Characterization of the Archaeal Thermophile Sulfolobus Turreted Icosahedral Virus Validates an Evolutionary Link among Double-Stranded DNA Viruses from All Domains of Life

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Characterization of the Archaeal Thermophile Sulfolobus Turreted Icosahedral Virus Validates an Evolutionary Link among Double-Stranded DNA Viruses from All Domains of Life
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  J OURNAL OF  V IROLOGY , Aug. 2006, p. 7625–7635 Vol. 80, No. 150022-538X/06/$08.00  0 doi:10.1128/JVI.00522-06Copyright © 2006, American Society for Microbiology. All Rights Reserved. Characterization of the Archaeal Thermophile  Sulfolobus  TurretedIcosahedral Virus Validates an Evolutionary Link amongDouble-Stranded DNA Viruses from All Domains of Life Walid S. A. Maaty, 1  Alice C. Ortmann, 2 Mensur Dlakic´, 3 Katie Schulstad, 1 Jonathan K. Hilmer, 1 Lars Liepold, 1 Blake Weidenheft, 2 Reza Khayat, 4 Trevor Douglas, 1 Mark J. Young, 2 and Brian Bothner 1 *  Department of Chemistry and Biochemistry, Montana State University, Bozeman, 1  Departments of Microbiology and Plant Sciences, Montana State University, Bozeman, 2  and Department of Microbiology, Montana State University, Bozeman, 3  Montana 59717, and Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037  4 Received 13 March 2006/Accepted 17 May 2006 Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life,leading to speculation about a common viral ancestor that predates the divergence of   Eukarya ,  Bacteria , and  Archaea . This suggestion is supported by the shared general architecture of this group of viruses and thecommon fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis.  Sulfolobus  turreted icosahedral virus (STIV),isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal hostto be described. Here we present a detailed characterization of the components forming this unusual virus.Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins srcinates from a reading frame lacking a consensus start site.The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins fromother dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggestthat they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presenceof an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presentedstructural models in conjunction with the protein, lipid, and carbohydrate information reported here revealthat STIV is strikingly similar to viruses associated with the  Bacteria  and  Eukarya  domains of life, furtherstrengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life. In comparison to viruses with eukaryotic and bacterial hosts,little is known about the viruses that infect  Archaea . This isdue, in part, to the relatively recent delineation of the archaealdomain of life but, more significantly, to the challenges of isolating and culturing the host organisms (42). The extremeenvironments favored by many archaeal species and limitedknowledge about their biochemistry and biology exacerbatethis problem. Often, it is through the study of host-virus inter-actions that insights to the biology of the host are elucidated.The recent discovery of   Sulfolobus  turreted icosahedral virus(STIV) presents an opportunity to expand our knowledge of  virology, study host biology, and investigate the evolutionaryrelationship of viruses from all three domains of life. Studieson the structure of STIV have revealed similarities with pro-karyotic and eukaryotic viruses that suggest a common ances-try for icosahedral double-stranded DNA (dsDNA) viruses(30, 38).STIV was isolated from  Sulfolobus  enrichment cultures that were established from a high-temperature acidic hot spring(  80°C, pH  3) in Yellowstone National Park (38). The virus was subsequently shown to infect virus-free isolates of   Sulfolobus solfataricus  strain P2, for which the complete genome has beensequenced. The electron cryomicroscopy (cryo-EM) recon-struction revealed a capsid with pseudo-T  31 icosahedral sym-metry that is composed primarily of a 37-kDa major capsidprotein, plus at least three additional capsid proteins at thefivefold vertices (38). Two features in particular stand out: theturrets, which are proposed to function in host recognition andDNA translocation, and two electron-dense layers sandwichedbetween the protein capsid and the packaged genome that maybe composed of lipids (30) (Fig. 1). While not common, inter-nal lipid layers are present in a number of dsDNA viruses.  Paramecium bursaria Chlorella  virus 1 (PBCV-1), which repli-cates in unicellular  Chlorella -like green algae (41, 46), and thebacteriophage PRD1 (13) have a lipid layer beneath the gly-coprotein capsid shell. The spherical halophilic euryarchaeon  Haloarcula hispanica  virus (SH1) also has an internal lipidlayer that is selectively acquired from the host (4).STIV’s dsDNA genome has 17,663 bp and 36 predicted openreading frames (ORFs) (38). To date, only three proteins havebeen characterized, and the remaining 33 ORFs representhypothetical proteins. Sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) coupled with protein N-ter-minal sequencing and peptide mass mapping identified themajor capsid protein, B345, from preparations of purified virus(38). The jelly roll fold of B345 is nearly identical to the majorcapsid protein of adenovirus, PBCV-1, and PRD1 (7, 30, 34).Structural models based on X-ray crystallography have beendetermined for two other STIV proteins, F93 and A197. F93 * Corresponding author. Mailing address: 308 Gaines Hall, Chem-istry and Biochemistry Department, Bozeman, MT 59715. Phone:(406) 994-5270. Fax: (406) 994-5407. E-mail: bbothner@chemistry.montana.edu.7625  is a winged-helix DNA binding protein (M. Lawrence, per-sonal communication), and A197 is a glycosyltransferase-like protein (31).Characterization of a virus necessarily involves the identifi-cation and analysis of the components that assemble to formthe particle. Proteomics-based approaches are a powerful toolfor dissecting macromolecular complexes such as virus parti-cles (44). In this report STIV particle composition was char-acterized using mass spectrometry. Nine viral and two hostproteins in purified preparations of STIV were identified bymass spectrometry. One of the viral proteins srcinates from anoncanonical reading frame, confirming that standard transla-tion rules are not sufficient to generate the entire proteome of archaeal organisms. Even though primary sequence-basedsearches failed to find homologous proteins, fold recognition-based searches suggested potential roles for many of the virion-associated proteins. Structural prediction indicates that fourof the proteins are likely to be part of the turrets, includingan ATPase involved in DNA packaging. We confirm thepresence of an internal lipid layer and show that the virusselectively incorporates a subset of total host lipids. We alsodemonstrate that the major capsid protein (B345) is glyco-sylated. MATERIALS AND METHODS Virus purification.  A single-colony isolate of a  Sulfolobus  sp. producing anicosahedral virus-like particle was established by previously described methods(37). This isolate was designated YNPRC179, while the particle was described asSTIV (38). STIV was purified from infected cultures using a modification fromthe srcinal method. Viruses were purified from a culture of YNPRC179 andinoculated into virus-free  Sulfolobus solfataricus  P2 cells. Production of the virus was carried out by growing the  S. solfataricus  P2 cells in 1 liter of medium 182(www.dsmz.de/microorganisms/html/media/medium000182.html) at pH 2.5 untilthe cultures reached early log phase. Previously purified STIV was used toinoculate the cultures, and the inculcated cultures were monitored for virusproduction using an immuno-dot blot assay for the major capsid protein. Cul-tures were harvested between 72 to 96 h postinfection. Cells were removed bycentrifugation (  6,000   g   for 10 min), and the supernatant was filtered througha 0.22-  m Seritop filter (Millipore). The filtrate was concentrated over Amiconmembranes (Millipore) with a molecular mass cutoff of 100 kDa until the re-tained volume was   15 ml. The filter retentate was subsequently loaded ontoCs 2 SO 4  (39%, wt/vol) and centrifuged at 247,600   g   for 16 h. Virus bands wereremoved and dialyzed against 50 mM citrate buffer (pH 3.2). Purity was gaugedby banding characteristics on Cs 2 SO 4  gradients, negative-stain transmission elec-tron microscopy, and UV-visual spectroscopy. SDS-PAGE analysis and in-gel digestion for protein identification.  Purified virus was denatured and separated on 4 to 20% SDS-PAGE gels and stained withCoomassie brilliant blue R250 (Bio-Rad), and all visible protein bands wereexcised. The gel slices were destained using 50% acetonitrile in 50 mM ammo-nium bicarbonate (pH 7.9) and vacuum dried. Samples were rehydrated with 1.5mg/ml dithiothreitol (DTT) in 25 mM ammonium bicarbonate (pH 8.5) at 56°Cfor 1 h, subsequently alkylated with 10 mg/ml iodoacetamide (IAA) in 25 mMammonium bicarbonate (pH 8.5), and stored in the dark at room temperature for1 h. The pieces were subsequently washed with 100 mM ammonium bicarbonate(pH 8.5) for 15 min, washed twice with 50% acetonitrile in 50 mM ammoniumbicarbonate (pH 8.5) for 15 min each, vacuum dried, and rehydrated with 4  l of proteomics grade modified trypsin (100   g/ml; Sigma) in 25 mM ammoniumbicarbonate (pH 8.5). The pieces were covered in a solution of 10 mM ammo-nium bicarbonate with 10% acetonitrile (pH 8.5) and incubated at 37°C for 16 h.Solution digestion of viral capsid proteins using thermolysin and LysC followedby trypsin was also performed. The purified capsid buffer consisted of 15   l 50mM Tris (pH 8.8) and 10   l 1.5 M NaOH. This buffer was added to 200   l of  virus in citrate buffer for a final pH of 8.4, the optimum alkalinity for LysC. A  volume of 1   l of LysC (proteomics grade; Sigma) was added to 10   l of virussolution in the presence or absence of 8 M urea, DTT, and IAA; the reactionproceeded at 37°C for 12 h. A threefold volume of 50 mM Tris (pH 8.8) wasadded, followed by the addition of 2  l of trypsin and 1.4  l of 0.1 M CaCl 2 . Thesamples were incubated at 37°C for 2 h. MALDI analysis.  The matrix for matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry was prepared by mixing twice-recrystallized  -cyano-4-hydroxy cinnamic acid in a solution of 50% acetonitrile with 0.1% trifluoroacetic acid. Volumes of 1   l sample and 0.7   l matrix werespotted onto an aluminum MALDI-TOF plate and allowed to air dry. Calibra-tion was performed using bradykinin and insulin peptide standards. Spectra werecollected by using a Biflex III MALDI-TOF mass spectrophotometer (BrukerDaltonics, Billerica, MA) and internally calibrated by using known tryptic digestpeptides. Results were compared with theoretical trypsin digests of viral ORFs byusing a protein analysis work sheet from ProteoMetrics Inc. (http://65.219.84.5 /paws.html). Nanospray LC-MS/MS.  Peptides eluted from the in-gel digests were analyzedon an integrated Agilent 1100 liquid chromatograph (LC) (XCT-plus) mass-selective detector (MSD) controlled by ChemStation LC 3D (Rev A.10.02). The Agilent XCT-plus ion trap mass spectrometer is fitted with an Agilent 1100CapLC and nano-LC sprayer under the control of MSD trap control version 5.2Build no. 63.8 (Bruker Daltonic GmbH). Injected samples were first trapped anddesalted isocratically on a Zorbax 300 SB- C 18  precolumn (5   m, 5   300-  minside diameter; Agilent) for 5 min with 0.1% formic acid delivered by theauxiliary pump at 3   l/min. The peptides were then reverse eluted from aprecolumn and loaded onto an analytical C 18  capillary column (15-cm by 75-  minside diameter, packed with 3.5-  m Zorbax 300, SB C 18  particles; Agilent)connected in-line to the mass spectrometer with a flow of 300 nl/min. Peptides were eluted with a 15 to 50% acetonitrile gradient over 50 min. Data-dependentacquisition of collision induced dissociation tandem mass spectrometry (MS/MS) was utilized. Parent ion scans were run over the  m/z  range of 400 to 2,000 at 8,100  m/z -s. MGF compound list files were used to query an in-house database usingBiotools software version 2.2 (Bruker Daltonic) with 0.2-Da MS/MS ion masstolerance. Samples were run in parallel on a Waters Q-TOF Premier massspectrometer equipped with a nano-Acquity LC system and MassLynx 4.0 soft- ware under similar conditions. Homology searches for the STIV structural proteins.  The amino acid se-quences of the identified proteins were analyzed using BLAST and PSI-BLAST(40) homology searches against the nonredundant protein database at NCBI. Anautomated procedure implemented in SAM (28) was used to align all identifiedsequences and to generate profile hidden Markov models. Sensitive profile- FIG. 1. Model of an STIV particle. Shown is a cutaway view of theT  31 icosahedral capsid of STIV based on cryo-EM reconstruction(30). Extending from each of the fivefold vertices are turret-like pro- jections. The protein shell is blue and has been removed from onequarter of the particle to reveal the inner lipid layers (yellow). Thecapsid diameter is  70 nm, and the turrets extend 13 nm above thesurface.7626 MAATY ET AL. J. V IROL  .  profile comparisons (43) were used for alignments with known structures. Puta-tive transmembrane domains in each structural protein sequence were predictedby TMpred (http://www.ch.embnet.org/software/TMPRED_form.html) andDNASTAR (Lasergene Inc., Madison, WI). Signal peptide prediction for eachprotein was performed using SignalP 3.0 (http://www.cbs.dtu.dk/services /SignalP/) and Psort 2.0 (http://bioweb.pasteur.fr/seqanal/interfaces/psort2-simple.html) against the SwissProt database, with each search submission consisting of lessthan 80 amino acids taken from the N terminus of the protein. Sequence repeats were analyzed using EMBL-EBI rapid automatic detection and alignment of repeats(http://www.ebi.ac.uk/Radar/). Protein fold recognition was done using one-dimen-sional (1D) and three-dimensional (3D) sequence profiles coupled with secondarystructure information as implemented in the 3D position-specific scoring matrix (PSSM) (29) (http://www.sbg.bio.ic.ac.uk/   3dpssm/index2.html). Protein modeling and docking into the cryo-EM map.  Sequence profiles gen-erated as described above were used for profile-profile comparisons (43) with various databases of protein families, including the collection of protein struc-tures in PDB (15). Alignments obtained in this fashion were used for 3D mod-eling (39). Based on model evaluations (39), the alignments were adjustedmanually and used for additional cycles of model building until no furtherimprovements were observed. Structural models of B164 were docked into areconstructed cryo-EM map using 3SOM (11). Glycoprotein analysis.  The Pro-Q Emerald 488 glycoprotein gel and blot stainkit (P21875; Molecular Probes) were used to visualize glycosylated proteins in1D and two-dimensional (2D) gels. Briefly, purified virus solutions were adjusted with 1/4 volume of 4X Pro-Q buffer. The samples were then boiled for 2 min at100°C before being loaded onto a 4 to 20% SDS-PAGE gel. Molecular weightmarkers, the CandyCane glycoprotein molecular weight marker (MolecularProbes; C21852), and SDS-PAGE standard broad range (Bio-Rad) were used asstandards. The gel was run for 44 min at 120 V, rinsed in distilled H 2 O (twice for10 min), and fixed with a solution of 50% methanol and 5% glacial acetic acid(twice for 1 h). The gel was then washed twice with 3% glacial acetic acid andincubated in oxidizing solution (component C, Pro-Q Emerald glycoprotein geland blot stain kit) for 40 min. The gel was washed again (three times for 15 min).The gel was stained in the dark using 25 ml of Pro-Q Emerald 488 stainingsolution for 4 h, followed by two more 15-min washes. Glycoprotein gels wereanalyzed using a Typhoon scanner (blue laser, 488 nm) and stained using SyproRuby (Bio-Rad) and Coomassie brilliant blue R250 (Bio-Rad).For an analysis of potential N glycosylation 20  l of STIV and  S. solfataricus P2 was solubilized in 7 M urea–2 M thiourea–4% CHAPS {3-[(3-cholamidopro-pyl)-dimethylammonio]-1-propanesulfonate}–50 mM Tris-HCl (pH 8.8) buffer.Solubilized capsid proteins were acetone precipitated and resuspended into 40  lphosphate-buffered saline (PBS) buffer, pH 7.4. An equal volume of glycopro-tein-denaturing buffer (BioLabs) was added. The solution was boiled at 100°C for10 min. A 1/10 volume of NEBuffer for G7 (BioLabs) was added, followed by a1/10 volume of 10% NP-40 (BioLabs). Of this solution, 40   l was kept as theenzyme control. The remaining solution received 2  l of the enzyme PNGase F(BioLabs). This digestion solution was then split, with one half being incubatedat 37°C for 1 h. To test for the presence of O glycosylation, STIV and  S. solfataricus  P2 proteins were resuspended in 50 mM PBS, pH 7.4, mixed with 2  l of   O -glycosidase (Roche Applied Science), and incubated at 37°C overnight. Lipid analysis.  Viral and host cell lipids were extracted and analyzed asdescribed previously (18). Briefly, each sample was acetone precipitated and theair-dried pellet was solubilized with chloroform-methanol (1:3). Virus samples were diluted 100-fold and analyzed by direct infusion electrospray in both pos-itive and negative modes on a micro-TOF (Bruker Daltonics). Instrument set-tings were as follows: capillary, 4,000 V; capillary exit,   240 V; dry gas (N 2 ), 3liters/min; temperature, 200°C. The negative mode had a more intense andconsistent signal in the expected  m/z  range for tetraether lipids (900 to 2,000), aspreviously reported (33). RESULTSSDS-PAGE profile of purified viral capsid proteins.  Highlypurified samples of STIV reproducibly contained seven prom-inent bands when analyzed under denaturing conditions (Fig.2A). 2D gel analysis of the same sample revealed that a num-ber of the proteins were present in multiple forms (Fig. 2B).Twenty-three protein spots were resolved between 14 to 60kDa within the pI range of 3 to 10. The higher-molecular- weight proteins present on the 1D gel were not visualized inthe 2D analysis, suggesting that they may have extreme pIs orbe hydrophobic. The protein spots at  40 and 45 kDa on the2D gel (spots 17 to 20 and 8 to 15, respectively) representmultiple forms of two viral proteins (Fig. 2B). The isoformsdiffer in pI, but their molecular weights are the same or nearlythe same. Posttranslational modifications which alter the pIbut do not significantly change the molecular weight offer apossible explanation for the observed pattern. Nomenclaturefor STIV proteins is based on the reading frame (A, B, or C) FIG. 2. Analysis of STIV capsid proteins by SDS-PAGE. Purified virus was denatured and separated (A) on a 4 to 20% SDS-PAGE gel or(B) on isoelectric focusing (3 to 10 nonlinear) gel strips and then separated in the second dimension on an 8 to 18% SDS-PAGE gel. Gels stained with Coomassie brilliant blue and individual protein spots and bands were then excised and digested with thermolysin and/or trypsin, and thepeptides were analyzed using mass spectrometry. The unmodified major capsid protein expressed in  E. coli  runs similarly to 14 (  ).V OL  . 80, 2006 PROTEOMIC ANALYSIS OF STIV 7627  and predicted number of amino acids. To investigate this fur-ther, the major capsid protein of STIV, B345, was expressed in  Escherichia coli  and this protein was analyzed with a 2D gel(30). The  E. coli -expressed protein comigrated with the mostbasic isoform of B345 found in infectious virus particles (spot14). Analysis of the spot pattern using the protein modificationscreening tool ProMost (23) (http://prometheus.brc.mcw.edu /promost/) indicated that differential phosphorylation and/ormodifications to basic side chains were likely low-molecular- weight posttranslational modifications that could account forthe observed gel shifts. Alternative explanations include het-erogeneous glycosylation with sulfate or acidic carbohydratesand isoelectric focusing artifacts. Identification of STIV proteins by mass spectrometry.  In-geldigestion and mass analysis were conducted to determine theidentity of the protein bands and spots (Fig. 2). Seven bandsand 23 spots were excised from the gels for analysis. Proteinidentifications were based on peptide mass and fragmentationpattern matches in the NCBInr database and an in-house da-tabase that contained the STIV genome translated in all six reading frames and the complete genome of   S. solfataricus  P2.The in-house database contained all ORFs greater than 24nucleotides in the STIV genome and also included a pyrrol- ysine codon in place of the amber stop codon (12, 24, 26).In-gel digestion and nanospray MS/MS analysis using both anion trap and Q-TOF instruments led to the identification of nine STIV-encoded proteins (Table 1). The amino acid se-quence coverage ranged from 20 to 90%, and confidence in theidentifications was based on scores from MASCOT (Matrix Science Inc.) and MassLynx v 4.0 (Waters Inc.) (see supple-mentalmaterialatwww.chemistry.montana.bothner/jvi_stiv2006).MASCOT protein scores were highly significant for each of thereported proteins and ranged from 52 to 727 using  0.5-Da errortolerance (Table 1). Only peptides with individual scores of greaterthan40wereincludedintheproteinscoring.Theseresults were confirmed using MALDI-TOF analysis of in-gel digestionsamples. Additional LC-MS experiments used direct in-solutiondigestion of purified particles. In addition to the virus proteins,two host proteins were identified. Three independent viral prep-arations were used in these experiments, and the same two hostproteins were identified each time. One is small basic proteinSSO7D (7 kDa), of unknown function. The other virally associ-ated host protein, SSO0881 (25 kDa), is similar to the vacuolarsorting VPS24 domain that is found in both  Saccharomyces cer- evisiae  and mammals (48, 51) and is conserved across the  Sulfolo- bus  family.Of the nine STIV proteins identified here, only the majorcapsid protein (B345) had previously been confirmed in virusparticles (38). Seven of the nine proteins are ordered sequen-tially within the genome, suggesting coordinated transcriptionand translation (Fig. 3). Interestingly, the last protein in thisseries (A78) was not included in the srcinal annotation. Thepresence of this protein is based on a MASCOT score of 108from MS/MS sequence data and peptide mass mapping fromMALDI-TOF MS. Moreover, MassLynx reported a probabilityof 99.5% for two peptides with an average mass error of 4.9115ppm. The peptide closest to the N terminus begins with a valine whose codon is nine nucleotides downstream of the A55stop codon (see supplemental material at www.chemistry.montana.bothner/jvi_stiv2006). A78 may be a read-throughproduct from the A55 ORF or represent the use of an alter-native start codon.  Analysis of identified proteins.  Thirty-six ORFs were pre-dicted in the srcinal annotation of the STIV genome (38).Standard BLAST searches based on the predicted amino se-quence encoded by the ORFs failed to identify homologous TABLE 1. Virus-associated proteins Identified protein Mass (Da) pISample srcinPredicted structureor functionBand no. 2D spot no. Insolution STIVC557 58,574 5.5 6    Protein interactionC381 41,655 5.56 4, 7 17, 18, 19, 20    PRD1 P5 vertex proteinB345 (coat protein) 37,810 6.17 5 8, 9, 10, 11, 12, 13, 14, 15, 16    Major capsid protein A223 24,410 4.72 7 24    PRD1 P5 vertex proteinB164 19,025 9.29 3    Poxvirus ATPaseB130 13,768 4.97 1 27, 28    NSM  b B109 11,969 5.04 2, 5 17, 18, 25    NSM*A78 9,610 10.79 1    NSM A55 6,338 4.49 1    NSMHost7 DNA-binding protein-kDa (SSO7D) 7,735 9.52    DNA bindingConserved hypothetical proteinSSO088125,020 5.86 4    VPS24 vacuolar sortingproteinOthersPIC/E  a 31,540 5.07 17, 18    ASP  c PIC/E   17,000   8.5–9.5 29, 30    ASPPIC/E   17,000   4.7 26    ASPPIC/E   26,000   4–5 21, 22, 23    ASP  a PIC/E, protease inhibitor cocktail and endonucleases added during the sample preparation for analysis by 2D electrophoresis.  b NSM, no significant match.  c  ASP, added during sample preparation. 7628 MAATY ET AL. J. V IROL  .  proteins in the NCBI nonredundant database. In evolutionarilydistant proteins, structural homology is likely to be retainedeven when the primary sequences have diverged beyond de-tection. Therefore, as an alternative approach, structural pre-diction was used to identify potential functional roles for theseproteins (Table 1).Structural predictions of the first 347 N-terminal residues of C557 show high similarity to an ankyrin repeat (3D PSSMconfidence    95%) that mediates protein-protein interaction(16) and the presence of an   30-amino-acid repeat. The se-quence (VEKVIQKDITHPPKFYLPPVHLPNIHQIEAGIGH) is repeated three times between residues 32 and 130 (seesupplemental material at www.chemistry.montana.bothner/jvi_stiv2006). Each repeat contains a conserved YLP motif (un-derlined), which is involved in protein-protein interaction inmany organisms. For example, this motif has been found inreceptor tyrosine kinases and members of the ErbB family of human epidermal growth factor receptors (20, 50). The C-terminal region is marked by a proline- and serine-rich motif,the functional significance of which is currently unknown. In anattempt to determine relative capsid composition a densitom-etry analysis of the 1D gel using ImageJ was performed. Thisshows a 15:1 ratio between the major capsid protein (B345)and C557. However, this ratio may not be accurate due toposttranslational modifications (see below).PSI-BLAST (40) searches with B164 identified significantmatches to a large class of P-loop ATPases. Profile-profilecomparisons (43) showed the most significant similarity (  E   1  10  33 ) to poxvirus A32 proteins (Pfam accession numberPF04665; Fig. 4), which are thought to be involved in viralDNA packaging (10). This group of proteins, however, has noclose homologue of known structure. For that reason, severalless-related ATPases (with  E  values between 10  7 and 10  13 ) were used as templates for B164 model building in order of their match significance (39). After several cycles of manualrealignment and building, we generated models that were of good quality regardless of the starting template. It should bepointed out, however, that even though these models are likelyto have the correct overall fold, they are not expected to beatomic-quality models because the starting templates wereconsiderably different at sequence level from B164. Each of these models was fitted into the cryo-EM reconstruction of STIV (30) by surface overlap maximization (11). This proce-dure is specifically designed to place partial structures or mod-els into low-resolution density maps by maximizing surfaceoverlap. All models docked into the same general area at thebase of the turret on the fivefold axis of symmetry. Dependingon the template used for modeling, the surface overlap be-tween models and the map was between 0.6 and 0.78, with thecorresponding correlation coefficients in the 0.5 to 0.6 range.Importantly, when we attempted to dock more than 12 modelsat a time, the new solutions overlapped with the existing ones,thus indicating that the position at the base of turrets is theonly one compatible with B164 models.Two virion-associated proteins share predicted structural FIG. 3. Location of capsid proteins on the STIV genome map.ORFs are named according to frame (A to F) and number of predictedamino acids. Proteins identified by protease mass mapping (solid blackarrows), in general, cluster together. There are eight proteins fromannotated ORFs and one additional protein, A78. This protein lacks astart methionine and was not included in the srcinal annotation. Themap was created using Vector NTI Advance 10.1.1.FIG. 4. B164 alignment with several proteins from the family  Poxviridae . SwissProt accession numbers are separated by an underscore fromspecies abbreviations (FOWPV,  Fowlpox virus ; 9POXV,  Vultur gryphus poxvirus ; POXVV,  Vaccinia virus ; MCV1,  Molluscum contagiosum virus subtype 1; SWPV,  Swinepox virus ; YMTV,  Yaba monkey tumor virus ; RPOXV,  Rabbit fibroma virus ). Residues are shaded according to 90%consensus, with white letters on black background signifying perfect conservation. Abbreviations on the consensus line are as follows: h,hydrophobic; s, small; l, aliphatic, b, big; a, aromatic; c, charged.V OL  . 80, 2006 PROTEOMIC ANALYSIS OF STIV 7629
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