3D/3A intron, both in the wild-type as expected, and to a slightly lesser extent, in the HA-tagged

252

construct (Fig. 3A, compare lane 6 to lane 3). As can be seen in Fig. 3B, corresponding to the

253

decrease in splicing of the 3A/3D intron seen in the NP1-mutant constructs, the levels of

10

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

230

expression of the 84 kDa, and 50 kDa proteins were significantly reduced (the 40 kDa protein

255

was poorly expressed in this experiment), as detected by the anti-HA antibody at the carboxyl

256

termini of the NP1 exon (Fig. 3B, compare lane 3 to 2). Reduced expression of the 84 kDa and

257

50 kDa proteins was also seen in the NP1-mutant transfection when detected by the antibody to

258

the carboxyl-terminus of NS (which does not detect the 40 kDa protein; Fig. 3B, compare lanes

259

6 to 5). These results demonstrate that NP1, which is required for the excision of the 3D/3A

260

intron, governs the expression of the 84 kDa, 50 kDa (and we expect also the 40 kDa) proteins

261

which are derived from mRNAs from which the 3D/3A intron is excised.

262

The individual NS proteins exhibit a complex localization pattern. Generation of

263

vectors that individually expressed the various NS isoforms allowed a general determination of

264

their localization, and perhaps provide insights into their potential function during the viral life

265

cycle. At 48 hrs following transient transfection of replication competent pIMVC in WRD cells,

266

immunostaining, using the anti-NS antibody whose epitope is designated by the star in Fig. 1C,

267

demonstrated that when expressed together, the 100 kDa, 84 kDa, 66 kDa, and 50 kDa proteins

268

exhibited a diffuse distribution in the nucleoplasm (Fig. 4A panel 1). Replication-defective

269

pIMVC 1Am and pIMVC 1AmΔ3DA constructs which individually encode R1 and R2 transcripts,

270

that are translated into NS-100 and NS-84, respectively, exhibited both nuclear and cytoplasmic

271

NS localization. The isoforms have a partial-to-complete diffuse nucleoplasmic distribution with

272

multiple cytoplasmic aggregates (Fig. 4A, panels 2 and 3). NS-100 seemed to form more round

273

cytoplasmic aggregates while the NS-84 isoforms generated more of interlacing filamentous

274

aggregates in the nucleus and cytoplasm. WRD cells transiently expressing NS-66 alone

275

displayed a strong diffuse nucleoplasm distribution of NS (Fig. 4A, panel 4), while cells

276

expressing NS-50 has diffuse faint nuclear distribution, apparently excluding nucleoli and

277

multiple concentrated, discreet, punctate spherical bodies within the nucleoplasm (Fig. 4A,

278

panel 5). These results indicated that while NS-100, NS-84, NS-66 and NS-50 isoforms were

11

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

254

primarily nuclear when generated together from the complete pIMVC WT clone, when

280

generated individually, their distribution and localization were quite different. Whether these

281

differences are due to differential post-translational modifications of these proteins, or

282

interactions between the proteins themselves are currently being investigated.

283

As mentioned previously, the NS antibody utilized in the experiments just described

284

does not recognize NS-40. Thus, to determine the localization of individually expressed NS-40

285

we compared the localization of N-terminal FLAG-tagged NS-66 and NS-40 following

286

immunostaining (Fig. 4B) using the anti-FLAG antibody (also used for the experiment shown in

287

Fig. 2B). FLAG-tagged NS-66 was again seen to be diffusely nuclear (Fig. 4B, panel 2),

288

consistent with our observation shown in Fig. 4A panel 4; however, NS-40 was predominantly

289

cytoplasmic (Fig. 4B, panel 1). There is a potential bipartite nuclear localization signal

290

(PSRKRTSSDETINSPPKKQRK, nts 2414-2478) that is located in the carboxyl end of the NS-

291

100, NS-84, NS-66 and NS-50 ORFs but absent in NS-40. Thus, the lack of the bipartite nuclear

292

localization signal in NS-40 may account for its predominant cytoplasmic localization.

293

The NS proteins whose expression was governed by NP1 were required for MVC

294

replication in canine but not human 293T cells. Previous results have shown that mutations

295

that prevent the production of all the MVC NS proteins prevents virus replication following

296

transfection in both canine and 293T cells, and that neither the MVC 100 kDa nor 66 kDa NS

297

protein can sustain MVC replication alone (10, 20). Additional experiments have shown that, as

298

seen for certain other parvoviruses, mutations that prevent MVC VP1 and VP2 production still

299

allow wild-type levels of mRF production ((20); Fasina and Pintel, unpublished). To determine

300

the importance in replication of the NP1-ORF containing NS-84, NS-50 and NS-40 proteins, we

301

inserted into the MVC infectious clone a mutation which debilitated both the donor and acceptor

302

of the 3D/3A intron without changing the NS-100 and NS-66 amino-acid sequence (3DAm,

303

diagrammed in Fig. 5A). This mutation would also be expected to prevent capsid protein 12

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

279

production which requires mRNAs spliced using the A3 acceptor. As expected, this mutant

305

generated the NS-100 and NS-66 proteins, but not NS-84, NS-50 (Fig. 5B) or NS-40 (data not

306

shown) following transfection of 293T cells. A similar expression profile was seen in WRD cells,

307

which transfect poorly (data not shown). Following transfection, the pIMVC 3D/3A mutant

308

(3DAm) failed to replicate in both canine cells tested: WRD (Fig. 5C, compare lanes 3 and 6 to

309

lanes 2 and 5) or MDCK (Fig. 5D, compare lanes 3 and 6 to 2 and 5). In contrast, however, the

310

3DAm mutant replicated to similar levels as wild type pIMVC following transfection of 293T cells

311

(Fig. 5E compare lanes 4 and 6 to lanes 3 and 5 – 293T cells are not susceptible to direct MVC

312

viral (re)infection). These results indicated that while the NS-84, NS-50 and NS-40 proteins

313

were essential for replication of MVC in canine cells, these proteins were dispensable for

314

replication in 293T cells.

315 316 317

DISCUSSION In this manuscript we expand the identification of MCV NS proteins to five, and show

318

that three newly identified MVC NS proteins (84 kDa, 50 kDa and 40 kDa) are generated from

319

mRNAs spliced at the 3D/3A intron. In each case splicing of 3D/3A fuses the 3’ ends of the NS

320

proteins to the NP1 ORF. NP1, which is required for efficient splicing of the 3D/3A intron,

321

governs expression of these NS proteins by virtue of its enhancement of 3D/3A excision. Thus,

322

in addition to controlling capsid gene access via its role in alternative polyadenylation of the

323

internal polyadenylation site (pA)p (20), and splicing of the 3D/3A intron (19), NP1 also controls

324

expression of three of the five identified NS proteins via its role in splicing. Mutations which

325

prevent expression of these three proteins inhibit virus replication in a host dependent manner.

326

It is not yet fully clear if there is a difference in NS protein expression from RNAs that utilize

327

(pA)p or read-through to the distal polyadenylation site (pA)d.

13

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

304

328

Similarities between the 84 kDa, 50 kDa, and 40 kDa MVC NS proteins and the NS2 proteins of minute virus of mice (MVM) bear mentioning. Similar to the case for MVC, splicing of

330

the RNA generating MVM NS2 joins the amino terminal MVM NS1 ORF into a separate ORF at

331

the carboxyl end of the NS gene (24-27). Additionally, as is the case for the minor MVC NS

332

proteins, MVM NS2 has been shown to be required for replication of MVM in a host-dependent

333

manner. While restricted in murine host cells, MVM NS2 mutants replicate in a wide variety of

334

transformed cells from various species (28-31), and perhaps analogously, the MVC mutants that

335

do not produce the 3D/3A spliced NS proteins are restricted for growth in canine cells yet

336

replicate efficiently in human 293T cells. The extent to which host-range replication for these

337

mutants extends is currently being investigated. Additionally, the Tattersall lab has pointed out

338

that MVM NS2 and the human bocavirus HBoV NP1 both bear Crm1 binding and 14-3-3 binding

339

sites, and HBoV NP1 can complement certain early functions of MVM NS2 (32). Together,

340

these results suggest an intriguing similarity in the small NS proteins of parvoviruses.

341

Surprisingly, the MVC NS proteins migrate surprisingly differently on SDS-PAGE gels

342

than expected based on their molecular weight as predicted from their amino acid composition

343

(Fig. 1C). This led us previously to assume that the protein migrating at approximately 84 kDa

344

was generated from the uninterrupted NS ORF (c.f. Fig 1C). This also initially confounded our

345

mapping of the various MVC NS proteins until cDNAs of each were generated. Why the

346

migration of these proteins is so different than their predicted molecular weight is not yet clear.

347

The unexpected migrations observed may certainly be due to post-translational modifications;

348

the NS proteins generated by other parvovirus genera have been shown to be highly modified

349

by phosphorylation and acetylation (33-37).

350

The conserved N-terminal region of the MVC NS proteins encodes a histidine-

351

hydrophobic-histidine (HUH) endonuclease motif (38, 39), also conserved in other parvoviral

352

nonstructural proteins (13, 39), which is required for site-specific DNA cleavage and ligation 14

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

329

during rolling-hairpin replication (12, 40). The active site of the MVC HUH motif would be

354

predicted to be H122, L123 and H124 which are present in all the MVC NS isoforms (Fig. 4B).,

355

suggesting that all MVC NS isoforms may interact with the viral genome. As in other viral HUH

356

motif-containing proteins (12, 14, 38, 41) there is an SF3 helicase domain with characteristic

357

Walker A (FYGPASTGKTN aa 423-433), B (LICWWEEC aa 464-471) C (KCIMGGTQFRIDRK

358

aa 482-495) and C’ (PQTPLIISTNHNI aa 503-515) motifs (42) downstream of the HUH motif in

359

NS-100 and NS-84 proteins (amino acids 1-696 are identical in NS-100 and NS-84 ); however,

360

it is removed by alternative splicing and absent in the NS-66, NS-50 and NS-40 proteins,

361

suggesting distinct differences in function. And although NS-100 and NS-84 proteins contain

362

HUH endonuclease and SF3 helicase domains, they have distinct carboxyl terminal ends which

363

likely contribute to important differences. Furthermore, NS-84, NS-50 and NS-40 share a

364

conserved 19 amino-acid carboxyl terminal domain with NP1.

365

In-silico analysis also suggests a strong bipartite nuclear localization signal

366

(PSRKRTSSDETINSPPKKQRK – nts 2414-2478) that is located in the carboxyl end of all the

367

NS isoforms except the NS-40 protein. We find that the FLAG-tagged NS-66 displays a

368

diffusely nuclear localization, while the NS-40 protein is predominantly cytoplasmic, perhaps

369

suggesting that the lack of the bipartite nuclear localization signal may alter its cellular

370

localization.

371

It has recently been shown that interactions between intrinsically disordered domains of

372

viral and host proteins can modulate certain host cellular responses to virus infection, such as

373

the DNA damage response (43). In-silico analysis shows that MVC NS-84, NS-50 and NS-40

374

share a highly disordered domain in their carboxyl termini with NP1 (data not shown). It is

375

conceivable that these regions may play important roles in their function. Although the 3D/3A

376

mutant replicates well in 293T cells, virus is not produced because capsid protein production is

377

prevented. Mutants are currently being constructed which prevent the production of NS-84, NS15

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

353

378

50, and NS-40, yet allow capsid protein production. These mutations should aid further analysis

379

of the role of these NS proteins during virus infection.

381

expressed (44-51). The Bocaparvoviruses, which have a single promoter, make extensive use

382

of RNA processing to properly express their genetic information, and encode a genus-specific

383

protein, NP1, that participates in this processing (19, 20). In addition to enhancing access to the

384

capsid coding gene, MVC NP1, by virtue of its role in splicing, also controls expression of three

385

NS proteins that play important roles in virus replication. Thus MVC NP1 influences viral RNA

386

processing for expression of both its non-structural and structural genes, adding to the variety of

387

strategies utilized by parvoviruses to maximize their genome coding potentials.

388 ACKNOWLEDGEMENTS

389 390

We thank Lisa Burger for excellent technical assistance, and members of the lab for advice and discussion. We thank Jianming Qiu for information prior to publication.

391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Qiu J, Pintel D. 2008. Processing of adeno-associated virus RNA. Front Biosci 13:31013115. Cotmore SF, Tattersall P. 2014. Parvoviruses: Small Does Not Mean Simple. Annual Review of Virology 1:517-537. Kailasan S, Agbandje-McKenna M, Parrish CR. 2015. Parvovirus Family Conundrum: What Makes a Killer? Annu Rev Virol 2:425-450. Cotmore S, Agbandje-McKenna M, Chiorini J, Mukha D, Pintel D, Qiu J, SoderlundVenermo M, Tattersall P, Tijssen P, Gatherer D, Davison A. 2014. The family Parvoviridae. Archives of Virology 159:1239-1247. Decaro N, Carmichael LE, Buonavoglia C. 2012. Viral reproductive pathogens of dogs and cats. Vet Clin North Am Small Anim Pract 42:583-598, vii. Harrison LR, Styer EL, Pursell AR, Carmichael LE, Nietfeld JC. 1992. Fatal disease in nursing puppies associated with minute virus of canines. J Vet Diagn Invest 4:19-22. Carmichael LE, Schlafer DH, Hashimoto A. 1991. Pathogenicity of minute virus of canines (MVC) for the canine fetus. Cornell Vet 81:151-171.

16

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

Parvoviruses exhibit a surprisingly wide variability in how their genomes are

380

8. 9. 10. 11. 12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Macartney L, Parrish CR, Binn LN, Carmichael LE. 1988. Characterization of minute virus of canines (MVC) and its pathogenicity for pups. Cornell Vet 78:131-145. Schwartz D, Green B, Carmichael LE, Parrish CR. 2002. The canine minute virus (minute virus of canines) is a distinct parvovirus that is most similar to bovine parvovirus. Virology 302:219-223. Sun Y, Chen AY, Cheng F, Guan W, Johnson FB, Qiu J. 2009. Molecular characterization of infectious clones of the minute virus of canines reveals unique features of bocaviruses. J Virol 83:3956-3967. Lederman M, Patton JT, Stout ER, Bates RC. 1984. Virally coded noncapsid protein associated with bovine parvovirus infection. J Virol 49:315-318. Hickman AB, Ronning DR, Kotin RM, Dyda F. 2002. Structural unity among viral origin binding proteins: crystal structure of the nuclease domain of adeno-associated virus Rep. Mol Cell 10:327-337. Hickman AB, Ronning DR, Perez ZN, Kotin RM, Dyda F. 2004. The nuclease domain of adeno-associated virus rep coordinates replication initiation using two distinct DNA recognition interfaces. Mol Cell 13:403-414. Yoon-Robarts M, Blouin AG, Bleker S, Kleinschmidt JA, Aggarwal AK, Escalante CR, Linden RM. 2004. Residues within the B' motif are critical for DNA binding by the superfamily 3 helicase Rep40 of adeno-associated virus type 2. J Biol Chem 279:5047250481. Bardelli M, Zarate-Perez F, Agundez L, Linden RM, Escalante CR, Henckaerts E. 2016. Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface. J Virol 90:6612-6624. Tewary SK, Liang L, Lin Z, Lynn A, Cotmore SF, Tattersall P, Zhao H, Tang L. 2015. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding. Virology 476:61-71. Fasina O, Pintel DJ. 2013. The adeno-associated virus type 5 small rep proteins expressed via internal translation initiation are functional. J Virol 87:296-303. King JA, Dubielzig R, Grimm D, Kleinschmidt JA. 2001. DNA helicase-mediated packaging of adeno-associated virus type 2 genomes into preformed capsids. EMBO J 20:3282-3291. Fasina OO, Dong Y, Pintel DJ. 2015. NP1 Protein of the Bocaparvovirus Minute Virus of Canines Controls Access to the Viral Capsid Genes via Its Role in RNA Processing. J Virol 90:1718-1728. Sukhu L, Fasina O, Burger L, Rai A, Qiu J, Pintel DJ. 2013. Characterization of the nonstructural proteins of the bocavirus minute virus of canines. J Virol 87:1098-1104. Binn LN, Lazar EC, Eddy GA, Kajima M. 1970. Recovery and characterization of a minute virus of canines. Infect Immun 1:503-508. Venkatesh LK, Fasina O, Pintel DJ. 2012. RNAse mapping and quantitation of RNA isoforms. Methods Mol Biol 883:121-129. Farris KD, Fasina O, Sukhu L, Li L, Pintel DJ. 2010. Adeno-associated virus small rep proteins are modified with at least two types of polyubiquitination. J Virol 84:1206-1211. Pintel D, Dadachanji D, Astell CR, Ward DC. 1983. The genome of minute virus of mice, an autonomous parvovirus, encodes two overlapping transcription units. Nucleic Acids Res 11:1019-1038. Cotmore SF, Sturzenbecker LJ, Tattersall P. 1983. The autonomous parvovirus MVM encodes two nonstructural proteins in addition to its capsid polypeptides. Virology 129:333-343. Jongeneel CV, Sahli R, McMaster GK, Hirt B. 1986. A precise map of splice junctions in the mRNAs of minute virus of mice, an autonomous parvovirus. J Virol 59:564-573. 17

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459

27. 28. 29. 30. 31.

32. 33. 34. 35. 36. 37.

38. 39. 40. 41. 42.

Qiu J, Yoto Y, Tullis G, Pintel DJ. 2006. RNA processing strategies pp 253-270, Parvoviruses. Edited by Jonathan Kerr, Susan Cotmore, Marshall Bloom, Michael Linden and Colin Parrish, 1st ed. Hodder Arnold London, Great Britain. Naeger LK, Cater J, Pintel DJ. 1990. The small nonstructural protein (NS2) of the parvovirus minute virus of mice is required for efficient DNA replication and infectious virus production in a cell-type-specific manner. J Virol 64:6166-6175. Naeger LK, Salome N, Pintel DJ. 1993. NS2 is required for efficient translation of viral mRNA in minute virus of mice-infected murine cells. J Virol 67:1034-1043. Ruiz Z, D'Abramo A, Jr., Tattersall P. 2006. Differential roles for the C-terminal hexapeptide domains of NS2 splice variants during MVM infection of murine cells. Virology 349:382-395. Brownstein DG, Smith AL, Johnson EA, Pintel DJ, Naeger LK, Tattersall P. 1992. The pathogenesis of infection with minute virus of mice depends on expression of the small nonstructural protein NS2 and on the genotype of the allotropic determinants VP1 and VP2. J Virol 66:3118-3124. Mihaylov IS, Cotmore SF, Tattersall P. 2014. Complementation for an essential ancillary non-structural protein function across parvovirus genera. Virology 468-470:226237. Nuesch JP, Corbau R, Tattersall P, Rommelaere J. 1998. Biochemical activities of minute virus of mice nonstructural protein NS1 are modulated In vitro by the phosphorylation state of the polypeptide. J Virol 72:8002-8012. Nuesch JP, Dettwiler S, Corbau R, Rommelaere J. 1998. Replicative functions of minute virus of mice NS1 protein are regulated in vitro by phosphorylation through protein kinase C. J Virol 72:9966-9977. Corbau R, Duverger V, Rommelaere J, Nuesch JP. 2000. Regulation of MVM NS1 by protein kinase C: impact of mutagenesis at consensus phosphorylation sites on replicative functions and cytopathic effects. Virology 278:151-167. Nuesch JP, Christensen J, Rommelaere J. 2001. Initiation of minute virus of mice DNA replication is regulated at the level of origin unwinding by atypical protein kinase C phosphorylation of NS1. J Virol 75:5730-5739. Li J, Bonifati S, Hristov G, Marttila T, Valmary-Degano S, Stanzel S, Schnolzer M, Mougin C, Aprahamian M, Grekova SP, Raykov Z, Rommelaere J, Marchini A. 2013. Synergistic combination of valproic acid and oncolytic parvovirus H-1PV as a potential therapy against cervical and pancreatic carcinomas. EMBO Mol Med 5:15371555. Chandler M, de la Cruz F, Dyda F, Hickman AB, Moncalian G, Ton-Hoang B. 2013. Breaking and joining single-stranded DNA: the HUH endonuclease superfamily. Nat Rev Microbiol 11:525-538. Tewary SK, Zhao H, Shen W, Qiu J, Tang L. 2013. Structure of the NS1 protein Nterminal origin recognition/nickase domain from the emerging human bocavirus. J Virol 87:11487-11493. Musayev FN, Zarate-Perez F, Bishop C, Burgner JW, 2nd, Escalante CR. 2015. Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1. J Biol Chem 290:27487-27499. James JA, Aggarwal AK, Linden RM, Escalante CR. 2004. Structure of adenoassociated virus type 2 Rep40-ADP complex: insight into nucleotide recognition and catalysis by superfamily 3 helicases. Proc Natl Acad Sci U S A 101:12455-12460. Hickman AB, Dyda F. 2005. Binding and unwinding: SF3 viral helicases. Curr Opin Struct Biol 15:77-85.

18

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509

43.

44. 45. 46.

47. 48. 49. 50. 51.

Lou DI, Kim ET, Meyerson NR, Pancholi NJ, Mohni KN, Enard D, Petrov DA, Weller SK, Weitzman MD, Sawyer SL. 2016. An Intrinsically Disordered Region of the DNA Repair Protein Nbs1 Is a Species-Specific Barrier to Herpes Simplex Virus 1 in Primates. Cell Host Microbe 20:178-188. Schoborg RV, Pintel DJ. 1991. Accumulation of MVM gene products is differentially regulated by transcription initiation, RNA processing and protein stability. Virology 181:22-34. Qiu J, Pintel DJ. 2002. The adeno-associated virus type 2 Rep protein regulates RNA processing via interaction with the transcription template. Mol Cell Biol 22:3639-3652. Qiu J, Nayak R, Pintel DJ. 2004. Alternative polyadenylation of adeno-associated virus type 5 RNA within an internal intron is governed by both a downstream element within the intron 3' splice acceptor and an element upstream of the P41 initiation site. J Virol 78:83-93. Zhao Q, Schoborg RV, Pintel DJ. 1994. Alternative splicing of pre-mRNAs encoding the nonstructural proteins of minute virus of mice is facilitated by sequences within the downstream intron. J Virol 68:2849-2859. Li L, Qiu J, Pintel DJ. 2009. The choice of translation initiation site of the rep proteins from goose parvovirus P9-generated mRNA is governed by splicing and the nature of the excised intron. J Virol 83:10264-10268. Liu Z, Qiu J, Cheng F, Chu Y, Yoto Y, O'Sullivan MG, Brown KE, Pintel DJ. 2004. Comparison of the transcription profile of simian parvovirus with that of the human erythrovirus B19 reveals a number of unique features. J Virol 78:12929-12939. Qiu J, Cheng F, Pintel D. 2007. Distance-dependent processing of adeno-associated virus type 5 RNA is controlled by 5' exon definition. J Virol 81:7974-7984. Qiu J, Cheng F, Yoto Y, Zadori Z, Pintel D. 2005. The expression strategy of goose parvovirus exhibits features of both the Dependovirus and Parvovirus genera. J Virol 79:11035-11044.

537

538

Figure 1. MVC encodes multiple NS isoforms during infection of WRD cells and

539

transfection of 293T cells. (A) (Left panel) Immunoblots, using antibodies directed against the

540

NS ORF (epitope depicted as a star) or NP1 (epitope depicted as a circle), of cell lysates of

541

either mock infected, (M, lane 1), or WRD cells infected with MVC at a moi of 7 harvested 48hrs

542

post-infection (lane 2). (Right panel) Immunoblots, using antibodies directed against the NS

543

ORF (star), NP1 (circle), or tubulin, of 293T cell lysates harvested 48 hrs post-transfection with

544

either pIMVC WT (lane 4), pIMVC 427 TAA (terminating the NS ORF as described, lane 5), or

545

mock-transfected (lane 3). The bands correlating to NS isoforms (NS-100, NS-84, NS-66 and

546

NS-50), and NP1 are indicated on the right, and molecular weight (MW) markers in kilodaltons

547

(kDa) are depicted on the left. (B) Reverse-transcriptase PCR of total 293T cell RNA extracted

19

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536

48hrs post-transfection with pIMVC WT, primed with oligo-deoxythymidine (Oligo-dT) and

549

reverse transcribed with (lanes 1-3, each lane represents an individual complete experiment),

550

and without (lane 4) reverse transcriptase (RT). Amplicons (1683bp, 1224bp, 678bp, and

551

464bp) generated with MVC NS ORF-specific forward (nt 868) and NP1 ORF-specific reverse

552

(nt 3097) primers, depicted with horizontal arrows in the transcription profile schematic in panel

553

C, are shown on the right side of the panel. The size markers (M), in kilobases (Kb), are

554

depicted on the left. (C) The transcription profile of MVC showing the alternative RNA

555

processing events that generate MVC nonstructural protein (NS isoforms and NP1)-encoding

556

transcripts (R1-R6) and structural protein (VP1 and VP2)-encoding transcripts (R7 and R8). The

557

NS and NP1-encoding transcripts are illustrated as alternatively polyadenylated transcripts

558

using the proximal (pA)p (R1-R6s), or distal (pA)d (R1-R6l) polyadenylation sites. The P6

559

promoter, splice donors (D) and acceptors (A), and the proximal (pA)p and distal (pA)d

560

polyadenylation sites are shown, along with relevant nucleotide landmarks within the MVC

561

genome (GenBank accession number FJ2141101.1). The position of the NS1 termination

562

mutant (pIMVC 427 TAA) is shown with an “X”, while the position of the 5’ (nt 868) and 3’ (nt

563

3097) primers used to the RT-PCR analysis in panel B are depicted with the horizontal arrows.

564

The NS ORF (nt 403-2727) and NP1 ORF (nt 2537-3097) are indicated. The epitopes detected

565

by the antibodies used to detect the NS proteins (NS ORF amino acids 687-700:

566

PKKQRKTEHKVLID) and NP1 (NP1 amino acids 1-13: MSTRHMSKRSKARSR), are indicated

567

by the star and closed circle, respectively. The triangle indicates hemagglutinin (HA) epitope at

568

NP1 ORF carboxyl termini in pIMVC 3097HA WT and pIMVC 3097HA NP1m constructs used

569

for the experiments in Fig. 3. The predicted (pred. MW) and observed (obs. MW) molecular

570

weights of the NS isoforms (NS-100, NS-84, NS-50 and NS-40), NP1 proteins, and capsid

571

proteins (VP1 and VP2) are indicated with the corresponding number of amino acid residues

572

(774aa, 715aa, 439aa, 382aa, 309aa,186aa, 703aa and 571aa) encoded by their mRNAs.

20

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

548

573 574

Figure 2. MVC NS-84, NS-50 and NS-40 mRNAs are alternatively spliced using the 3A acceptor into the NP1 ORF. (A) Lysates of 293T cells taken 48 hrs following transfection of

576

pIMVC WT (lane 2), pIMVC 1Am (lane 3), R2 cDNA (lane 4), R3 cDNA (lane 5), R4 cDNA (lane

577

6) and R5 cDNA (lane 7) were analyzed by immunoblotting with antibodies directed against NS

578

(depicted as the star in Fig. 1C) and tubulin. Molecular weight markers (MW) are indicated on

579

the left. (B) Lysates of 293T cells taken 48 hours following transfection with pIMVC WT (lane 2)

580

or CMV-3XF constructs expressing R3 cDNA (lanes 3 and 6), R4 cDNA (lanes 4 and 7) and R5

581

cDNA (lanes 5 and 8) isoforms, were analyzed by immunoblot using either anti-NS antibody

582

(epitope depicted by star in Fig. 1C (lanes 1-5), or anti-FLAG (lanes 6-8). The size markers are

583

shown as described above. (C) Schematic of wild-type MVC nts 2524 to 2574 and pIMVC

584

NSNP1fus mutant. The “T” nucleotide (nt 2536) deleted in NS1 ORF to generate the NSNP1fus

585

mutant fusing the NS and NP1 ORFs is italicized and bolded. Reading frames are indicated by

586

lines above and below the sequences. (D) Lysates of 293T cells taken 48hrs post-transfection

587

with pIMVC WT (lane 2) and pIMVC NS1NP1fus mutant (lane 3), were immunoblotted using

588

antibodies to the NS ORF (depicted by the star in Fig. 1C). Size markers and the bands

589

correlating to each NS1 isoforms are indicated on the left and right side of the panel,

590

respectively.

591

Figure 3. MVC NP1 is required for the expression of NS-84, NS-50 and NS-40. (A)

592

RNAse protection of 20 µg of RNA extracted from 293T cells 48 hrs following transfection with

593

pIMVC WT (lane 2), pIMVC NP1m (lane 3), pIMVC 3097HA WT (lane 5), and pIMVC NP1m

594

3097HA (lane 6), using the 2A\3D probe that spans nts 2344-2550 (shown in Fig. 1C). The size

595

of the probe (243nt) and protected RNAs (206nt, 164nt and 105nt) are shown on the left. Bands

596

reflecting RNA species unspliced through this region (RT), spliced at the second intron acceptor

597

but not at the third intron donor (2Aspl/3DUnspl), and RNAs spliced at the second intron 21

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

575

acceptor and also the third intron donor (2Aspl/3Dspl), are indicated on the right. (B) Sample

599

taken from 293T cells transfected with pIMVC 3097HA WT (lanes 2 and 4), and pIMVC NP1m

600

3097HA (lanes 3 and 6) were subjected to immunoblot analysis using antibody directed against

601

HA (lanes 1-3), or antibody directed against NS (epitope depicted as a star in Fig. 1C, lanes 4-

602

6). Tubulin was monitored as a loading control. The bands that correspond to each NS isoform

603

are indicated on the right and the molecular weight (MW) size markers are shown on the left.

604

Figure 4. MVC NS isoforms exhibit differential localization and distinct

605

morphology in transiently transfected WRD cells. (A) WRD cells were transfected with

606

pIMVC WT, or pIMVC constructs expressing cDNAs individually encoding each NS1 isoform

607

(pIMVC 1Am-NS-100, pIMVC R2 1AmΔ3DA-NS-84, pIMVC R3-NS-66, and pIMVC R4- NS-50)

608

as indicated to the left, and stained 48 hrs later with anti-NS antibody (epitope depicted as a star

609

in Fig. 1C). Nuclei were visualized with DAPI staining. Representative images are shown. (B)

610

Immunofluorescence of WRD cells 48 hrs post-transfection with CMV-3XF pIMVC R5 encoding

611

NS-40 isoform and CMV-3XF pIMVC R3 encoding the NS-66 isoform, using anti- FLAG

612

antibodies. Nuclei were counter-stained with DAPI. Representative images are shown.

613

Figure 5. MVC NS-84, NS-50 and NS-40 are required for viral genome replication in

614

a host-cell dependent manner. (A) Schematic representation of MVC transcription profile as

615

described in Fig.1C. Locations of the third intron donor (3D) and acceptor (3A) mutations in

616

pIMVC 3DAm are designated by an “X” which are also underlined for clarity. (B) Lysates taken

617

from the 293T cells 48 hr following transfection with pIMVC WT (lane 2) and pIMVC 3DAm (lane

618

3) were subjected to immunobloting using antibodies directed against NS (epitope designated

619

by the star in Fig. 1C) and tubulin. The MVC NS isoforms are shown on the right and the

620

molecular weight markers are shown on the left. (C & D) Southern Blots of total DNA extracts

621

from canine WRD (C) or MDCK (D) cells taken 72 hrs post-transfection with pIMVC WT (lane 2

622

and 5), pIMVC 3DAm (lane 3 and 6), or infected with MVC virus (VI) at moi of 7 (lanes 1 and 4) 22

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

598

transferred from 1% agarose gels. DNA shown in lanes 4-6 were treated with Dpn I to

624

differentiate transfected input plasmid from monomer (mRF) and dimer (dRF) replicative

625

intermediates, designated on the left side of the panel. (E) Southern Blots of total DNA extracts

626

from human 293T cells taken 48 hr post transfection of pIMVC WT (lanes 3 and 5) or pIMVC

627

3DAm (lanes 4 and 6). Lysates from MVC infected WRD cells (lane 1) and untransfected 293T

628

cells (lanes 2 and &) were included as controls. Parallel DNA samples were treated with Dpn I

629

(lanes 5-7) to differentiate transfected input plasmid from monomer (mRF) and dimer (dRF)

630

replicative intermediates shown on the left side of the panel.

23

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

623

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND

Downloaded from http://jvi.asm.org/ on April 21, 2017 by UNIVERSITY OF NEW ENGLAND