Quantification of the Host Response Proteome after Herpes Simplex

Article pubs.acs.org/jpr

Quantification of the Host Response Proteome after Herpes Simplex Virus Type 1 Infection Alicia R. Berard,†,‡ Kevin M. Coombs,†,‡,§ and Alberto Severini*,†,∥ †

Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0J9 Manitoba Center for Proteomics and Systems Biology, University of Manitoba, Room 799 John Buhler Research Centre, Winnipeg, Manitoba, Canada R3E 3P4 § Manitoba Institute of Child Health, University of Manitoba, Room 641 John Buhler Research Centre, Winnipeg, Manitoba, Canada R3E 3P4 ∥ National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3P6 ‡

S Supporting Information *

ABSTRACT: Viruses employ numerous host cell metabolic functions to propagate and manage to evade the host immune system. For herpes simplex virus type 1 (HSV1), a virus that has evolved to efficiently infect humans without seriously harming the host in most cases, the virus−host interaction is specifically interesting. This interaction can be best characterized by studying the proteomic changes that occur in the host during infection. Previous studies have been successful at identifying numerous host proteins that play important roles in HSV infection; however, there is still much that we do not know. This study identifies host metabolic functions and proteins that play roles in HSV infection, using global quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomic profiling of the host cell combined with LC−MS/MS. We showed differential proteins during early, mid and late infection, using both cytosolic and nuclear fractions. We identified hundreds of differentially regulated proteins involved in fundamental cellular functions, including gene expression, DNA replication, inflammatory response, cell movement, cell death, and RNA post-transcriptional modification. Novel differentially regulated proteins in HSV infections include some previously identified in other virus systems, as well as fusion protein, involved in malignant liposarcoma (FUS) and hypoxia up-regulated 1 protein precursor (HYOU1), which have not been identified previously in any virus infection. KEYWORDS: herpes virus, host proteomics, virus−host interactions, SILAC, cell proteomics, LC−MS/MS

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INTRODUCTION Herpes simplex viruses are ubiquitous, have coevolved with their hosts over extremely long periods of time, and cause very common infections in humans. Although the clinical manifestations of herpes simplex virus (HSV) infection are usually mild or inapparent, serious diseases, such as encephalitis, may occur, and HSV establishes a life-long latency, from which it can reactivate, causing painful and harmful recurrences. HSV uses its many genes to establish complex relationships with the host cells, and in this work we have used a proteome approach to provide a simultaneous global view of these relationships. Here we describe which host proteins are regulated by the HSV infection, and we show how specific cellular functions are altered by HSV during the different immediate early, early, and late phases of infection. These results will identify pathways that deserve further study in the quest for an HSV vaccine or better targets to control HSV infections. Herpes simplex virus (HSV1 and HSV2) commonly infects human populations with prevalence in adults from © 2015 American Chemical Society

50 to nearly 100%. Although the vast majority of infections are asymptomatic, HSV can cause oropharyngeal lesions and genital herpes. Although HSV type 2 is most commonly associated with genital lesions, with the recent decline of HSV1 infections during early childhood, genital HSV1 has also become common. More rarely, HSV causes more serious diseases including keratitis, encephalitis, or meningitis and disseminated infections in the newborn and in the immunosuppressed patient.1 HSV1 is a dsDNA virus with a linear genome of about 152 kb coding for about 80 open reading frames. HSV1 employs an intricate infection strategy in the host, which consists of lytic replication followed by a life-long latent stage in the sensory ganglia, from which, in turn, lytic reactivations may occur.1d During lytic replication, HSV1 establishes replication/ transcription compartments in the nucleus of the infected cell, Received: December 1, 2014 Published: March 27, 2015 2121

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EXPERIMENTAL PROCEDURES General methods for preparation and sample analysis have been previously described by us for reovirus23b and for influenza virus.23a,24 In brief the following methods were used.

and the viral genes are expressed in three major kinetic classes, known as immediate early (IE), which do not require de novo protein synthesis, early (E), which require synthesis of the IE proteins, and late (L),2 which require (true late) or are enhanced by viral DNA replication (leaky late).3 The IE genes are expressed upon entry and do not require de novo synthesis of viral proteins. Their expression is enhanced by the virion protein VP16,4 and their synthesis peaks at 2−4 hpi.5 ICP0 is a multifunctional IE protein which is required for the degradation of the ND10 nuclear structures, the efficient assembly of the viral replication compartments, and the optimal activation of early and late genes, a function mediated by ICP0 ability to inhibit the host CoREST/REST repressor.6 The IE protein ICP4 is also required for efficient early gene transcription. Optimal transcription of late viral proteins requires IE proteins ICP4, ICP0, ICP27, and viral DNA replication.4,7 Synthesis of the early proteins is detectable as early as 3 hpi and continues up to 15 hpi.5 The majority of these gene products code for the proteins needed for viral DNA replication. L genes are then activated and proteins needed for maturation, encapsidation and virion egress are synthesized. Therefore, HSV infection results in a very complex, multiphasic interaction of the viral proteome with the host proteome. A great number of these interactions have been studied in detail using targeted cell biology approaches. For example, the IE protein ICP0 is an E2 ubiquitin ligase that affects dissociation of the host nuclear domains 10,8 DNA repair,9 histone processing,10 and host antiviral response;11 the virion host shut-off factor drives the degradation of most of the host mRNAs, reducing host protein synthesis for the benefit of viral replication;12 the viral proteins γ34.1 and US11 interact with the protein kinase R system to counteract the cell interferon response.13 Still undiscovered aspects of such a complex HSV/host interdependence could be brought to light using a proteomic approach. Studying changes in the proteome in the context of virus infection is becoming a powerful tool for generating an abundance of novel targets for antiviral research.14 Proteomic approaches to studying virus infections are becoming more accurate and effective, especially with the advantages of high throughput methodologies such as mass spectrometry.15 There are many different approaches to studying proteomics in virus infections, including proteomics of virus particles (structure or host proteins inside the virus), viral protein interactomics, and changes to the host cell proteome upon viral infection.15b,16 There has been a variety of HSV proteomic research to date, such as identifying host proteins that interact with specific viral proteins,17 identifying all of the virus proteins in a mature virus particle and the nonstructural proteins present in host cells during infection,18 identifying host proteins present in mature virion tegument,19 detecting host protein changes in specific cellular compartments such as ribosomes, microsomes, and the nucleus,20 examining the macrophage secretome of infected cells21 and finally host cell extract analysis.22 However, as was recently reviewed,16 there are still many gaps in our knowledge of the virus−host proteomic interaction, including global protein dynamics and quantitative profiling of host proteins during infection. In this study we address this gap by using the well-established stable isotope labeling by amino acids in cell culture (SILAC) method that we have previously utilized successfully in other viral systems.23 Using SILAC in combination with LC−MS/MS produced higher throughput results than previous gel-based systems. To obtain a more complete picture of the alteration of the host proteome following HSV infection, we analyzed separately the cytoplasmic and nuclear fractions at 4 h, 10 h, and 24 h postinfection.

Virus and Cells

Herpes simplex type 1 strain F (ATCC VR-733) was grown from laboratory stocks. The virus was grown and titrated in Vero cells in Dulbecco’s Modified Eagle Medium (D-MEM) supplemented with 10% FBS, and 1% each of L-glutamine, nonessential amino acids, and sodium pyruvate. For SILAC labeling human embryonic kidney (HEK293) cells were grown in D-MEM supplied in a SILAC phosphoprotein identification and quantification kit (Invitrogen Canada Inc.; Burlington, Ontario). D-MEM was supplemented with 10% dialyzed FBS, and either 100 mg/L “light” (normal, L) lysine and arginine or “heavy” (13C6-lysine and 13 C6-/15N4-arginine, H) isotopic forms. Cells were allowed to double seven times in L or H media prior to infection, and two separate biologic replicates were performed. Approximately 107 L-labeled cells in T75 flasks were infected with HSV1 at a multiplicity of infection (MOI) of 5 PFU per cell. Equivalent numbers of H cells were mock-infected as control. The flasks were incubated at 4 °C for 1 h, with gentle rocking every 10−15 min, to allow virus to adsorb and to synchronize infections. Infected cell cultures were then incubated at 37 °C for 4, 10, and 24 h. Infectious HSV1 titers were determined by standard plaque assays in confluent Vero cells, using 12-well plates.25 Cell Fractionation

At 4, 10, and 24 hpi, both L infected and H mock-infected cells were collected and counted. Aliquots of cultures were also saved for virus titration to confirm infection status. Equal numbers of L and H cells were mixed, washed 3×, and solubilized with 0.5% NP-40, and nuclei were pelleted by centrifugation. The cytosol (supernatant) was transferred to a fresh microfuge tube as the cytosolic fraction. Nuclear pellets were extracted as previously described.24b Briefly, nuclear pellets were washed 2×, extracted by suspension in high salt buffer solution with protease inhibitors, and then frozen. Samples were thawed, sonicated, and spun, and the supernatant was collected. The remaining pellet was resuspended into urea buffer, frozen, then subsequently thawed, sonicated, and spun. The supernatant was collected into the same microtube as high salt supernatant as the nuclear fraction. Sample Preparation, Fractionation, and Protein Quantitation

Protein concentrations in cytosolic and nuclear lysates were determined using a BCA Protein Assay kit (Pierce; Rockford, IL). Samples were reduced, alkylated, digested with sequencing grade trypsin (Promega, cat. no. V5111), and resolved by 2D-reversed-phase high pH−low pH HPLC−MS/MS as previously described.23b A QStar Elite mass spectrometer (Applied Biosystems, Foster City, CA) was used in the datadependent MS/MS acquisition mode with one-second survey MS spectra (m/z 400−1500), followed by MS/MS measurements of the three most intense parent ions (80 counts/s threshold, +2 - +4 charge state, m/z 100−1500 mass range for MS/MS). The manufacturer’s “smart exit” (spectral quality 5) settings were used, and previously targeted parent ions were excluded. Spectra were identified using Analyst QS 2.0 (Applied Biosystems) software, and raw MS/MS data were analyzed using Protein Pilot (ABSciex). Proteins were identified using this software based on SILAC settings for QStar instruments. The default setting for protein quantitation requires that peptides 2122

DOI: 10.1021/pr5012284 J. Proteome Res. 2015, 14, 2121−2142

Article

Journal of Proteome Research be unambiguously assigned to a single protein isoform. Proteins that were detected with at least two unambiguous L peptides and two unambiguous H peptides each at a >99% confidence were used for further analysis. These proteins were normalized using z-score analysis, as described previously.23a The false discovery rate (FDR) was determined at the protein level as described previously.26 Altogether, for each separate experiment at each time point, 1555−2857 proteins were identified with a false discovery rate of 1%. Bioinformatic Analyses

DAVID and Ingenuity pathway analyses were performed as previously described.23 Fischer’s exact test was used to calculate a p-value determining the probability that each biological function and/or disease assigned to that network was due to chance alone. Immunoblotting

Western blot analyses of infected HEK 293 cells were performed essentially as described previously.23 Cytosolic and nuclear proteins were resolved on 10% SDS-PAGE gels, proteins transferred to polyvinylidene difluoride (PVDF) membranes, transfers confirmed by Ponceau staining, and membranes probed for various targets. Primary antibodies were anti-HSV (Abnova, cat#PAB0883), GAPDH (Cell Signaling, cat#2118), Histone H3 (K27me3 monoclonal antibody; Abnova, cat#MAB10253), DPM1 (Abnova, cat#PAB11425), RPL10 (Abnova, cat#PAB17331), RPL14 (Abnova, cat#H00009045-B01P), SP100 (Abnova, cat#H00006672-M02), NucB2 (Abnova, cat#PAB14182), AKR1B10 (Abnova, cat#H00057016-M01), APOE (Rockland, cat#600-101-197), RBMX (Abnova, cat#PAB17364), and PRKDC (Abnova, cat#H00005591-M02). The secondary antibodies were the appropriate horseradish peroxidase (HRP)-conjugated rabbit antimouse or goat antirabbit (Cell Signaling, cat#7076 and cat#7074, respectively). Bands were detected by enhanced chemiluminescence using an Alpha Innotech FluorChemQ MultiImage III instrument.

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Figure 1. Venn diagrams of numbers of identified proteins in cytosolic and nuclear fractions of both biological replicate experiments at 4 hpi (a and d), 10 hpi (b and e), and 24 hpi (c and f). (g) A summary of the numbers of differentially regulated proteins found in either the cytosolic or nuclear fractions at each time point.

with a 59% overlap (1149 duplicate proteins) in the cytosolic fraction, and 2317 proteins with a 68% overlap (1573 duplicate proteins) were identified in the nuclear fraction (Figure 1c,f). Overall, a total of 3675 unique proteins were identified with 2587 found in the cytosol and 2712 in the nuclear fraction. To determine which proteins were differentially regulated after infection, all of the proteins identified with L:H ratios were normalized using z-score analysis as described previously.23 A 95% confidence level cutoff was applied to the data by z-score analysis, and the proteins that fell outside this population distribution were then considered up- or downregulated. This was performed separately for both biological replicates, and the z-scores were compared between replicates for reproducibility. Proteins are identified as up- or downregulated if at least one of the biologic z-score values is ≥1.960σ or ≤ −1.960σ, respectively, and there are no major disagreements (ratio is not more than one standard deviation in the opposite direction) between biological replicates. Differentially regulated proteins are listed in Tables 1 and 2, and a summary of the numbers of up- and downregulated proteins are shown in Figure 1g.

RESULTS

Herpes Simplex-1 Successfully Infects HEK293 Cells

HEK293 cells have been used numerous times in the literature as a model for HSV1 infection studies.27 We have previously performed and reported proteomic changes of mammalian reovirus T1L in these cells,23b so, in order to identify virusspecific differences in a common cell type, we initially confirmed that our HEK293 cells could be infected with our HSV strain. We obtained a burst size of approximately 50 infectious virus particles per cell by 48 hpi, which represents a ∼10 000-fold increase over the input MOI of 0.005 (data not shown). Protein Identification

We quantitatively examined protein alterations in HEK293 cells at 4, 10, and 24 h postinfection with HSV1. We performed two separate biological replicates of the infection for each time point, and identified host cellular proteins in both the cytosolic and nuclear fractions separately. At 4 hpi, 2178 host proteins were identified by two or more peptides and with confidence ≥99%, with 46% overlap between biological replicates (1001 proteins found in both experiments) in the cytosolic fraction, and 1874 proteins with 62% overlap (1161 duplicate proteins) were identified in the nuclear fraction (Figure 1a,d). At 10 hpi, 2099 host proteins were identified with 1145 duplicate proteins (55% overlap) in the cytosolic fraction, and 1645 proteins with 909 duplicate proteins (55% overlap) were identified in the nuclear fraction (Figure 1b,e). At 24 hpi, 1947 host proteins were identified

HSV Proteins Identified

As another means to confirm successful infection of the samples, HSV proteins were also identified for each experiment at each time point (Table 3). At 4 hpi only an immediate-early protein (ICP4)36,37 was identified. At 10 h post infection many of the HSV1 early proteins were detected (e.g., thymidine kinase and DNA polymerase processing subunit). At 24 hpi, most of the late HSV proteins are also apparent. 2123

DOI: 10.1021/pr5012284 J. Proteome Res. 2015, 14, 2121−2142

4504349 188536047 6678271 4505573 4885457 4505229 119395750 4502027 4504347 148596949 149999606 162809334 93102375 4504025 105990535 27597090 41350320 76880486 4557325 122114654 38524622 4758272 55743134 58761510 117553584 94721336 5901954 118344456 4506629 4504277 4504061 4506669 30795231 4557797 167614488 4503139 223468681 13129018 4502981 195972866 5032027 29788785

accession

HBB SMARCC1 TARDBP Arhgef7 smad4 Fadd KRT1 alb hba2 NOLC1 mogs PZP Fam114a2 GLRX F5 SUPT6H MAGED2 ASCC3 APOE Mett10d Fnbp1 ENSA RPS6KA1 yrdC Ap3d1 rufy1 FGFR1OP utp18 rpl29 HIST2H2BE GNS rplp1 Basp1 NME2 TBC1D10B CTSB relA Ggct COX4I1 KRT10 RBBP4 tubB

HGNC

name

Pep

beta globin 2 SWI/SNF-related matrix-associated actin- dependent regulator of chromatin c1 2 TAR DNA binding protein 2 PAK-interacting exchange factor beta isoform a mothers against 2 decapentaplegic homologue 4 2 Fas-associated via death domain 2 keratin 1 7 albumin preproprotein 6 alpha 1 globin 3 nucleolar and coiled- body phosphoprotein 1 2 mannosyl-oligosaccharide glucosidase isoform 1 2 pregnancy-zone protein 3 hypothetical protein LOC10827 4 glutaredoxin (thioltransferase) 2 coagulation factor V precursor 2 suppressor of Ty 6 homologue 2 melanoma antigen family D, 2 activating signal 9 cointegrator 1 complex subunit 3 isoform a 2 apolipoprotein E precursor 2 methyltransferase 10 domain containing 2 formin binding protein 1 3 endosulfine alpha isoform 3 2 ribosomal protein S6 kinase, 90 kDa, polypeptide 1 isoform b 6 ischemia/reperfusion inducible protein 2 adaptor-related protein complex 3, delta 1 subunit isoform 1 2 RUN and FYVE domain-containing 1 isoform a 2 FGFR1 oncogene partner isoform a 2 UTP18, small subunit processome component 2 ribosomal protein L29 10 histone cluster 2, H2be 13 glucosamine (N-acetyl)-6-sulfatase precursor 10 ribosomal protein P1 isoform 1 10 brain abundant, membrane attached signal protein 1 15 nonmetastatic cells 1, protein expressed in isoform b 40 TBC1 domain family, member 10B 4 cathepsin B preproprotein 6 A isoform 2 3 gamma-glutamyl cyclotransferase 8 cytochrome c oxidase subunit IV isoform 1 precursor 5 keratin 10 2 retinoblastoma binding protein 4 isoform a 17 tubulin, beta 141

Table 1. HEK293 Proteins Increased >95% Confidencea

b

52.7 52.7 52.7 52.7 52.7 52.7 34.8 12.4 8.6 5.3 4.4 4.1 3.3 2.8 2.8 2.6 2.5 2.4 2.3 2.2 2.2 2.1 2.1 2.0 2.0 1.9 1.9 1.9 1.8 1.8 1.8 1.7 1.7 1.6 1.6 1.5 1.5 1.5 1.5 1.4 1.4 1.4

Ave L:H

4 hpi c

24 hpi

4 hpi

10 hpi

nuclear 24 hpi

3.6 1.1 2.1 1.2 0.6 1.3 1.3 0.9

2.1 3.9 1.5 1.4 1.7 2.1 1.8 1.2 2.3 0.9 1.7 2.0 1.7

8 14 13 20 16 47 5 6 3 10 5 21 148

4.2 16.1 37.6 1.2 1.1 79.8 1.7 1.1 1.1 0.0 0.8

2 8 4 5 4 2 2 6 2 2 9 2 2 3 3 4 4 4 2

18.4 1.0 0.7

2 4 2

2124

10 7 11 15 12 38 3 6 2 10 4 3 13 129

2.0 3.4 1.7 1.2 2.0 1.9 2.1 1.2 1.2 0.9 4.6 4.2 1.5 1.0

1.2 1.1 1.5 0.4

1.1 1.3

4 9

5 3 4 2

0.9 1.3

4 5

1.1

1.9

4

4

16.1 48.3 44.3

100.0

7 8 7

2

1.3

9

1.1 1.1

1.2 1.5 1.3

17 17 18

14 104

1.2 1.3 1.5

0.8

9

5 2 28

21.1 0.9

12.0 20.8 42.9 1.1 1.0

1.0 1.1 0.8

2 18

4 3 5 2 9

21 12 2

11 75

12

9 15 19

4

3

5

6

7 2 8

21 12

0.9 1.1

1.2

1.1 1.3 1.2

0.8

0.8

0.8

1.3

14.0 19.6 66.0

0.9 1.0

28 156

1.1 1.6

1.5

0.8 1.4 1.5 1.0

5 32 30 20

10

0.8 1.9

0.9

1.1

0.9

12.0 0.8

12 4

6

2

7

2 22

8.2 19.1 74.4 1.0 1.2

0.7

2 11 7 11 5 8

86.3 1.0 1.0

4 32 35

Pep Ave L:H Pep Ave L:H Pep Ave L:H Pep Ave L:H Pep Ave L:H

10 hpi

cytosol

Journal of Proteome Research Article

DOI: 10.1021/pr5012284 J. Proteome Res. 2015, 14, 2121−2142

2125

14110407 31543906 5174449 24475861 21264341 4506019 157694494

HNRPDL Ube2q1 H1fx Phpt1 Sms Ppp2r2a mybbp1a

HGNC

Hist1h4a Npm1 VIM GAPDHL6 c11orf2 sqstm1 Pepd HNRNPA2B1 EIF3J G3BP2 nonO Hnrnpc rpl19 HSPA5 Hnrnpu fus HNRNPL Ptbp1 RBMX paics DDX21 sfrs7 Rsl1d1 BOLA2B EIF4A2 Hnrnpa3 hadh G6PD rps27 OAT VDAC1 Rbm12 AKAP12

accession

77539758 40353734 62414289 7669492 8393009 4505571 149589008 14043072 83281438 45359849 34932414 117190254 4506609 16507237 74136883 4826734 52632385 4506243 56699409 5453539 50659095 72534660 118498359 85797673 83700235 34740329 94557308 109389365 4506711 4557809 4507879 23510462 21493024

Table 1. continued

name

heterogeneous nuclear ribonucleoprotein D- like ubiquitin-conjugating enzyme E2Q H1 histone family, member X phosphohistidine phosphatase 1 isoform 3 spermine synthase alpha isoform of regulatory subunit B55, protein phosphatase 2 MYB binding protein 1a isoform 1

histone cluster 2, H4b nucleophosmin 1 isoform 2 vimentin glyceraldehyde-3-phosphate dehydrogenase chromosome 11 open reading frame2 sequestosome 1 isoform 1 peptidase D heterogeneous nuclear ribonucleoprotein A2/B1 isoform B1 eukaryotic translation initiation factor 3, subunit 1 alpha, 35 kDa Ras-GTpase activating protein SH3 domain- binding protein 2 isoform a non-POU domain containing, octamer- binding isoform 1 isoform b ribosomal protein L19 heat shock 70 kDa protein 5 heterogeneous nuclear ribonucleoprotein U isoform a fusion (involved in t(12;16) in malignant liposarcoma) heterogeneous nuclear ribonucleoprotein L isoform b polypyrimidine tract- binding protein 1 isoform a RNA binding motif protein, X-linked phosphoribosylaminoim idazole carboxylase DEAD (Asp-Glu-Ala-Asp) box polypeptide 21 splicing factor, arginine/serine-rich 7 ribosomal L1 domain containing 1 bolA-like protein 2B eukaryotic translation initiation factor 4A2 heterogeneous nuclear ribonucleoprotein A3 L-3-hydroxyacyl-coenzyme A dehydrogenase precursor glucose-6-phosphate dehydrogenase isoform a ribosomal protein S27 ornithine aminotransferase precursor voltage-dependent anion channel 1 RNA binding motif protein 12 A kinase (PRKA) anchor protein 12 isoform 2 14 3 4 6 14 8 2

30 26 67 104 3 2 12 23 8 9 10 24 11 81 48 8 6 14 8 15 10 6 2 9 24 15 8 23 5 23 7 13 37

Pep

b

1.1 1.1 1.1 1.0 1.0 1.0 1.0

1.4 1.4 1.4 1.4 1.4 1.4 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.2 1.2 1.2 1.2 1.2 1.2 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1

Ave L:H

4 hpi c

24 hpi

4 hpi

10 hpi

nuclear 24 hpi

1.2 1.3 0.9 1.0 48.1

3 8 9 5

1.1 1.2 1.8 1.3 1.5 1.5 1.5 1.3 1.4 1.4 1.3 1.3 1.4 1.6 1.1 1.4 1.4 1.4 1.2 47.2 1.1 1.4 1.0 1.7 1.2 1.9 0.8 1.1

6 9 26 9 11 12 22 10 99 49 9 5 12 10 28 8 5 3 7 28 12 14 24 13 25 7 17 40 16

1.7 1.4 1.5 1.5

25 23 66 133

2.5 2.3 4.6 0.9 0.9 1.0 4.1

2.5 1.9 0.9 1.3 1.3 2.5 1.0 1.0

18 10 27 9 22 5 15 42 13 2 4 8 14 9 4

2.5 2.4 2.6 1.6 1.2 0.9 0.9 2.7 1.6 1.1 3.3 3.3 1.6 2.0 2.6 1.7 3.5 1.8 4.9 0.9 2.4 4.0 3.8 1.0

16 24 44 103 3 5 11 26 9 10 8 18 6 110 39 10 3 16 7 29 13 6 5 7 1.2 1.0 1.3 1.1 1.5 1.0 1.3

47 25 5 34 16 17 4

1.1 52.4 2.3 1.0

14

1.0 13 2 2

26

1.1 2.3 2.5 1.2 1.4 1.2 1.2 1.4

1.1 0.9 1.3 1.1 1.2 1.0 1.1 1.1 1.2

116 5 11 75 51 3 57 82 24

80 4 5 3 7 23 8 8

1.7

1.1 1.1

238 33 4

1.0

62

10 2

1.1 0.8

0.9

1.3 1.2 1.1 1.1 1.4 3.2

4 2 8 20 10 3 25

1.0

1.1 1.0 1.8 1.0 1.5 0.9 1.7

1.1 1.0 1.6 1.1 1.1 2 1.1 1.1 1.3

1.5

1.0 1.1

1.0

61

35 21 6 31 10 18 4

56 57 25

107 4 8 57 47

2

210 25

56

49

8 3 2

49

92 6 6 3 15 34 10 3

60 30 17 51 26 29 4

148 6 14 82 55 1.2 66 120 41

275 58 2 10

65

0.9

1.1 1.2 0.9

1.0

1.1 1.2 1.2 1.1 1.2 1.3 3.8 1.0

3.8 1.1 1.2 1.0 1.6 0.9 1.6

1.5 1.1 1.2

1.1 0.9 1.3 1.3 0.9

1.2 1.7 100.0 1.2

1.2

Pep Ave L:H Pep Ave L:H Pep Ave L:H Pep Ave L:H Pep Ave L:H

10 hpi

cytosol

Journal of Proteome Research Article

DOI: 10.1021/pr5012284 J. Proteome Res. 2015, 14, 2121−2142

4503015 5729864 13489083 5453832 157388904 4502227 4505185 4758796 36796743 148612809 167614485 6912734 25777612 21264343 217416379 11067747 123173757 157739940 27436901 4507813 91176333 11596859 21362062 29825823 4507797 110349772 4502751 20270337 21359867 5803023 4501989 19913432 47132595 60499025 126723060 34577049 14043026 4557871 54607120

HGNC

HIST2H2AA3 histone cluster 2, H2aa3 CPNE3 hbs1l SAPS3 hyou1 heatr2 arl1 MIF DRG1 MTHFD1L WNK1 ACAA2 TNPO3 psmd3 safb hnrpll Cdc5l RAVER1 nudt19 mRpL12 UGDH LYRM7 MRPL17 Lzic c17orf28 ube2v2 COL1A1 CDKN2C LEO1 cyc1 Lman2 AFP atp6v0d1 SLC25A3 FECH Zc3h4 DST Vamp8 TF LTF

accession

4504251

Table 1. continued

name

copine III Hsp70 subfamily B suppressor 1-like protein isoform 1 SAPS domain family, member 3 hypoxia up-regulated 1 precursor HEAT repeat containing 2 ADP-ribosylation factor like 1 macrophage migration inhibitory factor developmentally regulated GTP binding protein 1 methylenetetrahydrofol ate dehydrogenase (NADP+ dependent) 1-like WNK lysine deficient protein kinase 1 acetyl-coenzyme A acyltransferase 2 transportin 3 proteasome 26S non- ATPase subunit 3 scaffold attachment factor B heterogeneous nuclear ribonucleoprotein L- like isoform 2 CDC5-like RAVER1 nudix (nucleoside diphosphate linked moiety X)-type motif 19 mitochondrial ribosomal protein L12 UDP-glucose dehydrogenase Lyrm7 homologue mitochondrial ribosomal protein L17 leucine zipper and CTNNBIP1 domain containing hypothetical protein LOC28398 ubiquitin-conjugating enzyme E2v2 alpha 1 type I collagen preproprotein cyclin-dependent kinase inhibitor 2C Leo1, Paf1/RNA polymerase II complex component, homologue cytochrome c-1 lectin, mannose-binding 2 precursor alpha-fetoprotein precursor ATPase, H+ transporting, lysosomal, V0 subunit d1 solute carrier family 25 member 3 isoform b precursor ferrochelatase isoform a precursor zinc finger CCCH-type containing 4 dystonin isoform 1eA precursor vesicle-associated membrane protein 8 transferrin lactotransferrin precursor

10 5 5 4 44 11 2 34 5 15 8 18 4 23 6 3 8 2 4 5 8 5 2 2

1.0

Pep

b

2126

2

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.8 0.8 0.8 0.8 0.3 0.0

10

Ave L:H

4 hpi c

24 hpi

4 hpi

10 hpi

nuclear 24 hpi

1.2 1.8 2.1 0.9 0.9 1.3 79.8 47.3 23.7 2.5 2.4 2.1 2.0 2 1.6 1.5 1.4 1.3 0.9 0.8

8 3 12 2 2 2 8 5 2 2 2 8 1.7 5 6 2 3 2 2

1.8 1.0 1.2 1.0 0.9 0.9 1.6 1.0 0.9 0.8 1.1 1.3 0.9 1.3

6

6

4 7 7 54 12 2 31 14 14 14 23 3 26 3

1.2

4.9

12.6 100.0 52.7

2 2 4

1.6 3.9 1.3

2 3 4 5.6 4

0.6

2.6

2 2

1.8 4.2 1.1

5 2 15

2.3

2.9 0.9 1.4 0.7 2.1 2.1 0.8 2.2

29 8 6 15 13 2 26 4 8

1.4 3.6 1.0 1.4 6.6

6 5 7 68 9

2.3

0.9 1.0 1.5 1.0 1.8 0.8 1.3 3.7 2.0

7 18 4 23 6 4 7 2 2

3 18 2 8 2 3

8 2 5

0.8 1.2 2.3 1.4 0.9 1.3

1.0 1.3 1.0

1.0

1.0

4

2

1.3 0.9 2.0 1.1 1.0 7.8 1.3 1.3 1.7

6 7 2 7 2 2 11 9 8

1.2 41.6 0.7

7 5

1.0

1.0

1.3 1.2

1.4 0.9 0.9 1.0 1.1

13

5

2

7 2

7 19 4 21 3

1.1

1.2 1.1 2.3 0.9 2.0 1.1

12 2 3 8 5 10 3

1.1

9.3

7

28

2

11

6 27

3.1

1.0

1.2 1.1

0.9 1.1 1.0

2.6

2

7 6 6

1.5

5.1 0.8 1.1 1.5 6.8 1.1 1.2 1.1 1.1 0.8 0.9 0.6

2 7 11 19 2 19 3 8 42 7 34 12 17

1.4

0.9 1.1 42

14 13

Pep Ave L:H Pep Ave L:H Pep Ave L:H Pep Ave L:H Pep Ave L:H

10 hpi

cytosol

Journal of Proteome Research Article

DOI: 10.1021/pr5012284 J. Proteome Res. 2015, 14, 2121−2142

2127

HGNC

nop2 sco1 Hist1h1d EDC3 ARMC9 Rprd1a ostf1 bcor SCAF1 KRT6C Akr1b10 aldh1a1 Krt7 Ogt WDR5 spin1 GIGYF2 GNG12 ZNF579 NUP35 ARL6IP4 Kif18b rnf2 RBM22 ANKHD1 AKAP12 A2M STAU2 UBQLN4 KRT9 vtn plg FOS KRT2 F2 Pard3

name

nucleolar protein 1, 120 kDa cytochrome oxidase deficient homologue 1 histone cluster 1, H 1d enhancer of mRNA decapping 3 armadillo repeat containing 9 regulation of nuclear pre-mRNA domain containing 1A osteoclast stimulating factor 1 BCL-6 interacting corepressor isoform a SR-related CTD- associated factor 1 keratin 6C aldo-keto reductase family 1, member B10 aldehyde dehydrogenase 1A1 keratin 7 O-linked GlcNAc transferase isoform 1 WD repeat domain 5 spindlin GRB10 interacting GYF protein 2 isoform c G-protein gamma-12 subunit zinc finger protein 579 nucleoporin 35 kDa SRp25 nuclear protein isoform 3 kinesin family member 18B ring finger protein 2 RNA binding motif protein 22 ANKHD1-EIF4EBP3 protein A kinase (PRKA) anchor protein 12 isoform 1 alpha-2-macroglobulin precursor staufen homologue 2 ataxin-1 ubiquitin-like interacting protein keratin 9 vitronectin precursor plasminogen v-fos FBJ murine osteosarcoma viral oncogene homologue keratin 2 coagulation factor II preproprotein partitioning-defective protein 3 homologue

Pep

b

Ave L:H

4 hpi c

24 hpi

4 hpi

10 hpi

nuclear 24 hpi

3 2 9 2 2 5 2 6.2 5.7 3.7 2.7 2.1 0.8 0.8

0.9

1.4 2.1 52.4 52.4 22.6 11.6 9.8 6.7 2.8 2.5 2.3 2.3 2.2 2.1 1.9 1.9 1.8 1.6 1.3 1.1 1.0

19

8 2 2 2 5 3 5 7 6 5 2 2 3 2 6 2 2 14 8 8 6

1.1

0.9 1.9 11.0 2.4 4.2 3.3 2.8 2.6

7 4 5 2 2 2 3 2

0.8

2

2

1.1

0.9

2 2

0.9

11

0.6 0.9 0.7 1.0

1.4 100.0 47.8 22.4 11.2 2.8 1.7

9 5 6 2 5 6 5

0.8 1.3 1.7

2 4 9

10 11 4 4

0.8

2.6

5 7

0.9

0.6

0.8

9

2

23

Pep Ave L:H Pep Ave L:H Pep Ave L:H Pep Ave L:H Pep Ave L:H

10 hpi

cytosol

Protein is included if at least one of the biologic z-score values are ≥1.960σ (indicated by bolding) and there are no major disagreements between biological replicates. bPep refers to the number of peptides used to identify the corresponding protein. cL:H ratio refers to the geometric mean of all log L/H values for each given gi number, expressed as a relative protein quantity in infected cultures.

a

76150625 4759068 4885377 215598561 156151430 21361709 166235148 21071037 32698750 155969697 223468663 21361176 67782365 32307148 16554629 112293285 156766047 51036603 110681708 31982904 50409738 122937289 6005747 8922328 37620163 21493022 66932947 7657625 40538799 55956899 88853069 4505881 4885241 47132620 4503635 21361831

accession

Table 1. continued

Journal of Proteome Research Article

DOI: 10.1021/pr5012284 J. Proteome Res. 2015, 14, 2121−2142

HGNC

Lzic PPM1A flot1 zfand6 SNX4 CSTF3 PLOD1 Gamt MRPL17 SAMHD1 DCK PATL1 TPD52 GRWD1 POLR2B AACS FKBP7 CDK16 ERO1LB cnot1 Tbc1d24 Fahd2a GET4 ube2r2 C1orf123 Hnrnph3 Prkag1 nudt9 lepre1 Stoml2 CRTAP RIC8A RPN1 GDI1 mtr upp1 NLN akt1s1 Ppm1f STX12

accession

21362062 193211600 5031699 21359918 4507145 4557495 32307144 4503909 11596859 38016914 4503269 189217919 70608172 31542862 4505941 31982927 31317231 5453860 31377735 42716275 89886453 156231349 38570062 22212943 8923541 14141159 4506061 37594457 186928835 7305503 5453601 27883866 4506675 4503971 169790923 4507839 14149738 148806906 7661862 28933465

leucine zipper and CTNNBIP1 domain containing protein phosphatase 1A isoform 3 flotillin 1 zinc finger, AN1-type domain 6 sorting nexin 4 cleavage stimulation factor subunit 3 isoform 1 lysyl hydroxylase 1 precursor guanidinoacetate N- methyltransferase isoform a mitochondrial ribosomal protein L17 SAM domain- and HD domain- containing protein 1 deoxycytidine kinase protein associated with topoisomerase II homologue 1 tumor protein D52 isoform 1 glutamate-rich WD repeat containing 1 DNA directed RNA polymerase II polypeptide B acetoacetyl-CoA synthetase FK506 binding protein 7 isoform a precursor PCTAIRE protein kinase 1 isoform 1 endoplasmic reticulum oxidoreductin 1-Lbeta CCR4-NOT transcription complex, subunit 1 isoform a TBC1 domain family, member 24 fumarylacetoacetate hydrolase domain containing 2A hypothetical protein LOC51608 ubiquitin-conjugating enzyme UBC3B hypothetical protein LOC54987 heterogeneous nuclear ribonucleoprotein H3 isoform b AMP-activated protein kinase, noncatalytic gamma-1 subunit isoform 1 nudix-type motif 9 isoform b leprecan 1 isoform 1 stomatin (EPB72)-like 2 cartilage associated protein precursor resistance to inhibitors of cholinesterase 8 homologue A ribophorin I precursor GDP dissociation inhibitor 1 5-methyltetrahydrofolate- homocysteine methyltransferase uridine phosphorylase 1 neurolysin AKT1 substrate 1 (proline-rich) protein phosphatase 1F syntaxin 12

name

Table 2. HEK293 Proteins Decrease >95% Confidencea

2 2 2 2 3 2 3 2 2 3 3 2 2 2 3 2 2 4 2 9 2 2 2 3 2 3 3 2 6 9 3 5 14 28 2 2 7 5 7 2

Pep

b

0.0 0.2 0.2 0.2 0.3 0.3 0.3 0.3 0.3 0.3 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.6 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7

Ave L:H

4 hpi c

1.0

0.8 1.8

0.4 1.2 0.3 0.6 0.9 0.9 0.5 0.8 1.2 1.0 0.3

3 2

7 9 2 3 18 33 4 4 5 4 3

1.5 0.7

3 2

4

0.4 0.5 1.3 1.0

2 2 2 3

0.6 0.5 0.7

0.7

2

4 3 10

0.9 0.6

Ave L:H

2 3

Pep

10 hpi

cytosol

2128

0.6 0.7 0.6 0.7

0.8 0.8 1.9 0.5 0.5 1.6 0.8 0.6

3 13 6 4 2 14 18 5 6 5 3 2

0.8

0.5 0.8 0.9 1.1

2 3 2 4

2

0.8

1.8 0.4 0.9

2.6

Ave L:H

3

3 4 4

2

Pep

24 hpi

34 5

8 13

1.0 1.1

0.9 1.2

1.2

0.6

4

18

1.0

3

0.9 0.8

2.0

2

7 9

0.8

0.8

Ave L:H

4

14

Pep

4 hpi

26

3 11

16

5

9

Pep

1.0

1.2 1.2

1.0

0.9

0.8

Ave L:H

10 hpi

nuclear

2

40 11

7 36

30

12 5

2

7 8

22

Pep

0.9

1.1 0.7

0.9 1.3

1.1

1.0 0.7

1.0

0.7 1.1

1.0

Ave L:H

24 hpi

Journal of Proteome Research Article

DOI: 10.1021/pr5012284 J. Proteome Res. 2015, 14, 2121−2142

HGNC

PRKDC Cdk1 HNRNPF hadhb nucb2 ZNF259 NAA10 EIF2B3 METAP1 THUMPD1 LYRM7 ACYP1 GBE1 PLOD2 MAPK3 pkn2 asnS Usp7 CMPK1 TSN Eif3f PSME2 RCN2 Fkbp10 sephs1 MMS19 RfC4 ERO1L DFFA wdr82 Arf1 SNX1 camk1 NCKAP1 Timm44 PPCS LSS REEP5 EIF3C Phax

accession

13654237 4502709 4826760 4504327 4826870 4508021 10835057 9966779 164420681 42476024 91176333 4557245 189458812 62739166 91718899 5453974 168229252 150378533 7706497 4759270 4503519 30410792 4506457 192448443 24797148 170763479 4506491 7657069 4758148 147904340 66879664 23111034 4502553 7305303 33636719 116875844 47933397 115430112 83700233 66392146

Table 2. continued

name

protein kinase, DNA-activated, catalytic polypeptide isoform 1 cell division cycle 2 isoform 1 heterogeneous nuclear ribonucleoprotein F mitochondrial trifunctional protein, beta subunit precursor nucleobindin 2 zinc finger protein 259 alpha-N-acetyltransferase 1A eukaryotic translation initiation factor 2B, subunit 3 gamma methionyl aminopeptidase 1 THUMP domain containing 1 Lyrm7 homologue acylphosphatase 1 isoform a glucan (1,4-alpha-), branching enzyme 1 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 isoform b precursor mitogen-activated protein kinase 3 isoform 1 protein kinase N2 asparagine synthetase ubiquitin specific peptidase 7 UMP-CMP kinase 1 isoform a translin eukaryotic translation initiation factor 3, subunit 5 epsilon, 47 kDa proteasome activator subunit 2 reticulocalbin 2 precursor FK506 binding protein 10 selenophosphate synthetase 1 MMS19 nucleotide excision repair homologue replication factor C 4 ERO1-like DNA fragmentation factor, 45 kDa, alpha polypeptide isoform 1 WD repeat domain 82 ADP-ribosylation factor 1 sorting nexin 1 isoform a calcium/calmodulin-dependent protein kinase I NCK-associated protein 1 isoform 1 translocase of inner mitochondrial membrane 44 phosphopantothenoylcysteine synthetase isoform a lanosterol synthase isoform 1 receptor accessory protein 5 eukaryotic translation initiation factor 3, subunit C RNA U, small nuclear RNA export adaptor (phosphorylation regulated)

105 13 10 10 6 6 7 7 5 7 5 6 2 3 12 4 12 12 15 13 11 7 11 9 8 4 9 9 9 2 9 6 4 6 13 2 5 4 25 4

Pep

b

0.7 0.7 0.7 0.7 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9

Ave L:H

4 hpi c

2129

0.6 0.7 0.8 0.8 0.8 0.9 1.0 1.0 0.6 0.7 0.9 0.9 0.5 0.9 0.8 0.8 0.9 1.0

5 10 2 11 10 15 16 11 8 11 20 9 6 8 11 12 2 12 0.4 0.8 1.2 0.4 0.8 0.9 0.9 0.6

0.5 0.9 0.8

8 2 5

4 7 12 2 5 5 30 7

0.7 1.0 1.2 1.3 0.8 0.8 0.8 0.9

Ave L:H

106 8 3 8 10 7 8 5

Pep

10 hpi

cytosol

1.5 0.8 0.6 0.5 0.7 0.6 1.0 0.4 0.9 0.8 0.8 0.9 1.0 0.9 0.6 0.9 1.0 1.8 0.7 0.5 0.3 0.8 0.5 1.3 0.8 0.3 1.3 0.8 0.9

2 2 2 10 5 16 11 12 14 8 4 12 18 8 3 8 10 11 10 3 4 2 7 2 2 2 36 2

0.6 0.6 0.5 1.9 0.9 0.6 0.7 0.9

Ave L:H

9

63 11 4 8 9 7 9 7

Pep

24 hpi

1.1 1.0

1.0 0.9 1.3

9 6

9 3 2

0.9 0.5

0.4 0.9 1.0

5 7

2 10 4

0.9 0.9

1.0

12

6 3

1.1 1.0 0.9 1.2

0.9

3

6 2 18 8

0.9 1.0 1.1 1.1 0.9 0.9 1.1

Ave L:H

105 15 44 12 10 3 8

Pep

4 hpi

11 7

5 5

0.6 1.0

1.1 1.0

1.1

0.2

2

8

0.8

1.1 1.0

0.9

1.3 1.1 0.9 1.2

8

10 4

11

2 3 13 6

1.2

1.2

6 2

0.9 1.1 1.0 1.1 0.6

Ave L:H

69 14 35 10 3

Pep

10 hpi

nuclear

9 3

2 15

5 13 5

11

21 11

10

24 6

5

2

10

151 12 69 16 7

Pep

0.8 0.9

1.6 1.0

0.5 1.0 1.2

0.9

1.1 1.2

1.0

0.7 1.5

1.1

2.6

1.1

0.7 1.1 1.0 1.2 0.8

Ave L:H

24 hpi

Journal of Proteome Research Article

DOI: 10.1021/pr5012284 J. Proteome Res. 2015, 14, 2121−2142

HGNC

LTV1 DCPS txndc9 RFC2 QKI Sar1B DNM2 Chchd2 GDI2 lig1 Rbm12 rpl10 LYPLA2 NENF msh6 vta1 Galk1 COPS7B RBM34 cpd TTC1 npepps Lrpap1

SERPINB1 GSTO1 HSPH1 PIN4

Aprt HNRNPA1 ARPC5 LCP1 GSPT1 ppil1 glrx5 nosip FGFR1OP SUPT6H ankrd52 CRELD2

accession

21361875 7661734 18104959 31563534 45827712 7705827 56549125 7705851 6598323 4557719 23397708 223890243 9966764 7019545 4504191 21361741 4503895 12232385 38016127 22202611 4507711 158937236 4505021

13489087 4758484 42544159 38679892

4502171 4504445 5031593 167614506 194097354 7706339 42516576 7705716 5901954 27597090 157743284 205360958

Table 2. continued

name

LTV1 homologue mRNA decapping enzyme thioredoxin domain containing 9 replication factor C 2 isoform 1 quaking homologue, KH domain RNA binding isoform HQK-7B SAR1a gene homologue 2 dynamin 2 isoform 4 coiled-coil−helix-coiled-coil−helix domain containing 2 GDP dissociation inhibitor 2 isoform 1 DNA ligase I copine I isoform a ribosomal protein L10 lysophospholipase II neuron derived neurotrophic factor precursor mutS homologue 6 Vps20-associated 1 homologue lgalactokinase 1 COP9 constitutivephotomorphogenic homologue subunit 7B RNA binding motif protein 34 carboxypeptidase D precursor tetratricopeptide repeat domain 1 aminopeptidase puromycin sensitive low density lipoprotein receptor-related protein associated protein 1 precursor serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 1 glutathione-S-transferase omega 1 heat shock 105 kDa protein (peptidyl-prolyl cis/trans isomerase) NIMA-interacting, 4 (parvulin) adenine phosphoribosyltransferase isoform a heterogeneous nuclear ribonucleoprotein A1 isoform a actin related protein 2/3 complex subunit 5 L-plastin G1 to S phase transition 1 isoform 2 peptidylprolyl isomerase-like 1 glutaredoxin 5 nitric oxide synthase interacting protein FGFR1 oncogene partner isoform a suppressor of Ty 6 homologue ankyrin repeat domain 52 cysteine-rich with EGF-like domains 2 isoform b

2130

10 32 3 42 26 2 10 3 2 2

3 14 37 5

9 3 3 2 2 5 20 2 46 8 10 24 4 9 10 9 11 3 2 6 5 44 6

Pep

b

1.1 1.1 1.1 1.2 1.2 1.2 1.3 1.6 1.9 2.6

1.1 1.1 1.1 1.1

0.9 0.9 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.1 1.1 1.1 1.1

Ave L:H

4 hpi c

1.4 1.3 1.8 1.0 1.2 1.0 0.5 1.1 0.0 0.0 0.3

13 28 3 41 30 4 8 2 2 2 2

0.4 1.0 1.0 1.2

1.0 1.1 1.1 2.0

7 11 46 3 2 17 38 5

0.7 1.1 0.9 0.6 0.7 0.8 0.9 1.1 0.9 0.8 1.0 0.9 1.0 1.1 0.9 0.9 1.0

Ave L:H

3 2 5 5 5 6 23 2 44 8 12 22 3 8 10 6 11

Pep

10 hpi

cytosol

1.2 2.2 0.9 1.0 0.0 0.5 0.3 0.4 1.1 0.5

37 32 2 7 2 2 4 3

0.9 0.9 0.8

0.8 0.9 1.0 1.0

0.9 0.5 0.6 0.7 1.0 2.0 0.4 1.6 0.9 0.6 1.0 0.9 0.4 1.0 0.9 1.1 0.9 0.4

Ave L:H

12 29

22 41 2

6 5 48 8

9 2 5 2 3 6 20 2 35 9 12 25 5 11 8 3 9 6

Pep

24 hpi

0.9 0.9 1.0 0.5 0.6 1.0 1.4 0.6 0.4 0.7 0.6 1.0 0.7 0.8

3 8 6 3 11 6 12 4 2 2 5 8 10 6

0.9

0.9

2 18

1.0 0.9 1.0

0.6 1.0 7 9 7

2 62

0.7 0.8

0.9

5

5 16

0.7

Ave L:H

8

Pep

4 hpi

5

2 54 4 8 13 4

6 17

0.8

1.2 0.9 0.7 0.8 1.0 0.9

0.5 1.2

0.5 1.1 1.0 0.4

0.5

4

4 5 7 3

0.8 0.7

0.9 0.6

1.0

0.9

1.5

0.8

Ave L:H

3 7

3 8

9

6

2

7

Pep

10 hpi

nuclear

22

3 18 6 7 2

5 92

4 25 2

5 12 4 10 11

2 21 5 6

2 16

13 13

6

12

7

Pep

0.8

0.0 1.3 0.9 1.3 0.9

1.2 0.0

0.6 1.3 0.4

0.4 0.9 0.4 1.0 0.8

1.3 1.3 0.8 1.4

1.3 1.0

0.5 0.5

1.1

0.9

1.3

Ave L:H

24 hpi

Journal of Proteome Research Article

DOI: 10.1021/pr5012284 J. Proteome Res. 2015, 14, 2121−2142

HGNC

MARK1 TP53I3 SHPK nudt9 BAIAP2 GXYLT1 RANBP9 FLNC LAMC1 arsA Upf2 FAM114A1 KDM1A 15-Sep Mcat por akr1c3 Ctbp1 WDR33 Ankle2 C20orf117 LYPLA2 SUZ12 AATF Exosc3 Trove2 Gnas uaca prpf38a DPM1 POLR2G PKP3 SEC11A RRP9 KIAA0090 sfrs13b THOC3 TOR1AIP2 Kif22 KIAA1429 TACC1 mrps17

accession

153791472 22538446 74315356 13129010 9257197 153791362 39812378 188595687 145309326 6005990 18375676 29789373 58761546 42741648 51243059 127139033 24497583 4557497 56243590 148664230 66773344 9954875 197333809 7657013 50511943 108796056 117938759 59850762 24762236 4503363 4505947 6005830 7657609 4759276 22095331 148612890 14150171 21450775 6453818 33946282 170763517 7705425

Table 2. continued

name

MAP/microtubule affinity-regulating kinase 1 tumor protein p53 inducible protein 3 carbohydrate kinase-like nudix-type motif 9 isoform a BAI1-associated protein 2 isoform 1 glycosyltransferase 8 domain containing 3 isoform 1 RAN binding protein 9 gamma filamin isoform b laminin, gamma 1 precursor arylsulfatase A isoform a precursor UPF2 regulator of nonsense transcripts homologue hypothetical protein LOC92689 amine oxidase (flavin containing) domain 2 isoform b 15 kDa selenoprotein isoform 1 precursor mitochondrial malonyltransferase isoform a precursor cytochrome P450 reductase aldo-keto reductase family 1, member C3 C-terminal binding protein 1 isoform 1 WD repeat domain 33 isoform 1 ankyrin repeat and LEM domain containing 2 hypothetical protein LOC140710 WD repeat domain 46 joined to JAZF1 apoptosis antagonizing transcription factor exosome component 3 isoform 1 TROVE domain family, member 2 isoform 1 GNAS complex locus XLas uveal autoantigen with coiled-coil domains and ankyrin repeats isoform 1 PRP38 pre-mRNA processing factor 38 (yeast) domain containing A dolichyl-phosphate mannosyltransferase 1 DNA directed RNA polymerase II polypeptide G plakophilin 3 SEC11-like 1 RNA, U3 small nucleolar interacting protein 2 hypothetical protein LOC23065 serine-arginine repressor protein THO complex 3 torsin A interacting protein 2 kinesin family member 22 hypothetical protein LOC25962 isoform 1 transforming, acidic coiled-coil containing protein 1 isoform 1 mitochondrial ribosomal protein S17

Pep

b

Ave L:H

4 hpi c

2 2 2 5 2 2 4 8 4

Pep 0.3 0.4 0.4 0.4 0.5 0.5 0.6 0.6 0.9

Ave L:H

10 hpi

cytosol

3 23 2 2 2 2 3 2 2

Pep

0.3 0.7 0.5 0.2 0.2 0.4 0.5 0.5 1.7

Ave L:H

24 hpi

0.7 0.7 0.0 0.2 0.3 0.3 0.4 0.4 0.5 0.5 0.6 0.6 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.8 0.8 0.8 0.9 1.0

4 2 2 3 2 5 2 2 2 2 5 3 6 8 4 4 4 4 5 8 7 6 4 7 3 2

Ave L:H

2

Pep

4 hpi

0.6 1.1

1.5 1.1 0.5 0.4 0.8 0.6

0.8 0.6 1.0 0.6 0.4 0.6 0.6

2

2 2 2 2 3 6

5 5 5 5 2 2 4

Ave L:H

2

Pep

10 hpi

nuclear

2131

4 10 5 6

5 11 10

9 4

6 6

2 4 9 12

3 3 5

0.7 1.0 0.6 0.4

0.8 0.8 1.0

0.9 0.7

0.8 0.8

0.8 0.9 1.0 0.6

0.8 0.7 1.4

0.8 0.9 1.1 1.1

0.6

8

9 2 4 5

0.3

Ave L:H

3

Pep

24 hpi

Journal of Proteome Research Article

DOI: 10.1021/pr5012284 J. Proteome Res. 2015, 14, 2121−2142

Article

Protein is included if at least one of the biologic z-score values are ≤-1.960σ (indicated by bolding) and there are no major disagreements between biological replicates. bPep refers to the number of peptides used to identify the corresponding protein. cL:H ratio refers to the geometric mean of all log L/H values for each given gi number, expressed as a relative protein quantity in infected cultures.

Overall, 53 of the known 87 HSV1 gene products were detected,4 indicating a good sensitivity and reliability of our MS/MS for identifying proteins in HSV-1 infected cells. Validation

To confirm the validity of the SILAC process, we tested the differential regulation of select proteins for both cytosolic and nuclear fractions using Western blot, and compared them to the SILAC findings (Figure 2a,b). These proteins were chosen based on the availability of antibodies and the degree of differential regulation. Proteins that are more differentially regulated are more likely to be observed with the less sensitive Western blot assay. Cytosolic proteins confirmed with Western blot include RNA binding motif protein, X-linked (RBMX), ribosomal protein L14 (RPL14), nucleobindin 2 (NUCB2) and apolipoprotein E precursor (APOE). Nuclear proteins tested include NUCB2, dolichyl-phosphate mannosyltransferase 1 (DPM1), ribosomal protein L10 (RPL10), and ADP-ribosylation factor-like 1 (AKR1B10). All of these proteins showed a similar expression characteristic (up- or down-regulation) for both the SILAC and WB experiments. For loading controls, GAPDH was used in the cytosolic fraction and histone 3 was used in the nuclear fraction. These loading controls were used in densitometry analyses. In order to check for leakage of nuclear proteins in the cytosolic fraction during fractionation, histone 3 was also tested in the cytosolic fraction. There was no histone 3 present in the cytosolic fraction, indicating that the fractions were clean (data not shown). To confirm successful infection of our Western blot samples, an HSV tag antibody was used to determine the presence of HSV proteins in our infected or mock samples. This antibody is a synthetic antibody that recognizes the sequence QPELAPEDPED, an epitope of glycoprotein D, which is a late HSV envelope protein. By 24 h, there was a strong HSV signal in both the nuclear and cytosolic fractions, but not at earlier times, confirming the MS/MS results shown in Table 3. The mock samples (which were also harvested at 24 h) did not show HSV gD. To confirm that our HSV infections have similar characteristics to those reported in the literature, we tested our Western blot cell lysates for the down-regulation of protein kinase, DNA-activated, catalytic polypeptide (PRKDC) and SP100, two known proteins that decreased during HSV infection.28 Both of these proteins showed a decrease in abundance by 24 hpi in both the cytosolic and nuclear fractions. A summary of the densitometry analyses performed on representative blots for each protein shows the correlation of Western blot samples to the SILAC results (Figure 2c). Functional and Pathway Analyses

We used web-based proteomic tools to determine the cell metabolic pathways and functions that are altered during infection, by uploading our differentially regulated protein lists from either the cytosolic or nuclear fractions for each infection time point. Since mass spectrometry methods do not detect all cellular proteins in all experiments, pathways and functional analyses may still indicate the effects of HSV1 infection on cells even when not all the proteins of the pathway or function are detected by mass spectrometry. DAVID analysis29 (http://david.abcc.ncifcrf.gov/home.jsp) was utilized to determine the cellular functions that are enriched in our differentially regulated protein lists. Using a functional cluster tool, significantly altered gene ontology (GO) biological processes and molecular functions are grouped based on common metabolic pathways and are depicted in Figure 3.

a

0.4 0.7 0.0 0.0 0.6

Ave L:H Pep

7 4 2 53 4 0.7 0.8

Ave L:H Ave L:H Ave L:H Ave L:H name HGNC

SATB2 ARFGAP2 Pkp2 Npm1 CD59

accession

38016202 31543983 148664226 169162829 42761474

Table 2. continued

SATB homeobox 2 ADP-ribosylation factor GTPase activating protein 2 plakophilin 2 isoform 2b PREDICTED: similar to nucleophosmin 1 CD59 antigen preproprotein

Pep

b

4 hpi

c

Pep

10 hpi

cytosol

Pep

24 hpi

Pep

Ave L:H

Pep

10 hpi

nuclear

4 hpi

2 2

24 hpi

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Journal of Proteome Research Table 3. Number of Peptides Used in the Identification of HSV Proteins for All Time Points 4 hpi name transcriptional regulator ICP4 single-stranded DNA-binding protein multifunctional expression regulator ribonucleotide reductase subunit 1 regulatory protein ICP22 deoxyribonuclease major capsid protein DNA polymerase processivity subunit tegument protein VP22 thymidine kinase deoxyuridine triphosphatase

nucleus 13

10 hpi cytosol

nucleus

Proteins Identified from 4 hpi 2 18 Proteins Identified from 10 hpi 8 7 5 5 4 4 3 3 2

24 hpi cytosol

nucleus

cytosol

15

120

18

5 7 11

105 37 68 25 40 173 98 34 14 36

30 15 62 4 24 77 16 13 4 24

89 90 53 51 42 37 34 34 33 33 31 38 28 26 25 27 23 18 19 15 15 16 14 11 11 10 7 10 8 7 6 5 5 3 5 4 2 3 2

40 52 27 3 19 19 14 9 10 8 9 14 3 6 4 3 6 4 7 14 12 3 4 0 0 2 0 0 4 0 0 2 0 8 0 3 2 0 0 2 2 2

4 2 3

3 Protein Identified from 24 hpi

tegument protein VP13/14 large tegument protein tegument protein UL37 capsid maturation protease envelope glycoprotein B transactivating tegument protein VP16 capsid triplex subunit 1 tegument protein VP11/12 ubiquitin E3 ligase ICP0 envelope glycoprotein E DNA packaging tegument protein UL25 capsid triplex subunit 2 DNA packaging tegument protein UL17 envelope glycoprotein H small capsid protein nuclear egress membrane protein envelope glycoprotein C DNA polymerase catalytic subunit tegument protein UL21 envelope glycoprotein D tegument protein US11 envelope glycoprotein I virion protein US2 envelope glycoprotein M tegument host shutoff protein nuclear egress lamina protein envelope glycoprotein L serine/threonine protein kinase US3 tegument protein UL51 tegument protein UL16 capsid portal protein membrane protein UL45 tegument protein UL7 ribonucleotide reductase subunit 2 DNA replication origin-binding helicase membrane protein UL56 nuclear protein UL24 membrane protein US9 nuclear protein UL3 envelope glycoprotein G tegument host shutoff protein uracil-DNA glycosylase

The top five functional clusters are depicted in the legend for each chart. At the 4 hpi early time point, RNA processing/splicing was found in both cytosolic and nuclear

fractions (Figure 3a,b). Protein ubiquitination and cofactor binding clusters were also found in the nuclear fraction, while organelle, ribonucleoprotein complex, endocytosis, and 2133

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Figure 2. Western blot confirmation of selected differentially regulated proteins found in the (a) cytosolic fraction and (b) nuclear fraction of HSV infected HEK293 cells. (c) Densitometry analyses of the Western blot figures and comparison to SILAC-determined ratios. *Note: Western blot results shown are results from multiple different gels. Densitometry analysis was performed for each gel based on loading controls that were obtained separately for each gel. Mock lane is separate from the cytosolic fraction results due to omission of lanes, and rearranging blots, for organizational and spacesaving measures taken to compose the figure.

Using Ingenuity Pathway Analysis (Ingenuity Systems, www. ingenuity.com), we were able to generate networks of proteins based on the differentially regulated proteins found at each time point, for the nuclear and cytosolic fractions. This method of analysis differs from DAVID because the networks produced are based on direct protein−protein interactions that contain as many differentially regulated proteins (focus molecules) as possible, and provides biological functions based on the proteins in the network. DAVID, in contrast, identifies known biological pathways containing the most differentially regulated proteins. After IPA networks have been generated, the cellular functions and processes that are most relevant to the network are listed. The top networks of each sample are those that contain the most regulated focus molecules. The scoring of each network (p-score) is derived from the p-values determined for the network. The p-values are calculated by the probability of finding x number of focus proteins (up- or downregulated proteins)

regulation of apoptosis clusters were found enriched in the cytosolic fraction. At 10 hpi, enriched nuclear functions include ribosome, cytoskeleton, negative regulation of protein metabolic processes, organelle lumen, and DNA repair (Figure 3c). Enriched cytosolic functions include regulation of apoptosis, which started at 4 hpi, as well as response to oxidative stress, response to endogenous stimulus, and identical protein binding/protein homodimerization (Figure 3d). By the late time point of 24 hpi (Figure 3e,f), nuclear enriched functions include regulation of response to external stimulus, cytoskeleton, cell surface receptor linked signal transduction, protein complex assembly, and nuclear transport. Originally detectable in the nuclear fraction at 10 hpi, both ribosome and lumen functions are enriched at 24 hpi in the cytosol. The ribonucleoprotein complex is also enriched, which started at 4 hpi in the cytosol. As well, iron−binding, DNA packaging, and protein folding are also enriched. 2134

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Figure 3. DAVID cluster analysis of differentially regulated proteins (a−f). Top enriched cellular functions for each time point in both the cytosolic and nuclear fractions are listed to the right of each chart.

in the network size of n proteins (usually 35), calculated using Fisher’s exact test. The p-score concentrates on the exponent and is determined using the calculation:

At 10 hpi, nuclear processes include DNA replication and repair and inflammatory response (p-scores of 17 and 4, respectively), while cell death and survival dominate the cytosolic cellular process changes (p-score =13) (Figure 4c,d). Finally, at 24 hpi, nuclear processes include cellular movement and gene expression (p-scores of 19 and 16, respectively), and cytosolic processes include RNA post-transcriptional modification (p-score 21), gene expression (p-score 17), cell death (p-score 10) and cellular assembly (p-score 6) (Figure 4e,f). The networks that contain the most focus molecules were found at 24 hpi in the cytosol. The RNA post-transcriptional modification network contains 14 focus molecules out of 35 (Figure 5), and the gene expression, cell death, and survival network also contains 14 focus molecules.

p‐score = − log 10(p‐value)

As a specific example, the top network at 24 hpi in the cytosol (RNA post-transcriptional modification) scored 21 indicated that the p-value is 10−21 and very significantly regulated. Proteins in each network are color coded based on the regulation observed from the sample. Up-regulated proteins are indicated in red/pink, and the downregulated proteins are in green. Proteins that are not identified in the sample, but are interconnecting molecules in the network, are white. These white nodes are added to the network based on the most connection to the focus molecules. At 4 hpi, the nuclear proteins differentially regulated after infection pertain to gene expression (p-score =31), and the cytosolic proteins function in post-translational modification and gene expression (p-score =16) (Figure 4a,b, respectively).

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DISCUSSION We utilized SILAC to obtain lists of differentially regulated proteins that are affected after HSV1 infection, in the same manner that we previously analyzed mammalian subtype 1 Lang 2135

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Figure 4. (a−f) IPA top network analysis of differentially regulated proteins. Functions of the top networks generated with the most differentially regulated proteins for each time point in both the cytosolic and nuclear fractions are listed to the right of each chart.

reovirus infection23b and influenza virus infection.23a There have been a few previously published HSV proteomics studies, most dealing with viral proteins, and some examining host proteins. Most of the host proteomic literature focuses on cellular compartments such as ribosomes,20a microsomes,20b the nucleus,20c and the secretome of macrophages.21 Most recently one group identified host proteins incorporated into mature virions using mass spectrometry.19 One previously published study examined HEp-2 type cells only at 6 hpi by 2D-DIGE coupled with LC−MS/MS and found 103 proteins differentially regulated by HSV infection.22 That study found DNA replication, chromatin remodeling, mRNA stability, and ER stress response pathways were affected. In our study, we obtained a global proteomic profile of abundance changes at different times during HSV infection, using early (4 h), mid (10 h), and late (24 h) time points to represent immediate early, early, and late viral gene expression patterns. By using SILAC and 2D-LC-based rather than gel-based resolution,

we were able to identify thousands of proteins and hundreds of differentially regulated proteins. We used separate cytosolic and nuclear fractions to aid in identifying more proteins throughout the cell. This also allows for a more comprehensive analysis by analyzing the fractions separately, generating metabolic functions that are specific for cellular localization. The DAVID network analysis indicates some nuclear-based pathways highlighted in the cytosolic fraction, such as RNA splicing at 4 hpi. However, this does not necessarily indicate contamination between the nuclear and cytosolic fraction. For example, proteins that function in the RNA splicing pathway are transcribed in the cytosol and may be in the process of being up-regulated for functional purposes but have yet to translocate to the site of cellular activity. During Western blot analysis the use of histones show that the cytoplasmic fraction is not significantly contaminated by the nuclear fraction. In any case, the main goal of using two separate fractions for analysis was for the identification of more proteins than a whole cell analysis would provide. 2136

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Figure 5. IPA analysis. Top network generated in cytosolic fraction at 24 hpi (RNA post-transcriptional modifications). Red, gray, green, and white colored proteins represent up-, non-, and down-regulation, or lack of detection, respectively.

The HSV protein vhs is known to destabilize both host and viral mRNA with the specific purpose of stunting the antiviral immune response and alleviating the translational apparatus when the host is overloaded during virus infection, to facilitate viral gene expression.12b,30 It is generally thought that the vhs protein targets all host mRNA; however it cuts mRNAs at preferred sites, and mutations in these sites can lead to enhanced or severely decreased ability of vhs to cleave mRNA.31 This preference may also play a role in how effective the protein is at targeting one host protein mRNA over another, leading to differences in degradation levels. The vhs protein has been shown in some studies to enhance degradation of some host mRNA levels while stabilizing or delaying degradation of other host mRNA levels.32 This could lead to up-regulation of certain host proteins and is reflected in our current study that identified an approximate even number of up- and down-regulated proteins for each time point. The sets of regulated proteins that we identified show similarity to previously published host proteomic studies. In the study by Antrobus et al.,22 NONO (non-POU domain containing, octamer-binding) and pyridoxal kinase were found up-regulated at 6 hpi, while vimentin and methionine aminopeptidase 1 (METAP1) were found down-regulated. In our study, NONO was found up-regulated at 4 and 24 hpi in the cytosolic fraction, pyridoxal kinase and METAP1 were found down-regulated at 4 hpi, and vimentin was up-regulated at 24 hpi. Vimentin is a filament protein that may interact with a virus during infection.33 The up-regulation of vimentin found previously at 6 hpi in the Artrobus study22 and subsequent down-regulation of this protein in our study at 24 hpi may indicate that vimentin’s function changes in regard to virus interaction during infection. Pyridoxal kinase is a metabolic enzyme that is a cofactor of numerous pathways, including antiviral defense mechanisms.34

We found that this protein was down-regulated at 4 hpi, but it was up-regulated at 6 hpi in the study by Antrobus et al. This apparent discrepancy may be the result of a difference in cell type used or the fact that pyridoxal kinase was identified with only one peptide in the previous study. This difference in regulation may also be an indication that this protein is strictly regulated during virus infection. The regulation observed from both studies may be due to a change in protein function targeted by the virus in a time-dependent manner. NONO has a role in transcription, RNA processing, and DNA double-strand break (DSB) repair, and METAP1 functions in facilitating intracellular translocation of newly synthesized proteins from the ribosome. Both of these proteins may play important roles in HSV transcription.35 Lippe et al.36 identified numerous host proteins inside mature HSV virions. Of these proteins, keratin 1 and 10, macrophage migration inhibitory factor (MIF), a protein that has immunoregulatory functions in innate and adaptive immunity, were upregulated at 24 hpi in our study. Finally, Miettinen et al.21 characterized the secretome of HSV1-infected human primary macrophages. Of the proteins that are secreted at 18 hpi, heat shock protein 105 kDa, a protein known to independently prevent the aggregation of misfolded proteins37 and nucleophosmin, which has been recently shown to be critical for DNA DSB repair,38 were found in our study. Heat shock protein 105 kDa was down-regulated in the nucleus at 4 hpi, and nucleophosmin was up-regulated in the cytosol at 24 hpi. These proteins warrant further study to see if they play an important role in HSV infection, as they have previously been associated with HSV infection and have functional roles that are relevant to viral infections. IE, E, and L Phases

Using our up- and down-regulated proteins, we identified cellular metabolic pathways that are most affected during infection by 2137

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Figure 6. IPA analysis. RNA post-transcriptional modification pathway represented at the (a) 4 hpi immediate early (b) 10 hpi early and (c) 24 hpi late phases of infection. H2be (HIST2H2BE) and the beta hemoglobin (HBB) proteins are up-regulated at all three times post infection, whereas other proteins are only affected at one time point (such as RNA polymerase II at 6 hpi).

HSV1 in either the cytosolic or nuclear fractions, at each time point, with the aid of pathway analysis tools IPA and DAVID.29

DAVID analysis provides general gene ontology (GO) terms and known cellular pathways that are most enriched in our data set, 2138

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comparison to the T1L reovirus SILAC study indicating that host response is virus-specific. Specifically, apoptosis is seen regulated early in both infections, 6 hpi in reovirus and 4 hpi in HSV.23b However, RNA post-transcriptional modifications are observed early in reovirus infection, but are not regulated until the late 24 hpi time point in HSV infections. This is understandable because reovirus is an RNA virus that replicates in the cytosol; therefore, RNA post-transcriptional modification is utilized sooner during its life cycle.

whereas IPA allows a more detailed analysis by generating networks of interconnecting proteins (by direct relationships) that include as many differentially regulated proteins as possible and listing the top biological functions that pertain to the network as a whole. Even with the difference in analysis, we observed similar results. For the 4 hpi immediate early time point, IPA analysis of the nuclear fraction highlighted gene expression, cell growth, and proliferation, whereas RNA processing was a top DAVID enriched cellular function. These cellular functions are likely regulated by the virus to promote viral transcription. DAVID analysis also indicated up-regulation of protein ubiquitination and degradation, which is known to be promoted by the action of the IE protein ICP0.39 In the cytosol at 4 hpi, IPA analysis indicated cellular death is a major affected function, and DAVID analysis also indicated that apoptosis was affected. By the 10 hpi early phase, apoptosis regulation was still a factor in the cytosol for both IPA and DAVID analyses, whereas ribosome, DNA replication, recombination and repair topped the nuclear function list of both analyses. These functions are regulated because more viral genes are being expressed during this time in the virus life cycle and the cell is preparing for protein processing.4 In addition, the virus replicates by recombination and repair mechanisms. The inflammatory response observed in the nuclear fraction (IPA) and the response to stimulus occurring in the cytosolic fraction (DAVID) are due to the host responding to the infecting virus. Finally, at 24 hpi, the late phase of HSV infection included high expression of viral genes, RNA export and post-translational modifications and protein synthesis.4 DAVID analysis indicated the ribonucleoprotein complex and ribosomal subunit are utilized as well as DNA packaging functions, whereas IPA listed RNA post-transcriptional modification and gene expression as top metabolic functions.

Novel Proteins in RNA Post-Transcriptional Modification

Examination of the top cytosolic functions at 24 hpi (Figure 5) revealed that 12 of the total 35 proteins listed in the network have been previously mentioned in HSV literature studies to have a role in HSV infections. These are activating transcription factor 6 (ATF6), caspase 7 (CASP7), histone cluster 1 H4a (HIST1H4A), histone cluster 2 H2be (HIST2H2BE), heat shock 70 kDa protein 5 (HSPA5), lysine-specific demethylase 1A (KDM1A), non-POU domain containing, octamer-binding protein (NONO), p85, protein arginine methyltransferase 1 (PRMT1), RNA polymerase II, tumor necrosis factor (TNF) and vimentin (VIM). A portion of ATF6 was found to be homologous to the herpes simplex viral protein VP1641 and its regulation suggests a role in HSV gene expression. Inhibition of HSV-1-induced PI3K activity increases cleavage of CASP7.42 HIST1H4 is mobilized during infection with herpes simplex virus 1.43 Early transcription of HISTH2BE is detected in HSV1 infected corneas of 6-week-old female BALB/c mice44 in latency studies; however, it is also repressed by immediate early genes in vitro.45 HSPA5 levels in cells will either increase or decrease in HSV1 infection depending on the HSV1 infecting strain, which may lead to differences in the virus interaction with the host.46 KDM1A has been recently identified as a novel factor to regulate herpesviral gene expression47 as well as blocking replication of the virus and reactivation from latency.48 HSV is known to stimulate phosphatidylinositol 3-kinase signaling, which involves p85 interaction.49 PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.50 HSV utilizes RNA polymerase II for the transcription of viral genes. The immediate early protein ICP22 alters the RNA polymerase II protein to promote HSV infection.51 TNF-alpha is known to play a protective role in HSV-1 infection.52 Changes in the structure of a host cell has been observed during HSV infection, including the thinning and even distribution of vimentin fibers throughout the cell.53 NONO, as previously mentioned,22 has been found up-regulated after HSV infection in another study; however the role it plays during infection is unknown. For this same network, there are 14 proteins that we have shown to be regulated in our study, indicating they play a role in HSV infection. The HIST1H4A, HIST2H2BE, HSPA5, KDM1A, NONO, and VIM proteins in this network are previously mentioned in HSV literature to have a role in HSV infection (as mentioned above), but the other eight differentially regulated proteins have no known function in regards to HSV infection. However, some of these differentially regulated proteins have known roles in other virus infections. HBB is observed to be decreased in individuals that are infected with HIV.54 HNRNPA2B1 and HNRNPC proteins enhance pre-mRNA processing in Epstein−Barr virus infection,55 and HNRNPA2B1 also inhibits influenza A replication.56 HNRNPU is a potential HIV restriction factor during infection.57 NPM1 has an important role in Eptein-Barr virus infection of chaperoning the nuclear

Time Course Changes in Host Proteomic Response to Infection

Comparing the top functions from one time point to other time points in our study gives insight into the progression of the cellular changes during the life cycle of the virus. By overlaying the cytosolic 4 hpi or 10 hpi proteomic data onto the top 24 hpi cytosolic function (RNA post-transcriptional modification) in IPA, we are able to generate network pictures representing up- and down-regulated proteins for each time point in the same network (Figure 6). This network is largely up-regulated at 24 hpi; however both the histone cluster 2, H2be (HIST2H2BE), and the beta hemoglobin (HBB) proteins are up-regulated at all three times post infection. HIST2H2BE has been previously identified in HSV studies, and HBB has been identified in other viral studies. Comparison to Other Virus Proteomic Studies

Not surprising, there were differences in the data generated with HSV infected cells in comparison to our previous reovirus study. In T1L reovirus infection, there are more than twice as many up-regulated proteins than down-regulated proteins at the 6 h (132 and 68 proteins up- and down-regulated, respectively) and 24 h (104 and 49 proteins up- and down-regulated, respectively) time points.23b However, in this HSV SILAC study, up- and down-regulated proteins were observed in close to a 1:1 ratio (Figure 1g). A relatively greater percentage of down-regulated proteins during HSV infection was expected since HSV is known to effectively shut down host protein synthesis.40 Similarities and differences in the host pathways regulated after infection are observed in our HSV SILAC experiment in 2139

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(2) Cai, W.; Schaffer, P. A. Herpes simplex virus type 1 ICP0 regulates expression of immediate-early, early, and late genes in productively infected cells. J. Virol 1992, 66 (5), 2904−15. (3) Aubert, M.; Rice, S. A.; Blaho, J. A. Accumulation of herpes simplex virus type 1 early and leaky-late proteins correlates with apoptosis prevention in infected human HEp-2 cells. J. Virol. 2001, 75 (2), 1013− 30. (4) Wagner, E. K.; Sandri-Goldin, R. M. Herpes Simplex Viruses: Molecular Biology. In Encyclopedia of Virology, 3rd ed.; Mahy, B., Van Regenmortel, M. H. V., Eds.; Elsevier Inc.: Oxford, UK, 2008; pp 397− 405. (5) Lehman, I. R.; Boehmer, P. E. Replication of herpes simplex virus DNA. J. Biol. Chem. 1999, 274 (40), 28059−62. (6) Roizman, B. The checkpoints of viral gene expression in productive and latent infection: the role of the HDAC/CoREST/LSD1/REST repressor complex. J. Virol. 2011, 85 (15), 7474−7482. (7) Smith, M. C.; Boutell, C.; Davido, D. J. HSV-1 ICP0: paving the way for viral replication. Future Virol. 2011, 6 (4), 421−429. (8) Everett, R. D.; Maul, G. G. HSV-1 IE protein Vmw110 causes redistribution of PML. EMBO J. 1994, 13 (21), 5062−5069. (9) Lilley, C. E.; Chaurushiya, M. S.; Boutell, C.; Landry, S.; Suh, J.; Panier, S.; Everett, R. D.; Stewart, G. S.; Durocher, D.; Weitzman, M. D. A viral E3 ligase targets RNF8 and RNF168 to control histone ubiquitination and DNA damage responses. EMBO J. 2010, 29 (5), 943−955. (10) Cliffe, A. R.; Knipe, D. M. Herpes simplex virus ICP0 promotes both histone removal and acetylation on viral DNA during lytic infection. J. Virol. 2008, 82 (24), 12030−12038. (11) (a) Melroe, G. T.; Silva, L.; Schaffer, P. A.; Knipe, D. M. Recruitment of activated IRF-3 and CBP/p300 to herpes simplex virus ICP0 nuclear foci: Potential role in blocking IFN-beta induction. Virology 2007, 360 (2), 305−21. (b) Orzalli, M. H.; DeLuca, N. A.; Knipe, D. M. Nuclear IFI16 induction of IRF-3 signaling during herpesviral infection and degradation of IFI16 by the viral ICP0 protein. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (44), E3008−17. (12) (a) Esclatine, A.; Taddeo, B.; Evans, L.; Roizman, B. The herpes simplex virus 1 UL41 gene-dependent destabilization of cellular RNAs is selective and may be sequence-specific. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (10), 3603−3608. (b) Everly, D. N., Jr.; Feng, P.; Mian, I. S.; Read, G. S. mRNA degradation by the virion host shutoff (Vhs) protein of herpes simplex virus: genetic and biochemical evidence that Vhs is a nuclease. J. Virol. 2002, 76 (17), 8560−8571. (c) Zelus, B. D.; Stewart, R. S.; Ross, J. The virion host shutoff protein of herpes simplex virus type 1: messenger ribonucleolytic activity in vitro. J. Virol. 1996, 70 (4), 2411−9. (d) Smiley, J. R.; Elgadi, M. M.; Saffran, H. A. Herpes simplex virus vhs protein. Methods Enzymol. 2001, 342, 440−451. (13) (a) He, B.; Gross, M.; Roizman, B. The gamma(1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc. Natl. Acad. Sci. U. S. A. 1997, 94 (3), 843−848. (b) Mohr, I.; Gluzman, Y. A herpesvirus genetic element which affects translation in the absence of the viral GADD34 function. EMBO J. 1996, 15 (17), 4759−4766. (14) (a) Berkhout, B.; Coombs, K. M. Quantitative omics and its application to study virus-host interactions-a new frontier. Front. Microbiol. 2013, 4, 31. (b) Kilcher, S.; Mercer, J. Next generation approaches to study virus entry and infection. Curr. Opin. Virol. 2013, 4C, 8−14. (15) (a) Coombs, K. M. Quantitative proteomics of complex mixtures. Expert Rev. Proteomics 2011, 8 (5), 659−77. (b) Zhou, S.; Liu, R.; Zhao, X.; Huang, C.; Wei, Y. Viral proteomics: the emerging cutting-edge of virus research. Sci. Chin. Life Sci. 2011, 54 (6), 502−512. (16) Santamaria, E.; Sanchez-Quiles, V.; Fernandez-Irigoyen, J.; Corrales, F. Contribution of MS-Based Proteomics to the Understanding of Herpes Simplex Virus Type 1 Interaction with Host Cells. Front. Microbiol. 2012, 3, 107. (17) (a) Fontaine-Rodriguez, E. C.; Taylor, T. J.; Olesky, M.; Knipe, D. M. Proteomics of herpes simplex virus infected cell protein 27:

antigen 2 protein onto latency-associated membrane protein 1 during infection.58 Finally, RPL19 is known to regulate TLR3 signaling, which can affect the host cell response to virus infection,59 and some viruses require the use of RLP19 for host cell replication.60 Since they are already established in other virus systems, these proteins are good target protein candidates for HSV infection. To our knowledge, the remaining two differentially regulated proteins (FUS and HYOU1) in this network are novel with respect to virus infection and warrant further investigation because they may provide novel understanding of HSV-induced pathogenesis. FUS has been recently identified to target RNA for pre-mRNA alternative splicing as well as processing of long intron-containing transcripts,61 which may be important for replication of the virus. HYOU1 is known to function in apoptosis as well as chaperoning ER proteins.62

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CONCLUSION In conclusion, we have used SILAC to identify a number of additional novel candidate proteins, more focused study of which should provide greater understanding of HSV infection as well as a better understanding of how to use this virus as an effective research tool to study HSV virulence mutants (e.g., vector or vaccine candidates) or other herpesviruses. Specifically, we were able to characterize the host protein expression profile in regard to the different phases of the HSV1 life cycle, namely, immediate early, early, and late phases. The response of the host proteome reflects the virus gene expression and replication for each specific time point, giving us a substantial overview of the virus−host interaction evolution throughout the viral life cycle.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figure 1: Legend for IPA derived networks including path designer shapes and relationship status symbol shown in each network. Supplementary Table 1: HEK293 proteins increased >95% confidence including each experimental L:H and corresponding z-score. Supplementary Table 2: HEK293 proteins decreased >95% confidence including each experimental L:H and corresponding z-score. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: National Microbiology Laboratory, 1015 Arlington Street Winnipeg, MB, Canada, R3E 3R2. Tel.: 204 780 6022. Fax: 204 789 2140. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) (a) Bossi, P. [Genital herpes: epidemiology, transmission, clinic, asymptomatic viral excretion, impact on other sexually transmitted diseases, prevention, and treatment]. Ann. Dermatol. Venereol. 2002, 129 (4 Pt 2), 477−93. (b) Chentoufi, A. A.; Benmohamed, L. Mucosal herpes immunity and immunopathology to ocular and genital herpes simplex virus infections. Clin. Dev. Immunol. 2012, 2012, 149135. (c) Vyse, A. J.; Gay, N. J.; Slomka, M. J.; Gopal, R.; Gibbs, T.; MorganCapner, P.; Brown, D. W. The burden of infection with HSV-1 and HSV-2 in England and Wales: implications for the changing epidemiology of genital herpes. Sex. Transm. Infect. 2000, 76 (3), 183−7. (d) Grinde, B., Herpesviruses: latency and reactivation - viral strategies and host response. J. Oral Microbiol. 2013, 5. 2140

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