Vaccinia Virus Proteome: Identification of Proteins in Vaccinia Virus Intracellular Mature Virion Particles

0022-538X/06/$08.00

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doi:10.1128/JVI.80.5.2127–2140.2006

Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vaccinia Virus Proteome: Identification of Proteins in Vaccinia Virus

Intracellular Mature Virion Particles

Che-Sheng Chung,

† Chein-Hung Chen,

† Ming-Yi Ho,

Cheng-Yen Huang,

Chung-Lin Liao,

* and Wen Chang

*

Institute of Molecular Biology

and The Genomics Research Center,

Academia Sinica, Taipei, Taiwan, Republic of China

Received 25 October 2005/Accepted 5 December 2005

Vaccinia virus is a large enveloped poxvirus with more than 200 genes in its genome. Although many poxvirus

genomes have been sequenced, knowledge of the host and viral protein components of the virions remains

incom-plete. In this study, we used gel-free liquid chromatography and tandem mass spectroscopy to identify the viral and

host proteins in purified vaccinia intracellular mature virions (IMV). Analysis of the proteins in the IMV showed

that it contains 75 viral proteins, including structural proteins, enzymes, transcription factors, and predicted viral

proteins not known to be expressed or present in the IMV. We also determined the relative abundances of the

individual protein components in the IMV. Finally, 23 IMV-associated host proteins were also identified. This study

provides the first comprehensive structural analysis of the infectious vaccinia virus IMV.

Vaccinia virus is the prototype virus of the orthopoxvirus

genus in the family

Poxviridae

, which replicates in the

cyto-plasm of cells (57, 104) and encodes more than 200 open

reading frames (ORFs) in a 190-kb double-stranded DNA

genome. Vaccinia virus infection produces multiple forms of

infectious particles, namely, intracellular mature virions (IMV),

intracellular enveloped virions (IEV), cell-associated

envel-oped virions (CEV), and extracellular envelenvel-oped virions

(EEV). The IMV is the most abundant virion, with a single

membrane in cells; however, the origin of the membrane is

unknown (75, 77). A portion of the IMV is subsequently

wrapped with two layers of Golgi membrane to form an IEV,

which is transported through microtubules to the cell periphery

and loses one membrane during virion egress to become a

CEV (130, 140, 152, 153). The CEV remains associated with

the cell surface, where actin-containing microvilli are formed,

or can be released by host cell Src/Abl kinases into the medium

to become an EEV (41, 42, 61, 111, 156). The IMV is robust

and is known to be resistant to environmental and physical

changes, whereas the CEV and EEV are very fragile, and the

integrity of their outer membranes can be destroyed during

purification procedures (83).

Many of the poxvirus genomes, including those of different

strains of vaccinia virus, have been sequenced (http://www

.poxvirus.org/viruses.asp). The genome of the vaccinia virus

Western Reserve (WR) strain contains 218 potential ORFs;

however, the existence of an ORF does not necessarily reveal

the existence of a protein or the location of the protein. While

classical biochemical and genetic approaches have generated

significant knowledge of viral-gene functions, the physical

com-position of vaccinia virus IMV particles remains unknown.

Previous researchers have analyzed the protein composition

of the vaccinia virus IMV. Initial studies of the purified IMV

led to the identification on sodium dodecyl

sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) of 17 “regions”

con-taining 31 to 48 protein bands (103, 115, 139). Essani and

Dales increased the gel resolution and detected 56 polypeptide

bands on SDS-PAGE (54). Using different gel electrophoretic

conditions, Oie and Ichihashi found 84 protein spots that could

be assigned to 52 protein species in purified IMV (116).

Un-fortunately, the naming system for vaccinia virus proteins in

the above studies was based on the protein migration behavior

on gels, making it difficult to compare data generated in

dif-ferent gel electrophoresis systems. In addition, the majority of

viral genes encode proteins smaller than 50 kDa that tend to

cluster together and are difficult to resolve by SDS-PAGE.

Moreover, N-terminal sequencing of viral protein bands on

gels revealed that multiple protein bands can be derived from

a single viral gene as a result of posttranslational cleavage or

modification and that the 40 to 50 IMV bands seen on gels

were derived from only 12 viral genes (160). Similarly, Jensen

et al. found that the purified vaccinia virus core and membrane

contain 30 proteins encoded by 13 viral genes and 5 host genes;

additional, less abundant virion components were detected but

not identified (86).

Mass spectrometry (MS), in particular, tandem MS (MS/

MS), provides a powerful tool for proteome analysis because it

is much more sensitive than other methods, can deal with

protein mixtures, and offers a high throughput (120).

Wash-burn et al. identified 1,484 proteins in the yeast proteome using

gel-free liquid chromatography and tandem mass spectroscopy

(LC/MS/MS) (175). Similar analyses have been used to identify

virion proteins in human and murine cytomegalovirus,

Ep-stein-Barr virus, and Kaposi’s sarcoma-associated herpesvirus

(87, 92, 172, 191). The identification of virion proteins makes it

possible to start determining how the proteins interact, their

stoichiometry in the viral particle, and which cellular

compart-ments are involved in virion formation. In this study, we used

* Corresponding author. Mailing address for W. Chang: Institute

of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of

China. Phone: 886-2-2789-9230. Fax: 886-2-2782-6085. E-mail: mbwen

@ccvax.sinica.edu.tw. Mailing address for C.-L. Liao: The Genomics

Research Center, Academia Sinica, Taipei, Taiwan, Republic of China.

Phone: 886-2-2652-3926. Fax: 886-2-2782-9143. E-mail: clliao@gate

.sinica.edu.tw.

† Che-Sheng Chung and Chein-Hung Chen contributed equally to

this work.

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LC/MS/MS analysis to determine the viral and host proteins

that comprise each IMV particle. New components of vaccinia

virus IMV encoded by putative ORFs were discovered, and the

relative abundance of each component in the IMV was

deter-mined.

MATERIALS AND METHODS

Cells, virus, and reagents.HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Vaccinia virus (Western Reserve strain) was propagated in HeLa cells and purified as described previously (80, 86). In brief, the infected cells were harvested when the cytotoxic

pathological effect was complete. All stages of purification were performed at 4°C. The cells were centrifuged at 850⫻gfor 15 min to remove the medium and washed three times with phosphate-buffered saline. The pelleted cells were resuspended in TM buffer (10 mM Tris, pH 7.4, 5 mM MgCl2), and the

suspen-sion was passed 20 times in a Dounce homogenizer to break the cells. Nuclei were removed by centrifugation at 850⫻gfor 10 min, and the supernatant, containing virions, was laid on top of 36% sucrose solution and centrifuged at 45,000⫻gfor 80 min in a Beckman SW28 rotor. The virus pellet was resus-pended in TM buffer, sonicated, and further purified by centrifugation through a continuous 25 to 40% sucrose gradient at 27,500⫻gfor 40 min in a Beckman SW28 rotor. Fractions were taken, and the virion infectivity in each fraction was analyzed by a plaque formation assay. Fractions containing virions were diluted fourfold in TM buffer and centrifuged to pellet the IMV. The purity of the IMV

FIG. 1. (A) Purified IMV. Electron microscopy of purified IMV particles using negative uranyl acetate staining (left) and silver staining of IMV

proteins, 340 ng (lane 1) and 170 ng (lane 2), on 12% SDS-PAGE (right). (B) MS/MS spectrum of one tryptic peptide with a sequence identified

as HAFDAPTLYVK and with a Mascot score of 72. (C) Amino acid sequence of the putative E6R protein. Tryptic peptides detected by MS,

including the peptide in panel B, are underlined and give 53% sequence coverage. (D) MS/MS spectrum of one tryptic peptide with a sequence

identified as ADEDDNEETLK and with a Mascot score of 80. (E) Amino acid sequence of A27L envelope protein. Tryptic peptides detected by

MS, including the peptide in panel D, are underlined and give 71% sequence coverage.

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was confirmed by electron microscopy of negatively stained preparations. No detectable cellular debris was found in these preparations.

Tryptic and lysine C digestion of IMV particles.Purified IMV (9 to 50␮g) were incubated at 25°C overnight with 0.5 N cyanogen bromide (Fluka) in 90% formic acid (Fluka) to cleave the proteins into smaller polypeptides, and excess reagents were removed by vacuum drying. The polypeptides were denatured and reduced at 37°C for 1 h in 6 M urea (Sigma), 2 M thiourea (Riedel-de Hae¨n), and 10 mM dithiothreitol (Pharmacia Biotech), and then iodoacetamide (Sigma) was added to a final concentration of 20 mM and the sample was incubated at 37°C for 1 h in the dark. The peptide mixture was then diluted eightfold with 100 mM ammonium bicarbonate (Sigma), and sequencing-grade porcine trypsin (Pro-mega) or Lys-C (Wako) was added at a substrate-to-enzyme ratio of 20:1 (wt/wt), and the mixture was incubated at 37°C overnight. The peptides formed were desalted using a C18trap, lyophilized on a SpeedVac, and stored at⫺80°C.

Two-dimensional chromatography.The peptide mixture from 50␮g of IMV was fractionated by two-dimensional chromatography (strong cation-exchange chromatography [SCX]-reverse-phase liquid chromatography). The first dimen-sion, SCX, was eluted with a linear gradient of 0 to 300 mM KCl in 5 mM ammonium formate, pH 3.0. The peptides eluted from the SCX column were trapped in two C18reverse-phase traps operating alternately; the bound peptides

were eluted at 2-min intervals and collected on a fraction collector; a total of 22 fractions were collected. The amount of peptide in each fraction was estimated from the UV absorption at 214 nm.

Mass spectrometry. Mass spectrometric analysis was performed on a nanoscale LC-tandem mass spectrometry (quadrupole time-of-flight mass spec-trometer; QStar XL; Applied Biosystems). The instrument setup was as follows. The flow (150␮l/min) from the binary pump (Agilent 1100, with solvent A [100% deionized water] and solvent B [90% acetonitrile] [J. T. Baker], both solvents containing 0.1% formic acid) was split with two T-shaped connectors connected to a self-packed precolumn with an appropriate flow restrictor to give a column flow rate of 10␮l/min for sample loading and 200 to 300 nl/min for sample elution from the analytical column. The sample of 2 to 5␮g was injected onto the precolumn (15 mm long; 150-␮m internal diameter; C18) via a 20-␮l sample loop.

The analytical column (C18; 15 cm long; 75-␮m internal diameter) was connected

to a 15-mm electrospray emitter (10-␮m tip opening) by a 1-cm Teflon sleeve. The chromatographic separation was performed with a 120-min gradient profile as follows: 2% B (0 to 4.5 min), linear gradient of 2 to 10% B (4.5 to 5 min), 10 to 40% B (5 to 80 min), 40 to 50% B (80 to 100 min), 50 to 80% B (100 to 105 min), 80 to 2% B (105 to 106 min), and 2% B (106 to 120 min). The spectra of the eluted peptides were acquired in data-dependent mode by first acquiring a full MS scan fromm/z400 to 1900 for 1 second to determine the three most

intense peptide peaks with charge states above 2, and then three MS/MS scans betweenm/z100 and 2000 (1.5 s each) were performed for the MS-scanned parent ions with a threshold above 20 counts. Once sampled, each MS/MS precursor mass was excluded from further tandem experiments for 2 min.

The data files completed from the LC-MS runs were converted to Mascot generic-format files using the Mascot.dll script supplied with the Analyst QS software. The Mascot software package (Matrix Science) was used for database searching and protein identification using the modified vaccinia virus protein database. Peptide mass tolerance and fragment mass tolerance were set at 100 ppm and 0.25 Da, respectively, for the initial search. An alternative calibration algorithm based on Mascot protein identifications was applied to the raw data file to give mass accuracies within 20 ppm.

Proteomic analysis of IMV-derived peptides and database generation. Indi-vidual MS/MS spectra were submitted to analysis using MASCOT version 2.0 (Matrix Science Inc.) and searched against the Human International Protein Index protein sequence database (version 3.10; 57,478 protein sequences; Euro-pean Bioinformatics Institute [http://www.ebi.ac.uk/IPI/]) or against a modified vaccinia virus protein database. The modified viral-protein database combines 218 protein sequences of the WR strain of vaccinia virus obtained from http: //poxvirus.org, 64 nonredundant Copenhagen strain orthologs, and 12 additional peptide sequences predicted from WR viral genome sequences using two pro-grams, GeneMarkS (http://opal.biology.gatech.edu/GeneMark/) and fgenesV (http://softberry.com; 17). These databases allowed the identification of novel vaccinia virus proteins by an exhaustive search of all possible virus-derived peptide sequences. Proteins were scored using a probability-based MOWSE algorithm, and the scores were reported in the form⫺10⫻log(P), wherePis the probability that the observed match is a random event (123). Matches with scores higher than the 95% confidence level were regarded as significant. In all searches, phosphorylation, methionine oxidation, homoserine lactone, and car-bamidomethylation of cysteine residues were considered as possible modifica-tions. In order to increase the data confidence, the same data sets were submitted for a search against a “reverse database,” in which all peptide sequences were put in reverse order in silico; the percentage of peptides identified in the reverse database was less than 3.5%, confirming the specificity of the data search. Individual peptide interpretations were classed as significant if the MASCOT MOWSE score for the MS/MS spectra was greater than the cutoff value of 25.

Relative quantification of MS.Quantification of protein abundance in IMV was performed based on the criteria and equations defined previously (85). In brief, the relative abundances of the proteins were estimated using an exponen-tially modified protein abundance index (emPAI) approach, which is based on the correlation of peptides identified in MS/MS experiments with observable

FIG. 1—

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TABLE 1. Vaccinia virus IMV proteins identified by LC/MS/MS

VACV-WR ORF

Mass (Da)

VACV-CP

ORF Description

CNBr-trypsin digestion CNBr-LysC digestion

Reference(s) No. of

unipeptides

Sequence coverage (%)

No. of unipeptides

Sequence coverage (%)

VACV-WR_022 17,311 C6L Unknown 1 7

VACV-WR_035 48,842 K4L Phospholipase D-like 5 14 3 8 21, 24

VACV-WR_047 7,846 F8L Protein with iActA-like proline repeats not required for actin tail formation

3 46 76

VACV-WR_048 23,776 F9L S-S bond formation pathway thiol substrate 5 27 2 12 146

VACV-WR_049 52,098 F10L Ser/Thr kinase 12 31 7 22 98

VACV-WR_056 11,329 F17R Putative DNA-binding phosphoprotein in virus core

8 56 5 54 86, 90, 91, 121,

182, 190 VACV-WR_057 55,496 E1L Poly(A) polymerase catalytic subunit VP55 10 20 2 4 65 VACV-WR_060 29,808 E4L DNA-dependent RNA polymerase subunit rpo30,

intermediate-gene transcription factor VITF1 TFIIS-like

8 28 3 15 1, 31

VACV-WR_062 66,685 E6R Unknown 30 53 12 27

VACV-WR_064 31,868 E8R Membrane protein may help wrap virosome associated with IV/IMV and core F10L kinase substrate

11 52 3 13 51

VACV-WR_066 10,844 E10R S-S bond formation pathway sulfhydryl oxidase, substrates L1R/F9L

3 32 145

VACV-WR_067 14,890 E11L Virion core protein 2 20 1 10 174

VACV-WR_069 12,347 O2L Nonessential glutaredoxin not part of E10R-G4L S-S bond formation pathway

6 47 2 24 4, 88, 178

VACV-WR_070 35,819 I1L DNA binding core protein 6 18 3 11 148

VACV-WR_071 8,493 I2L IMV membrane protein required for membrane fusion

2 36 1 20 112

VACV-WR_072 29,978 I3L ssDNA-binding phosphoprotein 5 22 1 4 47, 168, 177

VACV-WR_074 8,738 I5L IMV protein VP13 1 12 1 12 84, 160

VACV-WR_076 48,979 I7L Viral core cysteine proteinase 12 24 5 9 10, 32, 33, 89

VACV-WR_077 77,550 I8R RNA-helicase, DExH-NPH-II DNA-helicase aided by L4R

16 29 7 14 13, 56, 93, 150

VACV-WR_078 67,997 G1L Insulin metalloproteinase-like has ENE and HLLEH inverse of HXXEX sites

16 29 3 7 74

VACV-WR_079 12,795 G3L Unknown 2 19 1 19

VACV-WR_081 13,978 G4L S-S bond formation pathway thioredoxin-like 3 29 2 12 72, 86, 178, 179

VACV-WR_083 7,283 G5.5R DNA-dependent RNA polymerase subunit rpo7 3 47 8, 106

VACV-WR_085 41,924 G7L Virion structural protein 16 38 9 27 107, 157, 158, 160

VACV-WR_087 38,761 G9R Myristylprotein 2 7 2 7 102

VACV-WR_088 27,262 L1R IMV membrane protein target of neutralizing antibody S-S bond formation pathway thiol substrate; myristylprotein

6 26 3 10 59, 86, 102, 126,

127, 146, 184

VACV-WR_090 40,595 L3L Unknown 11 29 4 10

VACV-WR_091 28,439 L4R Core protein vp8 microtubule-associated, ssRNA/ ssDNA-binding protein

21 66 9 31 86, 121, 124, 160,

181, 186 VACV-WR_092 15,034 L5R IMV membrane protein required for membrane

fusion

2 25 1 6 165

VACV-WR_093 17,910 J1R Virion protein required for morphogenesis 7 34 4 24 37, 38 VACV-WR_095 38,863 J3R Multifunctional poly-A polymerase subunit, cap

methyltransferase, and transcription elongation factor

13 48 5 21 65, 141

VACV-WR_096 21,328 J4R DNA-dependent RNA polymerase subunit rpo22 6 33 1 4 29, 79, 164 VACV-WR_098 146,740 J6R DNA-dependent RNA polymerase subunit rpo147 47 39 15 13 29, 53, 79, 164

VACV-WR_099 19,713 H1L Tyr/ser protein phosphatase 8 49 4 26 69, 100, 110

VACV-WR_100 21,529 H2R IMV membrane protein required for membrane fusion

1 6 1 3 142

VACV-WR_101 37,422 H3L IMV heparin binding surface protein involved in IMV maturation

20 54 10 40 44–46, 86, 97, 160

VACV-WR_102 93,574 H4L RAP94 tightly associated with DNA-dependent RNA polymerase, aids early-stage transcription, preinitiation, and termination

27 38 9 13 2, 5, 189

VACV-WR_103 22,287 H5R Morphogenesis-related, substrate of B1R kinase late gene transcription factor VLTF-4

4 28 2 15 14, 15, 16, 27, 94

VACV-WR_104 36,642 H6R Topoisomerase type IB 11 46 2 7 149,151

VACV-WR_106 96,673 D1R Large subunit of mRNA capping enzyme transcription termination factor VTF

30 39 16 24 109, 173

VACV-WR_107 16,935 D2L Virion core protein 5 36 3 21 52

VACV-WR_108 27,973 D3R Virion core protein 7 41 1 3 52

VACV-WR_111 73,784 D6R 70kDa small subunit of early gene transcription factor VETF

14 27 4 9 28, 66

VACV-WR_112 17,900 D7R DNA-dependent RNA polymerase subunit rpo18 1 6 1 8 3, 125 VACV-WR_113 35,424 D8L IMV membrane protein binds cell surface

chondroitin may effect viral entry

11 51 5 20 80, 86, 114

VACV-WR_116 72,264 D11L ATPase, nucleoside triphosphate phosphohydrolase-I, NPH-I transcription elongation, termination, release factor

14 25 6 14 30, 93, 131

VACV-WR_117 33,330 D12L Small subunit of mRNA capping enzyme transcription termination factor VTF

11 41 6 33 113, 173

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peptides obtained by in silico digestion, which allows the relative abundances of proteins in a complex protein mixture to be determined. The abundance of each protein is presented as the molar and weight percentages of the total protein molecules in the IMV particles.

RESULTS

Identification of IMV proteins.

Vaccinia IMV particles were

purified from the lysates of HeLa cells infected with the

vac-cinia virus WR strain, using two consecutive sucrose

purifica-tion procedures to achieve maximal purity. The purified IMV

particles were examined by electron microscopy to ensure that

the virions had normal virion morphology and that cellular

organelles and debris were undetectable. Proteins in purified

virions were separated on 12% SDS-PAGE and silver stained

(Fig. 1A). Gel electrophoresis of the purified virions followed

by in-gel digestion often causes loss of proteolytic fragments

generated from low-abundance proteins, and the gel matrix is

inherently not optimal for resolving proteins of low molecular

weight. Thus, a gel-free scheme with two-stage proteolysis was

adopted to facilitate the efficient extraction of peptides from

purified IMV particles. The purified IMV were completely

dissolved in 90% formic acid and subjected to cyanogen

bro-mide cleavage, followed by tryptic digestion in solution to

sequentially yield a mixture of peptides. Alternatively,

proteo-lytic digestion with Lys-C was performed in order to preserve

longer peptides for better quality in data acquisition. These

peptides were then analyzed by MS/MS using a modified

vac-cinia virus (WR) protein database constructed (see Materials

and Methods) to include all predicted vaccinia virus translation

products, as the commonly used vaccinia virus (WR) strain

TABLE 1—

Continued

VACV-WR ORF

Mass (Da)

VACV-CP

ORF Description

CNBr-trypsin digestion CNBr-LysC digestion

Reference(s) No. of

unipeptides

Sequence coverage (%)

No. of unipeptides

Sequence coverage (%) VACV-WR_118 61,852 D13L Rifampicin target associates with inner surface

immature virus membrane

1 2 2 3 11, 108, 161, 162

VACV-WR_121 8,922 A2.5L S-S bond formation pathway CxxxC links SH-oxidase E10R and thioredoxin G4L

4 36 3 42 147

VACV-WR_122 72,578 A3L p4b precursor of core protein 4b 46 65 15 34 121, 135, 160

VACV-WR_123 30,908 A4L 39-kDa core protein complexes with core protein p4a/4a

14 55 5 13 49, 86, 121

VACV-WR_124 18,984 A5R DNA-dependent RNA Polymerase subunit rpo19 5 45 2 12 6

VACV-WR_125 43,147 A6L unknown 5 14 6 23

VACV-WR_126 82,220 A7L 82-kDa large subunit of early gene transcription factor VETF

23 33 14 22 66

VACV-WR_128 12,102 A9L IMV membrane protein required for morphogenesis

1 11 187

VACV-WR_129 102,209 A10L Precursor p4a of core protein 4a, complexes with A4L

69 62 20 23 86, 121, 160, 171

VACV-WR_132 7,691 A13L IMV membrane protein 5 55 2 42 86, 160, 169

VACV-WR_133 9,987 A14L Phosphorylated IMV membrane protein required for morphogenesis

3 25 1 8 18, 86, 133, 134,

160, 167

VACV-WR_135 10,944 A15L Unknown 2 32 1 14

VACV-WR_136 43,396 A16L Soluble myristylprotein 7 25 1 5 102

VACV-WR_137 22,984 A17L IMV membrane protein undergoes phosphorylation and proteolytic processing; required for morphogenesis

5 21 3 17 50, 86, 95, 133,

160, 183 VACV-WR_138 56,679 A18R DNA helicase effects elongation and termination

of postreplicative viral transcription

8 17 1 4 93, 188

VACV-WR_140 13,636 A21L IMV membrane protein required for membrane fusion

1 10 166

VACV-WR_142 21,865 A22R Palmitylprotein; Holliday junction endonuclease; resolves viral DNA concatemers into unit length genomes

1 5 63, 67

VACV-WR_144 133,279 A24R DNA-dependent RNA polymerase subunit rpo132 35 34 14 13 9, 78 VACV-WR_148 84,299 A25L Gene fragment, cowpox A-type inclusion protein 34 44 13 19 62 VACV-WR_149 57,957 A26L p4c protein; IMV membrane protein required for

directing IMV into A-type inclusion body

23 49 10 23 105

VACV-WR_150 12,564 A27L IMV surface protein roles in IMV-cell attachment, fusion, and microtubule transport

10 71 3 24 39, 86, 101, 132,

138, 160 VACV-WR_151 16,319 A28L IMV membrane protein required for membrane

fusion

3 23 1 8 143, 144

VACV-WR_152 35,370 A29L DNA-dependent RNA polymerase rpo35 6 21 2 9 9

VACV-WR_153 8,740 A30L IMV protein for association of dense viroplasm with viral membranes during morphogenesis

2 54 107, 157, 158, 159

VACV-WR_154 14,200 A31R Unknown - 1 13

VACV-WR_167 15,043 A42R Profilin-like 8 57 2 12 20

VACV-WR_171 13,654 A45R Inactive Cu-Zn superoxide dismutase-like in virion

4 25 1 7 7

VACV-WR_172 27,618 A46R Toll/IL1-receptor [TIR]-like suppresses TIR-dependent signal transduction host defense modulator

2 7 25, 155

a

The data presented in Table 1 are compiled from three different IMV preparations. Tryptic digestions of different IMV preparations revealed 92% reproducibility, and Lys-C digestions revealed 87% reproducibility. Peptides generated by both trypsin and Lys-C are required to obtain a total of 75 viral proteins: 65 proteins (86.7%) were detected in peptides generated by either trypsin or Lys-C digestions, 9 proteins (12%) were detected only in trypsin-digested peptides, and 1 protein (1.3%) was detected only in Lys-C-digested peptides. VACV, vaccinia virus; CP, Copenhagen strain; ssDNA, single-stranded DNA; ssRNA, single-stranded RNA.

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protein database does not include all predicted viral proteins

or small proteins with fewer than 50 amino acids. As shown in

Fig. 1B, the MS/MS spectrum of a tryptic peptide was

inter-preted as HAFDAPTLYVK, one of 30 unique peptides

iden-tified in a putative protein, WR062/E6R, resulting in 53%

sequence coverage (Fig. 1C). Another tryptic peptide, ADED

DNEETLK, was identified in an envelope protein, A27L (Fig.

1D), that contains 71% sequence coverage, as shown in Fig.

1E. A total of three different IMV preparations were used in

five independent mass spectrometry analyses. Combining the

peptide data generated from trypsin and Lys-C digests, we

identified 75 viral proteins in the IMV (Table 1). Because the

gene names in the vaccinia virus WR strain are not as widely

used as their orthologs in the Copenhagen strain, the latter

gene names are used to refer to the viral translation products

we identified in this work. Of these, 69 were identified by at

least two trypsin- or Lys-C-generated peptides, while the other

6 (C6L, I5L, A9L, A21L, A22R, and A31R) were identified by

single peptide hits in the trypsin- or Lys-C-digested samples. A

further study using two-dimensional chromatographic

fraction-ation to separate peptides prior to MS/MS was performed

(SCX-LC/MS/MS), and at least one extra peptide (each) was

identified for C6L, A9L, A21L, and A31R (Table 2). All the

extra peptides identified in the SCX-LC/MS/MS study had

MASCOT scores of at least

⬎

28. No extra peptide was

iden-tified for I5L and A22R by SCX-LC/MS/MS, although the

same peptides as in one-dimensional LC/MS/MS were

repro-ducibly detected. Both proteins are included in Table 1,

be-cause they were previously detected in the IMV (63, 84, 160).

Of the 75 viral proteins identified, 65 were consistent with

previous reports of their presence in IMV particles (references

in Table 1); these were 22 membrane proteins (H3L, D8L,

H2L, L1R, L5R, A2.5, A9L, A13L, A14L, A17L, A21L, A25L,

A26L, A27L, A28L, E8R, E10R, F9L, I2L, I5L, J1R, and

D13L), 12 core proteins (A3L, A4L, A10L, A30L, F17R,

E11L, I1L, I3L, G7L, L4R, D2L, and D3R), 24 enzymes

[ki-nase (F10L), phosphatase (H1L), glutaredoxins (G4L and O2L),

topoisomerase (H6R), proteinases (G1L and I7L), ATPase

(D11L), poly(A) polymerase subunits (E1L and J3R), capping

enzymes (D1R and D12L), DNA-dependent RNA polymerase

complexes (A5R, A24R, A29L, J4R, J6R, H4L, E4L, G5.5R,

and D7R), helicases (I8R and A18R), and endonuclease

(A22R)], 3 viral transcription factors (A7L, H5R, and D6R),

and 4 proteins (A42R, A45R, A46R, and F8L) interacting with

host proteins. This shows that our proteomic data are reliable

and consistent with previous results. More importantly, the

proteomic data revealed 10 new viral proteins present in IMV,

consisting of the products of seven former putative ORFs

(E6R, G3L, L3L, A6L, A15L, A31R, and C6L) and three

proteins (K4L, G9R, and A16L) previously with unknown

lo-cations and functions. K4L shows homology with

phospho-lipase D but is nonessential for viral growth in cell cultures (21,

24, 122), while both G9R and A16L have been identified as

myristylated proteins in virus-infected cells (102).

Six viral proteins, I6L, G5R, A12L, A14.5, A32L, and B1R,

have been cited in the literature as being IMV associated but

were not identified in our analyses (Table 3) (19, 34, 43, 48, 68,

99, 160). All six proteins were not detected in LC/MS/MS using

different IMV preparations, although three proteins (A12L,

A32L, and B1R) were detected in SCX-LC/MS/MS, suggesting

that the abundances of these three proteins were below the

threshold of LC/MS/MS and that SCX chromatography was

necessary to reduce the sample complexity in order to enhance

the detection sensitivity (Table 3). Consistent with our

inter-pretation, A32L was previously shown to be difficult to detect

in purified IMV (34). The other three proteins (I6L, G5R, and

A14.5L) were not identified in either LC/MS/MS or SCX-LC/

MS/MS analyses. Several possible reasons may explain why

they were not detected in both MS/MS analyses. First, MS is

biased against small and hydrophobic proteins. A14.5L

con-tains 53 amino acids, producing at most two tryptic peptides

(19). It is also very hydrophobic, with a grand average of

hydropathicity index of 1.438 (96). These features of A14.5L

may have jeopardized its detection in MS analyses. Second,

MS, despite great sensitivity, can only detect peptides at

fem-tomole range. Third, peptides that are too close to be resolved

in MS will also be ignored by data acquisition software. We

speculate that these technical reasons might explain the lack of

detection of G5R and I6L.

Relative abundances of viral proteins in the IMV.

The

quan-tity of each viral component within the IMV particle is an

important issue, but it has been difficult to address in the past.

Crude estimation of the quantity of each protein by gel staining

intensity is not suitable for complex structure analysis. While

TABLE 2. Viral proteins identified by LC/MS/MS by a single tryptic peptide and confirmed by SCX-LC/MS/MS

VACVa -WR ORF

Reverse-phase LC/MS/MS SCX followed by reverse-phase LC/MS/MS

Reference for IMV association No. of

total hits

No. of unique peptides

Mascot score

Peptide sequence (CNBr-trypsin)b

No. of total

hits

No. of unique peptides

Mascot score

Peptide sequence (CNBr-LysC)

VACV-WR_022(C6L) 1 1 30 YYDGNIYELAK 3 2 66 ADSFSLESDSIK

32 IDAVRYYDGNIYELAK

VACV-WR_074(I5L) 2 1 39 VISGAALIVK 3 1 72 VISGAALIVK 160

VACV-WR_128(A9L) 2 1 35 LRPNSFWFVVVR 4 2 46 SCYTAILK 187

28 RNESSINSNSSPK

VACV-WR_140(A21L) 5 1 42 LFSYNFTTSGIK 6 4 40 NVPIPCSK 166

49 INEVNNNK 40 DVDTLYCDK

31 NFICVDDRLFSYNFTTSGIK

VACV-WR_142(A22R) 1 1 32 DNSVRVLDI(p)SK 1 1 34 DNSVRVLDI(p)SK 63

VACV-WR_154(A31R) 1 1 33 YLSGGGIYHDDLVVLGK 4 2 31 VTINNLK

33 YLSGGGIYHDDLVVLGK a

VACV, vaccinia virus. b

(p), predicted phosphorylated site.

on November 8, 2019 by guest

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recent advances in differential protein analyses have provided

a means for comparing relative protein expression between

two populations, there is a need for a method giving the

rela-tive quantification of a single protein in a complex population.

Although quantification of particular proteins of interest using

isotope-labeled synthetic peptides, termed Protein-AQUA, is

in principle applicable to comprehensive analyses (12, 64), it is

hampered by the high cost of isotope-labeled peptides and the

difficulty of quantitative digestion of proteins in gel (73).

Recently, Ishihama et al. demonstrated that it is feasible to

quantify label-free protein components using an emPAI

ap-proach (85), which fully utilizes the advantages of the wide

dynamic detection range provided by LC/MS analysis and

re-liable protein search algorithms. This approach to determining

the relative abundances of proteins in a complex protein

mix-ture is based on the correlation of peptides identified by

tan-dem MS with observable peptides obtained by in silico

diges-tion. The abundance of each protein determined in this study

is presented as the molar and weight percentages of the total

protein molecules in IMV particles (Table 4). The most

abun-dant viral-protein group (weight percentage,

⬎

5) consisted of

four core proteins (A4L, A10L, F17R, and A3L). The relatively

abundant protein group (5

⬎

weight percentage

⬎

1) consisted of

seven envelope proteins (A27L, A25L, A14L, H3L, A26L, D8L,

and A13L,), two core proteins (L4R and G7L), four proteins

involved in transcription (D11L, J6R, A24R, and D1R), and

one novel protein (E6R). Eleven of the above 18 abundant

proteins have been previously identified as abundant proteins

in IMV particles (86, 160). The intermediate-abundance

pro-tein group (1%

⬎

weight percentage

⬎

0.5%) contained 15

viral proteins, and the less abundant group (weight percentage,

⬍

0.5%) contained 41 viral proteins. The distribution of viral

proteins based on different molar or weight abundances in

IMV revealed a similar distribution (Fig. 2); the low

abun-dances of many proteins explain why they have escaped

previ-ous proteomic detection.

Identification of host proteins associated with the IMV.

The

host protein content of vaccinia virus IMV was determined by

comparing the detected peptides with a Human International

Protein Index protein sequence database, and 23 host proteins

associated with vaccinia virus IMV particles were identified

(Table 5). All were present at low abundance (intermediate

and less abundant categories) in virions and could be divided

into several categories, namely, calcium binding proteins

(an-nexin A1 and A2), chaperon proteins (cyclophilin A, Hsc71,

and Hsp90), cytoskeleton proteins (actin, tubulin, and myosin),

chromosome architecture proteins (histone and HMG1),

transla-tion components (eIF4A-1, eIF1

␣

-1-1, and 60S ribosomal

pro-tein), protein transport/vesicular-trafficking proteins (ADP

ribo-sylation factors1/3 and 4, Rab7, Rab10, and ubiquitin), and

proteins involved in redox regulation (thioredoxin and

peroxire-doxin 1). Of these host proteins, cyclophilin A,

␤

-actin, and

ubiquitin have been previously reported as components of

vac-cinia virus IMV particles (35, 86, 176), and another 13 have

been reported in other viruses (Table 5) (22, 87, 92, 172, 191).

Thus, seven host proteins (60S acidic ribosomal proteins,

66-kDa protein, ADP ribosylation factor 4, HMG1, peroxiredoxin

1, Rab10, and thioredoxin) were found to be associated with

vaccinia virus IMV and have not yet been detected in other

viruses.

DISCUSSION

Proteomic techniques are widely used to identify protein

components of large complexes that participate in different

biological processes. MS remains the most suitable tool, as it is

fast, sensitive, and widely applicable. Current high-sensitivity

methods using MS for protein identification have greatly

low-ered detection limits, making it easy to analyze proteins with

low cell copy numbers. In addition, the quality of

bioinformat-ics continues to improve, making the prediction of gene

prod-ucts deduced from genome information increasingly accurate

when MS data are matched to known protein sequences. The

use of nano-LC for the high-resolution separation of digested

peptides and nano-electrospray ionization/MS analysis allows

protein detection in the low femtomole range in routine

anal-ysis. This approach can also be used to complement traditional

biochemical methods of elucidating viral structures and

func-tions and determining the number and nature of proteins

within a virus. In this study, we identified 75 viral and 23 host

proteins associated with purified vaccinia virus IMV particles,

a significant increase from previous studies (86, 160). Ten

TABLE 3. Virion proteins cited in literature but not identified by LC/MS/MS

VACVa -WR ORF

LC/MS/MS SCX-LC/MS/MS _Reference

showing IMV association No. of

unique peptides

No. of total

hits

No. of unique peptides

Mascot score

Peptide sequence (CNBr-LysC)

VACV-WR_075(I6L)

0

0

0

48, 68

VACV-WR_082(G5R)

0

0

0

43

VACV-WR_131(A12L)

0

8

5

57

SSSSSTSASK

160, 180

41

NLLAQIGGDAAVK

56

DGQIVQAVTNAGK

40

VGEINHDLLGIDSVNAGK

25

NLAVRSSYDDYIETVNK

VACV-WR_134(A14.5L)

0

0

0

19

VACV-WR_155(A32L)

0

1

1

29

NCFQEK

34

VACV-WR_183(B1R)

0

4

3

41

NQWVVGPLIGK

99, 128

29

ASNIVLDQIDK

53

LYLVDYGLVSK

a

VACV, vaccinia virus.

on November 8, 2019 by guest

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TABLE 4. Relative viral protein abundances in vaccinia virus IMV

VACV-WR ORF

VACV-CP

ORF Description

Try-emPAI Reference for

previous proteomic identification Rank by

wt %

Average

emPAI Mol% Weight % VACV-WR_123 A4L 39-kDa core protein complexes with core protein p4a/4a 1 81.73 20.10 24.04 86 VACV-WR_129 A10L Precursor p4a of core protein 4a, complexes with A4L 2 7.75 1.91 7.54 86, 160 VACV-WR_056 F17R Putative DNA binding phosphoprotein in virus-core 3 69.00 16.97 7.44 86

VACV-WR_122 A3L p4b precursor of core protein 4b 4 8.04 1.98 5.55 160

VACV-WR_150 A27L IMV surface protein roles in IMV-cell attachment, fusion, and microtubule transport

5 34.21 8.41 4.09 86, 160

VACV-WR_091 L4R Core protein vp8 microtubule-associated, ssRNA/ssDNA binding protein

6 13.00 3.20 3.52 86, 160

VACV-WR_148 A25L Gene fragment, cowpox A-type inclusion protein 7 4.23 1.04 3.40 VACV-WR_133 A14L Phosphorylated IMV membrane protein required for

morphogenesis

8 33.77 8.31 3.21 86, 160

VACV-WR_101 H3L IMV heparin binding surface protein involved in IMV maturation

9 8.65 2.13 3.08 86, 160

VACV-WR_149 A26L Gene fragment, cowpox A-type inclusion protein 10 5.11 1.26 2.82 VACV-WR_116 D11L ATPase, nucleoside triphosphate phosphohydrolase-I, NPH-I

transcription elongation, termination, release factor

11 3.76 0.93 2.59

VACV-WR_113 D8L IMV membrane protein binds cell surface chondroitin, may effect viral entry

12 7.09 1.74 2.39 86, 160

VACV-WR_062 E6R Unknown 13 2.78 0.68 1.77

VACV-WR_132 A13L IMV membrane protein 14 23.42 5.76 1.71 86, 160

VACV-WR_098 J6R DNA-dependent RNA polymerase subunit rpo147 15 1.04 0.25 1.45 VACV-WR_144 A24R DNA-dependent RNA polymerase subunit rpo132 16 1.00 0.25 1.27 VACV-WR_106 D1R Large subunit of mRNA capping enzyme transcription

termination factor VTF

17 1.14 0.28 1.05

VACV-WR_085 G7L Virion structural protein 18 2.52 0.62 1.01 160

VACV-WR_167 A42R Profilin like 19 5.88 1.45 0.84

VACV-WR_078 G1L Insulin metalloproteinase-like; has ENE and HLLEH inverse of HXXEX sites

20 1.16 0.28 0.75

VACV-WR_064 E8R Membrane protein may help wrap virosome; associates with IV/IMV and core F10L kinase substrate

21 2.40 0.59 0.73

VACV-WR_071 I2L Unknown 22 9.00 2.21 0.73

VACV-WR_077 I8R RNA helicase, DExH-NPH-II DNA helicase aided by L4R 23 0.90 0.22 0.66 VACV-WR_126 A7L 82-kDa large subunit of early gene transcription factor

VETF

24 0.83 0.20 0.65

VACV-WR_117 D12L Small subunit of mRNA capping enzyme transcription termination factor VTF

25 2.02 0.50 0.64

VACV-WR_124 A5R DNA-dependent RNA polymerase subunit rpo19 26 3.45 0.85 0.62

VACV-WR_108 D3R Virion core protein 27 2.30 0.57 0.61

VACV-WR_099 H1L Tyr/Ser protein phosphatase 28 3.22 0.79 0.60

VACV-WR_095 J3R Multifunctional poly(A) polymerase subunit, cap methyltransferase, and transcription elongation factor

29 1.59 0.39 0.59

VACV-WR_093 J1R Virion protein required for morphogenesis 30 3.34 0.82 0.57 VACV-WR_102 H4L RAP94 tightly associated with DNA-dependent RNA

polymerase, aids early-stage transcription preinitiation and termination

31 0.63 0.16 0.56

VACV-WR_103 H5R Morphogenesis-related, substrate of B1R kinase late-gene transcription factor VLTF-4

32 2.38 0.59 0.50

VACV-WR_076 I7L Viral core cysteine proteinase 33 1.07 0.26 0.50

VACV-WR_048 F9L S-S bond formation pathway thiol substrate 34 1.95 0.48 0.44 VACV-WR_069 O2L Nonessential glutaredoxin not part of E10R-G4L S-S bond

formation pathway

35 3.22 0.79 0.38

VACV-WR_104 H6R Topoisomerase type IB 36 1.05 0.26 0.37

VACV-WR_107 D2L Virion core protein 37 2.18 0.54 0.35

VACV-WR_111 D6R 70-kDa small subunit of early-gene transcription factor VETF

38 0.49 0.12 0.34

VACV-WR_151 A28L Unknown, putative signal peptide 39 2.06 0.51 0.32

VACV-WR_083 G5.5R DNA-dependent RNA polymerase subunit rpo7 40 4.60 1.13 0.32 VACV-WR_153 A30L IMV protein for association of dense viroplasm with viral

membranes during morphogenesis

41 3.77 0.93 0.31

VACV-WR_049 F10L Ser/Thr kinase 42 0.62 0.15 0.31

VACV-WR_090 L3L Unknown 43 0.67 0.16 0.26

VACV-WR_137 A17L IMV membrane protein undergoes phosphorylation and proteolytic processing; required for morphogenesis

44 1.18 0.29 0.26 86, 160

VACV-WR_060 E4L DNA-dependent RNA polymerase subunit rpo30, intermediate-gene transcription factor; VITF1 TFIIS like

45 0.86 0.21 0.24

VACV-WR_057 E1L Poly(A) polymerase catalytic subunit VP55 46 0.41 0.10 0.22

VACV-WR_035 K4L Phospholipase D like 47 0.46 0.11 0.21

VACV-WR_152 A29L DNA-dependent RNA polymerase rpo35 48 0.63 0.15 0.21

VACV-WR_079 G3L Unknown 49 1.70 0.42 0.21

VACV-WR_135 A15L Unknown 50 1.98 0.49 0.21

VACV-WR_096 J4R DNA-dependent RNA polymerase subunit rpo22 51 1.00 0.25 0.20

VACV-WR_072 I3L ssDNA binding phosphoprotein 52 0.64 0.16 0.18

VACV-WR_070 I1L DNA binding core protein 53 0.49 0.12 0.17

VACV-WR_171 A45R Inactive Cu-Zn superoxide dismutase like in virion 54 1.24 0.30 0.16

VACV-WR_125 A6L Unknown 55 0.37 0.09 0.15

VACV-WR_088 L1R IMV membrane protein target of neutralizing antibody S-S bond formation pathway thiol substrate; myristyl protein

56 0.57 0.14 0.15 86

Continued on facing page

on November 8, 2019 by guest

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novel viral proteins were identified in IMV (Table 1).

EEV-specific viral proteins, such as F13L, A33R, A34R, A36R, and

B5R, and viral nonstructural proteins, such as A11R, were not

detected in our analyses, supporting the idea that the 10 novel

viral proteins are not just copurified lysate contaminants (129,

137). If we include the additional six virion proteins cited from

the literature (Table 3), there are a total of 81 viral proteins in

IMV.

Furthermore, the relative abundance of each viral and host

protein in IMV was determined by calculating the emPAI

value, providing important quantification information for

pro-teomic experiments. The most abundant viral proteins were in

general agreement with previous reports (86), validating our

quantification methods. In addition, our analysis also revealed

that a significant proportion of virion proteins were present at

low abundance. It is worth noting that the quantification

method based on the emPAI value is fairly new, and the

rel-ative abundance would be better utilized to compare proteins

of different abundance groups (Fig. 2) rather than to compare

proteins within the same low-abundance group.

In addition to the viral proteins, 23 IMV-associated host

proteins were identified. Compared with the data

reproduc-ibility of 75 viral proteins obtained from different IMV

prep-arations, the host proteins indeed revealed somewhat more

variation from one preparation to another. Several, such as

ARF1/3, ARF 4, Rab-7, and Rab-10, are involved in transport

and vesicle trafficking, and their association with IMV particles

may be due to the intracellular route taken by the IMV during

virion egress. Some host proteins, such as tubulin, actin,

an-nexin A2, cyclophilin A, Hsc71, and Hsp90, have been

identi-fied in other virions, such as human cytomegalovirus, murine

cytomegalovirus, and adenovirus (Table 3). It is worth noting

that our previous study (82) showed a transient association of

Hsp90 with viral factories in cells, while no Hsp90 was detected

by immunoblot analysis in purified IMV. Since MS/MS is much

more sensitive than immunoblot detection, this discrepancy

could be due to the low abundance of Hsp90 associated with

viral particles. Although we cannot rule out the possibility that

some of the cytoskeleton and chaperon proteins could be

for-tuitously associated with viral particles during IMV

prepara-tion, as previously shown by Franke and Hruby (58), other

FIG. 2. Distribution of viral proteins with different abundances in

IMV. All the viral proteins identified in this study were quantified as

described in Materials and Methods and divided into four categories

based on the protein contents in IMV, as shown below the graph. The

black columns show the molar percentages of the total proteins in

IMVs, while the white columns show the weight percentages.

TABLE 4—

Continued

VACV-WR ORF

VACV-CP

ORF Description

Try-emPAI Reference for

previous proteomic identification Rank by

wt %

Average

emPAI Mol% Weight %

VACV-WR_136 A16L Soluble myristyl protein 57 0.32 0.08 0.13

VACV-WR_081 G4L S-S bond formation pathway; thioredoxin like 58 0.93 0.23 0.12 86

VACV-WR_066 E10R S-S bond formation pathway sulfhydryl oxidase, substrates L1R/F9L

59 1.03 0.25 0.11

VACV-WR_121 A2.5L S-S bond formation pathway CXXXC links SH oxidase E10R and thioredoxin G4L

60 1.20 0.30 0.10

VACV-WR_112 D7R DNA-dependent RNA polymerase subunit rpo18 61 0.58 0.14 0.10 VACV-WR_138 A18L DNA helicase effects elongation and termination of

postreplicative viral transcription

62 0.18 0.05 0.10

VACV-WR_074 I5L IMV protein VP13 63 1.15 0.28 0.10 160

VACV-WR_047 F8L Protein with iActA-like proline repeats not required for actin tail formation

64 1.24 0.30 0.09

VACV-WR_087 G9R Myristyl protein 65 0.23 0.06 0.08

VACV-WR_100 H2R Unknown 66 0.38 0.09 0.08

VACV-WR_092 L5R Putative membrane protein 67 0.39 0.10 0.06

VACV-WR_142 A22R Palmityl protein; Holliday junction endonuclease; resolves viral DNA concatemers into unit-length genomes

68 0.26 0.06 0.05

VACV-WR_128 A9L IMV membrane protein required for morphogenesis 69 0.46 0.11 0.05

VACV-WR_067 E11L Virion core protein 70 0.33 0.08 0.05

VACV-WR_118 D13L Rifampin target associates with inner surface immature virus membrane

71 0.08 0.02 0.05

VACV-WR_140 A21L Unknown 72 0.33 0.08 0.04

VACV-WR_022 C6L Unknown 73 0.26 0.04

VACV-WR_172 A46R Toll/IL1-receptor (TIR) likesuppresses TIR-dependent signal transduction host defense modulator

74 0.15 0.04 0.04

VACV-WR_154 A31R Unknown 75

a_{Data presented in Table 4 are compiled from three different IMV preparations. VACV, vaccinia virus; CP, Copenhagen strain; ssRNA, single-stranded RNA;} ssDNA, single-stranded DNA.

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TABLE

5.

Host

proteins

identified

in

vaccinia

virus

IMV

Protein content (mol%)

No. of unipeptides Sequence coverage (%) emPAI

Protein content (mol%)

IPI00008530 34,252 60S acidic ribosomal protein P0 2 6 0.52 0.13 IPI00333428 66,265 66-kDa protein 1 1 0.06 0.02 1 1 0.06 0.07 IPI00215914 20,684 ADP ribosylation factor 1 and/or 3 1 5 0.29 0.07 1 9 0.58 0.68 HCMV 172 IPI00215918 20,367 ADP ribosylation factor 4 2 9 0.67 0.16 IPI00218918 38,559 Annexin A1 2 8 0.33 0.08 HCMV, MCMV 92, 172 IPI00455315 38,449 Annexin A2 7 26 0.51 0.13 HCMV, KSHV 172, 185, 191 IPI00021439 41,710 Actin, cytoplasmic 1 8 30 0.57 0.14 2 5 0.12 0.14 ASFV, HCMV, HIV, KSHV, MCMV, VV 55, 86, 92, 119, 172, 191 IPI00639885 50,109 Elongation factor 1-alpha 1 9 21 0.63 0.16 2 5 0.12 0.14 HCMV, HIV, MCMV 40, 92, 172 IPI00025491 46,125 Eukaryotic initiation factor 4A-I 2 4 0.23 0.06 3 8 0.27 0.31 HCMV 172 IPI00003865 70,854 Heat shock cognate 71-kDa protein 16 28 0.90 0.22 5 11 0.45 0.52 HCMV, HIV 71, 172 IPI00382470 98,052 Heat shock protein HSP 90-alpha 2 3 10 0.22 0.05 2 9 0.25 0.30 DHBV, HCMV, KSHV, 81, 172, 191 IPI00334775 84,790 Heat shock protein HSP 90-beta 11 16 0.86 0.21 6 9 0.32 0.37 DHBV, HCMV, KSHV, 81, 172, 191 IPI00419258 24,747 High-mobility group protein 1 3 20 1.51 0.37 IPI00003935 13,781 Histone H2B.q 1 7 0.47 0.12 1 6 0.58 0.68 MCMV, SV40 36, 92 IPI00019502 226,392 Myosin-9 (myosin heavy chain, nonmuscle IIa) 7 5 0.22 0.05 1 1 0.02 0.03 HCMV, KSHV 172, 191 IPI00419585 17,870 Peptidyl-prolyl cis-trans isomerase A (cyclophilin A) 3 21 0.78 0.19 2 13 0.67 0.78 HCMV, HIV, KSHV, SIV, VSV, VV 23, 26, 35, 60, 163, 172, 191 IPI0000874 22,096 Peroxiredoxin 1 5 23 0.76 0.19 IPI00016513 22,527 Ras-related protein Rab-10 2 11 0.52 0.13 1 6 0.23 0.27 IPI00016342 23,475 Ras-related protein Rab-7 3 16 0.70 0.17 1 8 0.33 0.39 KSHV 191 IPI00216298 11,599 Thioredoxin 1 7 0.47 0.12 1 7 0.47 0.55 IPI00387144 50,120 Tubulin alpha-ubiquitous chain 9 26 0.63 0.16 5 13 0.36 0.42 ASFV, HCMV 55, 172 IPI00011654 49,727 Tubulin-beta-2 chain 7 19 0.48 0.12 5 13 0.61 0.71 ASFV 55 P62988 (Swiss- Prot ID) 8,565 Ubiquitin 5 30 4.44 1.09 3 29 2.70 3.15 ASFV, baculovirus, HIV, HSV, MuLV, SIV, VV 55, 70, 86, 117, 118, 176 aTwenty-three host proteins are compiled from both trypsin-digested and Lys-C-digested virion preparations. The data reproducibility among dif fe rent virus preparations reached 67% for trypsin digestions and 87% for Lys-C digestions. bASFV, African swine fever virus; DHBV, duck hepatitis virus; HCMV, human cytomegalovirus; HIV, human immunodeficiency virus; HSV, herpes simplex vi rus; KSHV, Kaposi’s sarcoma-associated herpesvirus; MCMV, murine cytomegalovirus; MuLV, murine leukemia virus; SIV, simian immunodeficiency virus; SV40, simian virus 40; VSV, vesicular stomatitis vi rus; VV, vaccinia virus.

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abundant host proteins, such as myosin and vimentin, were not

detected in purified IMV. Also, we did not detect host proteins

that are known to be on vaccinia virus EEV, i.e., CD46, CD59,

CD29, CD71, CD81, and MHC-1 (170). We also did not detect

CD55, moesin, and cofilin, which were frequently identified in

other viruses, such as human T-cell leukemia/lymphoma virus

type 1, human immunodeficiency virus, and human

cytomega-lovirus, implying that host protein association with vaccinia

virus IMV has some selectivity (92, 119, 136, 154, 172).

Inter-estingly, six host proteins were uniquely identified in vaccinia

virus, and their roles in vaccinia virus biology need to be

stud-ied in the future.

ACKNOWLEDGMENTS

This work was supported by grants from the Academia Sinica and

the National Science Council (NSC94-2627-M-001-005 and

NSC91-3112-P-001-057-Y), Taiwan, Republic of China.

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