1

Functional dissection of an alternatively spliced herpesvirus gene by splice site 1

mutagenesis 2

3

Tim Schommartza, Stefan Lorochb, Malik Alawia,c, Adam Grundhoffa, Albert Sickmannb, 4

Wolfram Brunea,§ 5

6

a Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany 7

b Leibniz-Institut für Analytische Wissenschaften–ISAS, Dortmund, Germany 8

c Bioinformatics Core, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 9

10

§ Address correspondence to: wolfram.brune@hpi.uni-hamburg.de 11

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Running Head: Splice site mutagenesis of M112/113 13

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Word count: 250 (abstract), 5961 (text) 15

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JVI Accepted Manuscript Posted Online 24 February 2016J. Virol. doi:10.1128/JVI.02987-15Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Abstract 17

Herpesviruses have large and complex DNA genomes. The largest among the herpesviruses, the 18

cytomegaloviruses, encode over 170 genes. Although most herpesvirus gene products are 19

expressed from unspliced transcripts, a substantial number of viral transcripts are spliced. Some 20

viral transcripts are subject to alternative splicing, which leads to the expression of several 21

proteins from a single gene. Functional analysis of individual proteins derived from an 22

alternatively spliced gene is difficult as deletion and nonsense mutagenesis, both common 23

methods used in the generation of viral gene knockout mutants, affect several or all gene products 24

at the same time. Here we show that individual gene products of an alternatively spliced 25

herpesvirus gene can be inactivated selectively by mutagenesis of the splice donor or acceptor 26

site and by intron deletion or substitution mutagenesis. We used this strategy to dissect the 27

essential M112/113 gene of murine cytomegalovirus (MCMV), which encodes the MCMV Early 28

1 (E1) proteins. The expression of each of the four E1 protein isoforms was inactivated 29

individually, and the requirement of each isoform for MCMV replication was analyzed in 30

fibroblasts, endothelial cells, and macrophages. We show that the E1 p87, but not the p33, p36, 31

and p38 isoforms, are essential for viral replication in cell culture. Moreover, the presence of one 32

of the two medium-sized isoforms (p36 or p38) and the presence of intron 1, but not its specific 33

sequence, are required for viral replication. This study demonstrates the usefulness of splice site 34

mutagenesis for the functional analysis of alternatively spliced herpesvirus genes. 35

Importance 36

Herpesviruses encode up to 170 genes on their DNA genomes. The functions of most viral gene 37

products remain poorly defined. The construction of viral gene knockout mutants has thus been 38

an important tool for functional analysis of viral proteins. However, this strategy is of limited use 39

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when viral gene transcripts are alternatively spliced leading to the expression of several proteins 40

from a single gene. In this study we showed, as a proof of principle, that each protein product of 41

an alternatively spliced gene can be eliminated individually by splice site mutagenesis. Mutant 42

viruses lacking individual protein products displayed different phenotypes, demonstrating that the 43

products of alternatively spliced genes have non-redundant functions. 44

45

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Introduction 46

Herpesviruses are large DNA viruses that replicate their genomes and transcribe their genes 47

within the host cell nucleus. Although more than 90% of cellular transcripts consist of exons and 48

introns and require splicing to become mature protein-coding mRNAs (1, 2), the majority of 49

herpesvirus proteins are translated from unspliced transcripts. As unspliced transcripts are 50

exported with lower efficiency from the nucleus to the cytosol, herpesviruses therefore express 51

mRNA export factors that increase nuclear export and translation of unspliced viral transcripts 52

(3). Nevertheless, a substantial number of herpesviral proteins are expressed from spliced 53

transcripts. Interestingly, many of these are transcribed during latency or at the beginning of the 54

lytic cycle. 55

Human cytomegalovirus (HCMV) is an opportunistic pathogen that can cause severe 56

disease in immunocompromised individuals (4). HCMV is the archetype of the β-herpesvirinae 57

and has the largest genome of all human herpesviruses, comprising more than 170 protein coding 58

open reading frames (5). It has been known for a long time that HCMV expresses several proteins 59

from spliced transcripts. In fact, a more recent analysis of the HCMV transcriptome by deep 60

sequencing identified a surprisingly large number of splice junctions that were predicted to affect 61

58 genes, indicating that the splicing of viral transcripts is more common than previously 62

recognized (6). The number and diversity of viral proteins is further extended by alternative 63

splicing, which allows a single gene to code for several protein products. Two prominent 64

examples for alternatively spliced genes are the HCMV UL122/123 locus encoding the major 65

immediate-early (MIE) proteins and the UL112/113 gene encoding the Early 1 (E1) proteins. The 66

MIE and E1 proteins are conserved in cytomegaloviruses of animals and have homologs in two 67

other human β-herpesviruses, namely human herpesviruses 6 and 7. 68

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While the MIE protein IE1 and IE2 (a.k.a. IE-72 and IE-86) have been studied extensively 69

(reviewed in (7)), relatively little is known about the functions of the UL112/113 proteins or their 70

homologs in other β-herpesviruses. The HCMV UL112/113 and murine cytomegalovirus 71

(MCMV) M112/M113 genes are found at homologous positions within the HCMV and MCMV 72

genomes respectively, and share a similar splicing pattern (8-10). Alternative splicing accounts 73

for the expression of four protein products with apparent molecular masses of 34, 43, 50, and 84 74

kDa in the case of HCMV (8) and 33, 36, 38, and 87 kDa in the case of MCMV (9, 10). 75

Posttranslational modifications such as phosphorylation are thought to be responsible for 76

differences between the predicted and the apparent molecular masses of these proteins (9, 11, 12). 77

The E1 proteins are expressed shortly after the MIE proteins (9, 13), suggesting that very little IE 78

protein is necessary to activate their promoter. Together with the MIE proteins, the E1 proteins 79

can enhance the expression of other viral genes in transient transfection assays (14, 15). The E1 80

proteins may additionally have a role in viral DNA replication as they can bind DNA and 81

accumulate in viral replication compartments within the host cell nucleus as well as interact with 82

the viral DNA polymerase processivity factor (13, 16-18). Moreover, the UL112/113 gene is 83

necessary for the transient complementation of HCMV oriLyt-dependent DNA replication (19), 84

and antisense RNA to UL112/113 transcripts inhibits viral DNA replication (20). The large E1 85

p87 isoform of MCMV was shown to interact with the major transactivator protein IE3 and 86

relieve its repressive effect on its own promoter (21). Moreover, point mutations affecting the E1 87

p87 isoform facilitate MCMV replication in human cells, which are usually nonpermissive for 88

MCMV replication (22). 89

The different E1 protein isoforms are thought to have non-redundant functions, but their 90

specific functions and their importance for the viral replication cycle have remained largely 91

undefined. Due to the complex splicing pattern, it is impossible to inactivate E1 protein isoforms 92

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individually by deleting the coding sequence or introducing nonsense mutations as it has been 93

done routinely for other viral genes. In this study we use a combination of splice site 94

mutagenesis, intron deletion and substitution, and nonsense mutagenesis for a detailed mutational 95

analysis of the alternatively spliced MCMV M112/113 locus. The expression of each of the four 96

E1 protein isoforms was inactivated individually and the requirement of each isoform for MCMV 97

replication was analyzed. We show here that the E1 p87, but not the p33, p36, and p38 isoforms, 98

are essential for viral replication in cell culture. In addition, the presence of one of the two 99

medium-sized isoforms (p36 or p38) and the presence of intron 1, but not its specific sequence, 100

are necessary for viral replication. To our knowledge, this is the first comprehensive mutational 101

analysis of a complex alternatively spliced herpesvirus gene. 102

103

Material and Methods 104

Cells and viruses. NIH-3T3 fibroblasts (CRL-1658), SVEC4-10 endothelial cells (CRL-1658) 105

and RAW 264.7 macrophages (TIB-71) were obtained from the ATCC. The GFP-expressing 106

MCMV Smith strain (MCMV-GFP) and the MCMV K181 strain have been described (23, 24). 107

MCMV was propagated on NIH-3T3 cells and titrated by the median tissue culture infective dose 108

(TCID50) method following standard protocols (25). Infections were done with centrifugal 109

enhancement (1,000 × g, 30 min) unless stated otherwise. For replication kinetics, cells in six-110

well dishes were infected without centrifugal enhancement. After 4 hours, cells were washed 111

twice with PBS and new medium was added. Supernatants were harvested at different times 112

postinfection for virus titration. All replication kinetics experiments were done in triplicates. 113

RNA isolation and sequencing. Total RNA was isolated from NIH-3T3 cells infected with 114

MCMV at a multiplicity of infection (MOI) of 5 TCID50/cell using an innuPREP RNA Mini Kit 115

(Analytic Jena). After DNase treatment (Turbo DNA-free Kit, Ambion) total RNA was subjected 116

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to RNA sequencing. mRNA was extracted using the NEBNext Poly(A) mRNA Magnetic 117

Isolation module (New England Biolabs) and RNA-Seq libraries were generated using the 118

NEXTflex Directional RNA-Seq Kit (dUTP based) for Illumina (Bioo Scientific) according to 119

the manufacturer´s instructions. Proper fragment size distribution and quality of the libraries were 120

verified on a BioAnalyzer High Sensitivity Chip (Agilent). Diluted libraries were multiplex-121

sequenced on the Illumina HiSeq 2500 instrument (2 × 125 bp run) with 61 and 70 million reads 122

per sample, respectively. 123

For PCR-based splicing analysis, cDNA was synthesized from total RNA using RevertAid H 124

Minus Reverse Transcriptase, oligo[dT]18, and RNase inhibitor (Thermo Fisher Scientific). 125

M112/113 fragments were PCR-amplified from cDNA or BAC DNA, separated by agarose gel 126

electrophoresis, and visualized by ethidium bromide staining. 127

RNA-Seq data analysis. In silico splice site prediction was performed using Hbond scoring 128

(http://www.uni-duesseldorf.de/rna/html/hbond_score.php). For the analysis of the RNA-Seq data 129

Trimmomatic (26) was employed to remove adapters and low quality (phred quality score < 15) 130

bases from the 3' ends of sequencing reads. A split read alignment was performed by aligning the 131

trimmed reads to the MCMV Smith NCBI reference sequence (NC_004065.1) or the MCMV 132

K181 Genbank entry (AM886412.1) using the software segemehl (27). The positions of splice 133

junctions were obtained from the alignments following the procedure described in segemehl's 134

manual. All genomic positions refer to the MCMV Smith or MCMV K181 reference sequence 135

(GenBank accession numbers NC_004065 and AM886412, respectively). Splice junctions are 136

depicted with the last position of the upstream exon and the first position of the downstream exon 137

separated by a circumflex (^). 138

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Plasmids. The M112/113 coding sequence was PCR amplified from the MCMV Smith genome 139

using primers containing BglII and HpaI restriction sites, respectively. The PCR product was 140

cleaved with BglII and HpaI and inserted into expression plasmid pcDNA3 (Invitrogen) digested 141

with BamHI and EcoRV. Point mutations were introduced following the GeneArt Site-Directed 142

Mutagenesis protocol (Thermo Fisher Scientific). Deletion of the first intron was performed by 143

PCR-driven overlap extension (28). Transfection of NIH-3T3 cells with expression plasmids was 144

done using Polyfect (Qiagen). 145

Virus mutagenesis and sequencing. Mutations within the M112/113 locus were introduced by 146

en passant mutagenesis (29) into the MCMV-GFP bacterial artificial chromosome (BAC), which 147

was used as parental wildtype (wt) genome. Revertants were constructed using the same method. 148

For construction of the MCMV SynI1 mutant, a sequence containing the synthetic intron of the 149

pCI-Neo (Promega) expression vector was ordered from GeneArt Gene Synthesis (Thermo Fisher 150

Scientific). The synthetic intron was inserted by en passant mutagenesis into the MCMV Δp33 151

BAC at the position of the deleted intron 1. For the construction of a mutant expressing p33 form 152

an ectopic location, the p33 ORF was PCR amplified and inserted between the BamHI and NheI 153

sites of pReplacer (30). Insertion of a kanamycin resistance marker and the p33 ORF driven by a 154

phosphoglycerate kinase (PGK) promoter into the m02-m06 locus was done as essentially 155

described (30). To reconstitute infectious virus from MCMV BACs, purified BAC DNA was 156

transfected in NIH-3T3 cells using Polyfect (Qiagen). To analyze the M112/113 sequence, the 157

M112/113 locus was PCR-amplified either from BAC or viral DNA. PCR products were either 158

sequenced directly by DyeDeoxy sequencing (GATC Biotech) or first cloned in pCR4-TOPO 159

using a TOPO TA Cloning Kit (Thermo Fisher Scientific) and then sequenced. 160

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Antibodies and immunoblotting. A polyclonal rabbit antiserum against MCMV E1 (10) was 161

provided by Julie A. Kerry (Eastern Virginia Medical School, Norfolk, VA). Mouse monoclonal 162

antibodies against the MCMV IE1 (CROMA101) and M44 (3B9.22A) were provided by Stipan 163

Jonjic (University of Rijeka, Rijeka, Croatia) and Lambert Loh (University of Saskatchewan, 164

Saskatoon, Canada), respectively. Antibodies recognizing MCMV gB (M55.01) and β-actin (AC-165

74) were purchased from CapRi.and Sigma, respectively. For immunoblot analysis, cells were 166

infected at an MOI of 5 and cell lysates were harvested at the indicated time points. Protein 167

samples were separated by SDS-PAGE and transferred onto positively charged nitrocellulose 168

membranes (Amersham). Proteins of interest were detected by using protein-specific primary 169

antibodies and horseradish peroxidase-coupled secondary antibodies (Dako Cytomation) via 170

enhanced chemiluminescence. 171

172

Results 173

RNA-Seq analysis of the MCMV M112/113 region. 174

Previous studies have shown that the M112/113 region encodes 4 proteins derived from 175

differentially spliced transcripts (9, 10). We wanted to verify the previously predicted splicing 176

pattern (Fig. 1A) and determine the relative abundance of the individual M112/113 transcripts in 177

MCMV-infected NIH-3T3 fibroblasts by whole transcriptome shotgun sequencing (also called 178

RNA-Seq) and identification of splice junctions using Hbond score (31). The identified splice 179

junctions largely confirmed the previously predicted splicing pattern with two notable exceptions: 180

we could not detect the predicted splice acceptor site A3 (Fig. 1A) but identified instead a new 181

splice donor site D3 at nucleotide position 164084 (Fig. 1B) resulting in a revised splicing pattern 182

of the M112/113 region in the MCMV Smith strain (Fig. 1C). The same splice junctions were 183

identified by RNA-Seq of NIH-3T3 cells infected with the MCMV K181 strain (Fig. 1B), 184

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indicating that the revised splicing pattern is not unique to the MCMV Smith strain. The relative 185

abundance of spliced and unspliced transcripts was analyzed at different times post infection. The 186

ratio of spliced to unspliced transcripts for those spanning intron 1 did not change significantly 187

over the course of the infection (Fig. 1D). The relative abundance of unspliced transcripts 188

retaining intron 2 also remained largely unchanged, however the relative abundance of D2^A2 189

and D3^A2 spliced transcripts changed over time (Fig. 1E). The percentage of unspliced 190

transcripts was very low (less than 3%) for both introns. Other splice junctions using different 191

donor and/or acceptor sites or other combinations of the described donor and acceptor sites have 192

also been detected. Most of these splice junctions were not conserved between MCMV Smith and 193

K181 and were detected in only few sequence reads. Hence these low-abundant splices were not 194

further considered for this study. 195

196

Verification of M112/113 splicing pattern by plasmid mutagenesis. 197

In order to verify the splicing pattern predicted by the RNA-Seq data we cloned the M112/113 198

coding region in the plasmid expression vector pcDNA3 and subsequently introduced specific 199

mutations in order to prevent the expression of individual E1 protein isoforms (Fig. 2A). Intron 1 200

was removed in the Δp33 mutant, leading to a fusion of exons 1 and 2, while in the Δp36 mutant 201

the splice donor D3 was mutated by two nucleotide exchanges that do not affect the amino acid 202

sequence encoded by the unspliced transcript. In the same manner, splice donor D2 was mutated 203

to generate the Δp38 mutant. We also introduced a single nucleotide mutation into the splice 204

acceptor A2 in order to eliminate expression of both medium-sized isoforms (mutant Δp36Δp38). 205

Unfortunately, there was no way to completely eliminate the large p87 isoform without affecting 206

other isoforms as well. Therefore, two stop codons were introduced shortly after splice donor D2, 207

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which should lead to a mutation containing a truncated p87 protein lacking the last 292 amino 208

acids. 209

To determine whether the mutations lead to a loss of individual E1 protein isoforms, the 210

mutated plasmids were transfected into NIH-3T3 cells and their cell lysates analyzed by 211

immunoblot using an E1-specific antiserum recognizing all four E1 isoforms (10). As expected, 212

cells transfected with the M112/113 Δp33, Δp36, and Δp87 plasmids expressed all E1 protein 213

isoforms except for those intentionally deleted (Fig. 2B). NIH-3T3 cells transfected with the 214

M112/113 Δp38 or Δp36Δp38 neither expressed the deleted isoforms nor the p33 isoform (Fig. 215

2B). However, a weak expression was detected by immunoblot in transfected 293A cells (not 216

shown), suggesting that p33 expression from these two plasmids was weak but not absent. It is 217

also noteworthy that a truncated p87 protein was not observed in cells transfected with the Δp87 218

plasmid (Fig. 2B). However, since the predicted size of the truncated p87 protein is similar to that 219

of the p38 isoform, it is possible that a band representing the truncated p87 protein is hidden 220

within the bands representing the p38 isoform. In summary, the results of the M112/113 plasmid 221

mutagenesis confirm the splicing pattern prediction based on the RNA-Seq data. 222

223

Essential and nonessential roles of individual E1 isoforms for MCMV replication. 224

To investigate whether the loss of E1 isoforms influences the ability of MCMV to replicate in 225

cell culture MCMV mutants lacking individual E1 isoforms were constructed. The same 226

mutations as described for the M112/113 plasmids (Fig. 2A) were introduced into a BAC clone 227

of MCMV-GFP, i.e. wt MCMV (Smith strain) expressing GFP. The MCMV-GFP backbone was 228

chosen for easier detection of poorly replicating virus mutants after BAC transfection. In addition 229

an E1 stop mutant was constructed in which codons 6, 8 and 9 of M112 were changed to stop 230

codons. Mutant BACs were analyzed by restriction digestion and gel electrophoresis and by 231

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sequencing of the M112/113 region. The mutant MCMV BACs were then transfected into NIH-232

3T3 fibroblasts to reconstitute mutant viruses. 233

In spite of numerous attempts, the E1 stop mutant could not be reconstituted. However, 234

the revertant virus, in which the stop mutations had been changed back to the original sequence, 235

replicated like the wt virus (data not shown). This finding is consistent with a previous 236

publication reporting that an MCMV M112/113 deletion mutant was unable to replicate in 237

cultured fibroblasts (22) and suggests that the E1 proteins are essential for MCMV replication. 238

However, the possibility that individual E1 isoforms are nonessential remained to be investigated. 239

We could not detect viral replication and spread upon transfection of BAC DNA of the 240

MCMV Δp87 mutant into NIH-3T3 cells. However, the corresponding revertant virus (Rev p87) 241

replicated with wt kinetics (Fig. 3A) suggesting an essential role of the p87 isoform for MCMV 242

replication. 243

The MCMV Δp36 and Δp38 mutants were easily reconstituted and replicated well in 244

NIH-3T3 fibroblasts. While the MCMV Δp36 replicated with wt kinetics in NIH-3T3 cells (Fig. 245

3B), the Δp38 mutant replicated to titers 10 to 100-fold lower compared to the parental virus. The 246

corresponding revertant (Rev p38) replicated with wt kinetics (Fig. 3C), indicating that the 247

replication defect of the Δp38 mutant was the result of the splice donor mutation rather than an 248

accidental mutation elsewhere in the viral genome. Immunoblot analysis of cells infected with the 249

Δp36 and Δp38 mutants showed the absence of the deleted E1 isoforms while the remaining 250

isoforms were expressed at similar levels as in cells infected with wt MCMV (Fig. 3D). To 251

further investigate the phenotype of the Δp38 mutant, we analyzed viral protein expression up to 252

48 hrs after low-MOI infection. In fibroblasts infected with the Δp38 mutant, expression of the 253

viral IE1, E1, and M44 proteins was similar to the one observed wt or Rev m38-infected cells, 254

but expression of the late protein gB was delayed (Fig. 3E). Since late gene expression occurs 255

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only after viral DNA replication, the delayed gB expression indicates impaired viral DNA 256

replication and/or reduced late gene expression. 257

As the E1 p36 and p38 isoforms were not essential for MCMV replication, we wondered 258

whether a virus lacking both of these isoforms could replicate. As shown in the plasmid 259

mutagenesis experiment mutation of the A2 splice acceptor site (Fig. 2) resulted in a loss of the 260

p36 and p38 isoforms. When a mutant MCMV BAC carrying the same mutation (Δp36Δp38) 261

was transfected into NIH-3T3 cells, replicating virus could not be recovered in several attempts. 262

By contrast, a revertant (Rev p36p38) replicated with the same kinetics as the parental wt virus 263

(Fig. 4). Only on one occasion a single plaque was observed after transfection of the MCMV 264

Δp36Δp38 BAC, suggesting that a compensatory mutation might have occurred in this one 265

instance. The absence of both p36 and p38 isoforms was confirmed following analysis of lysates 266

of infected cells by immunoblot. Interestingly, the p33 and p87 isoforms were expressed at higher 267

levels than in cells infected with wt MCMV, and an unexpected additional protein with an 268

apparent molecular mass of approx. 60 kDa was detected by the E1-specific antiserum (Fig. 4B). 269

To investigate the origin of this additional protein, we sequenced the M112/113 gene of this 270

mutant (named Δp36Δp38mut). In addition to the splice acceptor mutation, a G-to-T mutation 271

was detected at position 164420. This mutation was apparently not present in all viral genomes as 272

the sequencing electropherogram showed two peaks (G and T) of equal amplitude at this position 273

(Fig. 4C). This result suggested a mixed infection with Δp36Δp38 and a mutant thereof carrying 274

a G-to-T mutation, which leads to a premature stop and the expression of a truncated p87 protein. 275

This hypothesis was confirmed when we PCR-amplified a part of the M112/113 gene, cloned the 276

PCR product in a plasmid vector, and sequenced 9 plasmid clones: 5 clones carried the wt 277

sequence and 4 carried the G-to-T mutation, indicating that the two mutant viruses were present 278

in similar amounts. Attempts to separate the two mutants by limiting dilution failed, suggesting 279

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that the two mutants complement each other and that neither of the two mutants can replicate on 280

its own. However, the mix of mutants (Δp36Δp38mut) replicated to slightly lower titers than the 281

wt virus (Fig. 4D). To rule out the possibility that a compensatory second-site mutation rather 282

than the G-to-T mutation within M112/113 was responsible for the replication of Δp36Δp38mut, 283

we introduced the G164420T mutation into Δp36Δp38 by BAC mutagenesis. This mutant, 284

Δp36Δp38/p60, should express only p33 and the truncated p60 protein, but not p36, p38, or p87. 285

The Δp36Δp38 and Δp36Δp38/p60 BACs were transfected into murine fibroblasts, separately 286

and in combination. As expected, neither the Δp36p38 nor the Δp36Δp38/p60 BAC alone was 287

able to produce replicating virus. However, co-transfection of both BACs resulted in plaque 288

formation (data not shown). This result confirmed that the G164420T mutation leading to a 289

truncated protein (p60) is sufficient to restore virus replication. 290

Taken together the results obtained with the MCMV Δp36, Δp38, and Δp36Δp38 mutants 291

indicate that the loss of one isoform, p36 or p38, but not both is compatible with MCMV 292

replication. However, a truncated p87 protein can compensate for the loss of both of these 293

isoforms. 294

295

The presence of the M112/113 intron 1 is necessary for MCMV replication. 296

Surprisingly, the MCMV Δp33 mutant (which lacks intron 1) could not be reconstituted. This 297

result was unexpected because the p33 protein is almost identical to the amino terminal part of 298

p36, p38, and p87 and contains only one lysine residue at its carboxy-terminus that is not present 299

in the other E1 isoforms. We therefore decided to investigate the role of the p33 isoform and 300

intron 1 in more detail. 301

First we tested whether the p33 protein was required for MCMV replication. To do this, 302

we inserted the E1 p33 ORF driven by a PGK promoter into the nonessential m02 to m06 region 303

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of the MCMV Δp33 BAC (Fig. 5A). This strategy of cis-complementation has previously been 304

used successfully for other MCMV mutants (32-34). However, transfection of the mutant BAC 305

did not result in virus reconstitution, suggesting that ectopic expression of p33 is not sufficient to 306

rescue the MCMV Δp33 mutant. 307

Next we tested whether the presence of M112/113 intron 1 is required for MCMV 308

replication. To do this, we inserted a synthetic intron derived from the commonly used plasmid 309

expression vector pCI-Neo (Promega) into the MCMV Δp33 BAC at the position of the authentic 310

intron 1 (Fig. 5B). The synthetic intron 1 (SynI1) is slightly larger than the authentic intron 1 and 311

has a completely different sequence. An unspliced transcript containing SynI1 would not code for 312

p33, but a larger protein with 34 additional amino acids and a predicted mass similar to p36 or 313

p38 (Fig. 5B). Strikingly, transfection of the MCMV SynI1 BAC resulted in the recovery of a 314

mutant virus that replicated with wt kinetics in NIH-3T3 fibroblasts. As predicted, the MCMV 315

SynI1 virus did not express p33 (Fig. 5C and D). However, we could not detect a new E1 protein 316

product encoded by exon 1 and intron 1, suggesting that such a protein product is either not 317

present or hidden within the band representing the p36 or the p38 isoform. To discern between 318

these two possibilities we prepared oligo-dT-primed cDNA from cells infected with either with 319

wt MCMV or the SynI1 mutant, both containing PCR amplified intron 1 as well as flanking 320

sequences with primers binding within exons 1 and 2. The same PCR reaction was done using the 321

wt MCMV-GFP BAC (containing the authentic intron 1), the MCMV SynI1 BAC (containing the 322

synthetic intron), and the MCMV Δp33 BAC (lacking intron 1) as templates. As shown in figure 323

5E, wt MCMV infected cells contained both spliced and unspliced transcripts. By contrast, only 324

spliced transcripts were detected in cells infected with the MCMV SynI1 mutant, indicating that 325

the synthetic intron is removed with much greater efficiency than the authentic intron 1. Hence 326

the predicted p33* protein resulting from a read-through into the synthetic intron (Fig. 5B) is 327

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probably made at very low levels or not at all. Taken together the results strongly suggest that the 328

presence of an intron 1 rather than the expression of the E1 p33 isoform is essential for MCMV 329

replication. 330

331

Replication of MCMV M112/113 mutants in different cell types. 332

In NIH-3T3 fibroblasts, the MCMV Δp38 mutant had a modest replication defect (Fig. 3C), but 333

the Δp36 mutant and the SynI1 mutant (that lacks p33) replicated with wt kinetics (Figs. 3B and 334

4C). These data suggested that p33 and p36 are completely dispensable for replication in this cell 335

type. To test the importance of the E1 p33, p36, and p38 isoforms in other cell types we analyzed 336

viral replication kinetics in two additional cell types permissive for MCMV replication. In 337

SVEC4-10 endothelial cells, the mutant MCMVs replicated with similar kinetics as in NIH-3T3 338

fibroblasts. The Δp36 and SynI1 mutants attained titers comparable to the parental wt virus, but 339

the Δp38 mutants had a modest replication defect (Fig. 6A). In RAW267.4 macrophages only 340

MCMV Δp36 replicated to wt titers (Fig. 6B) while MCMV Δp38 displayed a pronounced 341

replication defect. A similar replication defect was, surprisingly, also observed for the MCMV 342

SynI1 mutant, suggesting an important role for either the E1 p33 isoform or the authentic intron 1 343

for MCMV replication in macrophages. In lysates of infected SVEC4-10 and RAW267.4 cells 344

expression of the expected E1 protein isoforms was detected by immunoblot analysis (Fig. 6C 345

and D). Interestingly, an additional band was detected above the band representing the p33 346

isoform in SVEC4-10 endothelial cells (Fig. 6C). The presence of this band correlated with the 347

presence of the p33 isoform, suggesting that a posttranslational modification of p33 occurring in 348

SVEC4-10 but not in NIH-3T3 cells might be responsible. A similar protein band was detected 349

by the E1 antiserum in RAW267.4 macrophages. However, the same band was present in mock-350

infected macrophages, indicating that this band does not represent an MCMV protein. 351

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We concluded that the E1 p38 isoform is important for efficient replication in all cell 352

types tested, whereas the E1 p36 appears to be dispensable in these cell types - at least in vitro. 353

Although dispensable for MCMV replication in fibroblasts and endothelial cells the E1 p33 354

isoform or the authentic M112/113 intron 1 are required for efficient replication in RAW264.7 355

macrophages. 356

357

Discussion 358

Compared to most other viruses, herpesviruses have very large and complex genomes, upon 359

which they encode a large number of viral proteins. The complexity is further increased by the 360

fact that a subset of viral transcripts is subject to splicing or even alternative splicing. Recent viral 361

transcriptome analyses by RNA-Seq have demonstrated that splicing of herpesviral transcripts is 362

more common than previously thought (6, 35-38). Consequently there is an increasing need for 363

strategies to investigate the function of individual protein products of alternatively spliced genes. 364

We show here, using the MCMV M112/113 gene as a paradigm, that all known protein products 365

of an alternatively spliced herpesvirus gene can be inactivated individually by splice donor and 366

acceptor mutagenesis, intron deletion and substitution mutagenesis, and the introduction of stop 367

codons (nonsense mutagenesis). Although mutagenesis of a splice donor or acceptor site has been 368

done before on a few occasions, e.g. for the inactivation of the stable introns derived from the 369

HSV-1 latency associated transcript or CMV immediate-early transcripts (39-41), this is the first 370

comprehensive mutational analysis of a differentially spliced herpesvirus gene. 371

The introduction of nucleotide exchanges to inactivate a splice donor or acceptor site is 372

probably the smallest possible alteration one can introduce. Nevertheless, such a small mutation 373

may still have an impact on the expression of proteins derived from the remaining splice variants. 374

In this study, inactivation of the p33, p36, or p38 isoform did not result in strong changes in the 375

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abundance of the remaining protein isoforms (Figs. 2B and 3D ). However, inactivation of the 376

two most abundant isoforms, p36 and p38, by mutation of the shared splice acceptor site did 377

result in a strong increase of the p33 and p87 isoforms, which are usually expressed only at low 378

levels (Fig 4B). This observed shift in abundance was not unexpected as a previous study have 379

found that elimination of MCMV IE1 transcript by fusion of exons 3 and 5 results in an enhanced 380

expression of IE3 (42). 381

Surprisingly, the deletion of M112/113 intron 1, which was done to eliminate the smallest 382

E1 isoform, was not compatible with MCMV replication. The p33 protein is translated from 383

transcripts retaining intron 1 and differs only by a single C-terminal lysine residue from the N-384

terminal parts of p36, p38, and p87. Viral replication was not rescued by ectopic expression of 385

p33, but by insertion of a synthetic intron in place of intron 1. The MCMV SynI1 mutant 386

replicated to wt levels in NIH-3T3 fibroblasts and SVEC4-10 endothelial cells (Figs. 5D and 6A), 387

but did not express p33 (Fig. 5C), nor were unspliced mRNA transcripts detected (Fig. 5E). 388

Therefore, it is the presence of intron 1 rather than the expression of p33 that is essential for 389

MCMV replication. Which essential function intron 1 fulfills is currently unknown. One possible 390

function of intron 1 could be to ensure sufficient expression of the large p87 isoform. In the 391

absence of intron 1, the p87 protein is encoded by an unspliced transcript. Unspliced transcripts 392

are known be exported with lower efficiency from the nucleus to the cytosol, leading to a reduced 393

translation (43). The fact that an E1 expression plasmid without intron 1 expresses robust 394

amounts of p87 (Fig 2B) does not refute this hypothesis. Expression plasmids with very strong 395

promoters lead to high-level protein expression even from unspliced transcripts, and this might 396

mask a reduced export of unspliced transcripts. Another possible explanation for the requirement 397

of intron 1 is an effect on splicing of the second intron, leading to an altered ratio of the p36, p38, 398

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and p87 isoforms. Indeed, in cells transfected with the M112/133 Δp33 plasmid, expression of 399

p38 was strongly reduced (Fig. 2B). 400

Inactivation of the p87 isoform proved to be most difficult. The only mutation that 401

prevents p87 expression but leaves the coding sequence of the other isoforms intact is a nonsense 402

mutation within intron 2 (Fig. 2A). This mutation should lead to the expression of a substantially 403

truncated p87 protein, however such a protein was not identified in cells transfected with a 404

mutant M112/113 expression plasmid (Fig. 2B). The truncated protein might be unstable or 405

masked by the p36 or p38 proteins, which are similar in size. The most obvious explanation for 406

the inability of the MCMV Δp87 to replicate is an essential function of p87. As a matter of fact, 407

C-terminal truncation of the UL112/113 p84 isoform, the homolog of p87 in HCMV, also 408

resulted in a non-replicating virus (18). This replication defect is probably due to the inability of 409

the truncated p84 protein to recruit the DNA polymerase accessory protein, pUL44, to viral pre-410

replication compartments (18). Although such a function has not yet been described for MCMV 411

p87, this protein was reported to control the repressive effect of IE3 on the MIE promotor (21), 412

and there might be additional functions exclusive to p87. Unfortunately, in silico protein domain 413

predictions did not yield in any informative results for the unique C-terminal part of p87. In any 414

case, the possibility that the truncated p87 protein prevents MCMV replication by a dominant 415

negative effect cannot be ruled out entirely. As the truncated p87 protein differs from p38 only by 416

12 amino acids, this possibility appears rather unlikely. 417

The importance of the large p87 isoform for viral replication was also confirmed by the 418

emergence of two MCMV E1 mutants, which complemented each other (Fig. 4B, C and D). 419

These mutants were derived from the inactivation of the two medium-sized isoforms, p36 and 420

p38. One mutant virus expressed a truncated version of p87 apparently compensated for the loss 421

of the two medium-sized isoforms, whereas the other mutant expressed full-length p87. The fact 422

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that these two mutants could neither be separated nor reconstituted from BACs individually 423

supports the concept that the full-length p87 as well as one medium-sized isoform are required 424

for MCMV replication. 425

The four E1 protein isoforms of HCMV and MCMV were identified more than 25 years 426

ago (9, 11). It would therefore not be surprising if deep sequencing of the MCMV transcriptome 427

would have revealed previously undetected splice sites within M112/113. However, the RNA-428

Seq data largely confirmed the 4 known isoforms, even though one splice junction differed from 429

the previously described ones (Fig 1B and C). Other unknown splice junctions were detected at a 430

very low frequency, comprising less than 1% of the splices in this region (supplemental table S1). 431

Most of these rare splices were not conserved between the MCMV strains Smith and K181. 432

However, one putative unknown intron of 214 nucleotides for MCMV Smith and 217 nucleotides 433

for MCMV K181 was detected 180 bp downstream of the A2 acceptor site. However, the used 434

splice donor and acceptor sites do not match the minimal consensus sequences in both strains. 435

Thus it remains unclear whether this is a true non-canonical intron used at a low frequency or 436

rather an artifact. The latter is being supported by the fact that the predicted size of this intron 437

differs between the two MCMV strains, which was not observed for the introns of the confirmed 438

isoforms. In an RNA-Seq analysis of the HCMV genome such atypical splice junctions were 439

classified as probable artifacts (6). 440

Apart from isoforms derived from differently spliced transcripts additional protein 441

variants could be generated by posttranslational modifications. In fact, two recent papers reported 442

the detection of 3 small UL112/113 proteins, which were apparently generated by calpain-443

mediated cleavage (44). In immunoblots using the anti-E1 antiserum we also detected additional 444

protein bands of low intensity (Fig. 2B), some of which were not detected in lysates of control 445

cells. We do not know whether these bands represent functional post-translationally modified E1 446

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proteins or rather unspecific degradation products. It is also possible that additional splice 447

variants or post-translationally modified protein variants are expressed in cell types other than 448

fibroblasts. Indeed, an additional protein variant most likely representing a modified p33 protein 449

was detected in SVEC4-10 endothelial cells (Fig 6C), but its functional importance remains to be 450

determined. 451

Why is a subset of herpesvirus genes spliced whereas most viral proteins are translated 452

from unspliced transcripts? And why are many of the spliced transcripts expressed during latency 453

or at the very beginning of the lytic cycle? These intriguing questions have not yet been resolved. 454

It is conceivable that herpesviruses use splicing to ensure the expression of critical proteins at 455

times when viral mRNA export factors are not yet present. Another possibility is that 456

herpesviruses use (alternative) splicing as a means to regulate the levels or activities of viral 457

proteins. The use of RNA-seq combined with splice junction analysis provides a more 458

comprehensive view of viral transcript splicing, and the selective elimination of individual 459

spliced transcripts by splice site mutagenesis sets the stage for a comprehensive functional 460

dissection of differentially spliced genes. 461

462

Acknowledgments 463

We thank Daniela Indenbirken for deep sequencing, Julie Kerry, Stipan Jonjić, and Lambert Loh 464

for providing antibodies, and Theodore Potgieter for critical reading of the manuscript. This work 465

was supported by the DFG (grant BR 1730/4-1 to W.B.). 466

467

References 468

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592

Figure legends 593

594

Figure 1. Analysis of the intron-exon structure of the MCMV M112/113 gene. (A) Schematic 595

representation of the M112/113 locus and the four E1 proteins according to Ciocco-Schmitt et al. 596

Splice donor and acceptor sites are marked with D and A respectively. Nucleotide positions of 597

exon boundaries are indicated. (B) Splice junctions were detected by RNA-Seq of cells infected 598

with MCMV Smith or MCMV K181. (C) Schematic representation of the revised M112/113 599

locus based on RNA-Seq data. (D) Relative abundance of spliced and unspliced transcripts for 600

the M112/113 intron 1 (D) and intron 2 (E). 601

602

Figure 2. Mutagenesis of the M112/113 locus. (A) Schematic view of the MCMV M112/113 603

locus and the four proteins expressed from this locus by alternative splicing. Mutations 604

introduced to inactivate the expression of individual protein isoforms are shown. Splice donor 605

and acceptor sites are indicated by arrow heads, and the minimal consensus sequences are shown 606

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in bold. Nucleotide and amino acid changes are underlined. (B) NIH-3T3 cells were transfected 607

with empty vector or expression plasmids containing the wt or mutant M112/113 sequence. E1 608

protein expression was detected by immunoblotting using an E1-specific antiserum. Actin was 609

detected as a loading control. 610

611

Figure 3. Replication kinetics and E1 protein expression of MCMV M112/113 mutants. (A) 612

NIH-3T3 cells were infected at an MOI of 0.02 TCID50/cell with wt MCMV and Rev p87. Virus 613

released into the supernatant was quantified by titration. Replication kinetics of (B) wt and Δp36 614

and (C) wt, Δp38, and Rev p38 were determined as described above. Means ± SEM of 615

experiments done in triplicate are shown. DL, detection limit. (D) NIH-3T3 cells were infected at 616

an MOI of 3 TCID50/cell with wt and mutant MCMVs. E1 protein expression was determined by 617

immunoblotting. IE1 was used as infection control and β-actin as loading control. (E) NIH-3T3 618

cells were infected with MCMV wt, Δp38 or Rev p38 at an MOI of 0.5 TCID50/cell. Cells lysates 619

were harvested at different times post infection. Expression levels of the viral proteins IE1, E1, 620

M44, and gB were analyzed by immunoblot. The upper part of the E1 immunoblot is shown as a 621

longer exposure for better visualization of the p87 isoform. 622

623

Figure 4. Characterization of a spontaneously adapted Δp36Δp38 mutant. (A) NIH-3T3 cells 624

were infected at an MOI of 0.02 TCID50/cell with wt MCMV or Rev p36p38. Viral replication 625

kinetics were determined as described in the previous figure. Means ± SEM of experiments done 626

in triplicate are shown. DL, detection limit. (B) NIH-3T3 cells were infected at an MOI of 3 627

TCID50/cell with wt MCMV or an MCMV mutant that spontaneously emerged in fibroblasts 628

transfected with the MCMV Δp36Δp38 BAC (Δp36Δp38mut). E1 protein expression was 629

analyzed by immunoblotting. An asterisk marks a new E1 isoform expressed by the 630

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spontaneously occurring MCMV mutant. (C) Identification of a mutation by sequencing the exon 631

2 to exon 3 region. The nucleotide position of the mutation is given above. (D) Viral replication 632

kinetics of wt MCMV and Δp36Δp38mut were determined as described in the previous figures. 633

634

Figure 5. Deletion and substitution mutagenesis of the M112/113 intron 1. (A) Schematic 635

showing the deletion of the M112/1113 intron 1 and insertion of the p33-coding ORF into the 636

nonessential m02-m06 region of the MCMV genome. (B) Schematic of the MCMV SynI1 mutant 637

carrying a synthetic intron in place of the authentic intron 1. An unspliced transcript is predicted 638

to encode an E1 protein significantly larger than p33 (termed p33*). (C) NIH-3T3 cells were 639

infected at an MOI of 3 TCID50/cell with wt MCMV or the SynI1 mutant. E1 protein expression 640

was analyzed by immunoblotting. IE1 was used as infection control and β-actin as loading 641

control. (D) NIH-3T3 cells were infected at an MOI of 0.02 TCID50/cell with wt MCMV and the 642

SynI1 mutant. Virus release into the supernatant was determined by titration. Means ± SEM of 643

experiments done in triplicate are shown. DL, detection limit. (E) An M112/113 fragment was 644

PCR-amplified from cDNA of wt MCMV or SynI1-infected cells. The same PCR amplification 645

was done using different MCMV BACs as templates. The sizes of unspliced (us) and spliced (s) 646

transcripts are shown. 647

648

Figure 6. Replication of MCMV M112/113 mutants in endothelial cells and macrophages. 649

(A) SVEC4-10 endothelial cells were infected with an MOI of 0.1 TCID50/cell, (B) RAW264.7 650

macrophages were infected with an MOI of 0.5 TCID50/cell with MCMV mutants. Viral 651

replication kinetics were determined as described above. (C) SVEC4-10 cells or (D) RAW264.7 652

were infected at an MOI of 3 TCID50/cell with the indicated viruses. E1 protein expression was 653

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determined by immunoblotting. IE1 was used as infection control and β-actin was used as a 654

loading control. 655

656

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