Microarray Analysis of Paramecium bursaria Chlorella Virus 1 Transcription

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Microarray Analysis of Paramecium bursaria Chlorella Virus 1 Transcription
  J OURNAL OF  V IROLOGY , Jan. 2010, p. 532–542 Vol. 84, No. 10022-538X/10/$12.00 doi:10.1128/JVI.01698-09Copyright © 2010, American Society for Microbiology. All Rights Reserved. Microarray Analysis of   Paramecium bursaria  ChlorellaVirus 1 Transcription  † Giane M. Yanai-Balser, 1 Garry A. Duncan, 2 James D. Eudy, 3 Dong Wang, 4 Xiao Li, 5 Irina V. Agarkova, 1 David D. Dunigan, 1,6 and James L. Van Etten 1,6 *  Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68583-0722 1  ; Biology Department, Nebraska Wesleyan University, Lincoln, Nebraska 68504-2794 2  ; Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska 68198-5455 3  ; Statistics Department,University of Nebraska, Lincoln, Nebraska 68583-0963 4  ; Biomedical Engineering and Biotechnology,University of Massachusetts, Lowell, Massachusetts 01854 5  ; and Nebraska Center for Virology,University of Nebraska, Lincoln, Nebraska 68583-0900 6 Received 12 August 2009/Accepted 7 October 2009  Paramecium bursaria  chlorella virus 1 (PBCV-1), a member of the family  Phycodnaviridae , is a large double-stranded DNA, plaque-forming virus that infects the unicellular green alga  Chlorella  sp. strain NC64A. The330-kb PBCV-1 genome is predicted to encode 365 proteins and 11 tRNAs. To monitor global transcriptionduring PBCV-1 replication, a microarray containing 50-mer probes to the PBCV-1 365 protein-encoding genes(CDSs) was constructed. Competitive hybridization experiments were conducted by using cDNAs from poly(A)-containing RNAs obtained from cells at seven time points after virus infection. The results led to the followingconclusions: (i) the PBCV-1 replication cycle is temporally programmed and regulated; (ii) 360 (99%) of thearrayed PBCV-1 CDSs were expressed at some time in the virus life cycle in the laboratory; (iii) 227 (62%) of the CDSs were expressed before virus DNA synthesis begins; (iv) these 227 CDSs were grouped into two classes:127 transcripts disappeared prior to initiation of virus DNA synthesis (considered early), and 100 transcripts were still detected after virus DNA synthesis begins (considered early/late); (v) 133 (36%) of the CDSs wereexpressed after virus DNA synthesis begins (considered late); and (vi) expression of most late CDSs isinhibited by adding the DNA replication inhibitor, aphidicolin, prior to virus infection. This study provides thefirst comprehensive evaluation of virus gene expression during the PBCV-1 life cycle.  Paramecium bursaria  chlorella virus 1 (PBCV-1), the proto-type of the genus  Chlorovirus  (family  Phycodnaviridae ), is alarge, icosahedral (190 nm in diameter), plaque-forming virusthat infects the unicellular, eukaryotic green alga  Chlorella  sp.strain NC64A. The PBCV-1 virion has a lipid membrane lo-cated inside an outer glycoprotein capsid. The 330-kb genomeis a linear, nonpermutated, double-stranded DNA (dsDNA)molecule with covalently closed hairpin ends that has approx-imately 365 protein encoding genes (CDSs), as well as 11tRNA encoding genes (reviewed in references 34, 39, and 40).The CDSs are evenly distributed on both strands and inter-genic space is minimal (typically fewer than 100 nucleotides);the exception is a 1,788-bp sequence in the middle of thegenome that encodes the tRNA genes. Approximately 35% of the 365 PBCV-1 gene products resemble proteins in the publicdatabases.PBCV-1 initiates infection by attaching rapidly and specifi-cally to the cell wall of its host (22), probably at a unique virus vertex (4, 26). Attachment is immediately followed by host cell wall degradation by a virus-packaged enzyme(s) at the point of contact. After wall degradation, the viral internal membranepresumably fuses with the host membrane, causing host mem-brane depolarization (9), potassium ion efflux (25), and anincrease in the cytoplasm pH (2). These events are predicted tofacilitate entry of the viral DNA and virion-associated proteinsinto the cell. PBCV-1 lacks a gene encoding a recognizableRNA polymerase or a subunit of it, and RNA polymeraseactivity is not detected in PBCV-1 virions. Therefore, viralDNA and virion-associated proteins are predicted to migrateto the nucleus, and early viral transcription is detected 5 to 10min postinfection (p.i.), presumably by commandeering a hostRNA polymerase(s) (possibly RNA polymerase II) (14, 29).Virus DNA synthesis begins 60 to 90 min p.i., followed by virusassembly at 3 to 5 h p.i. in localized regions of the cytoplasm,called virus assembly centers (21). At 6 to 8 h p.i., virus-induced host cell lysis occurs resulting in release of progeny virions (  1,000 viruses/cell,   25% of which are infectious).These events are depicted in Fig. 1.To initiate PBCV-1 transcription, the host RNA poly-merase(s), possibly in combination with a virus transcriptionfactor(s), must recognize virus DNA promoter sequences. Re-cently, three short nucleotide sequences were identified in pu-tative virus promoter regions (150 bp upstream and 50 bpdownstream of the ATG translation site) that are conserved inPBCV-1 and other  Chlorovirus  members (7). PBCV-1 CDSsare not spatially clustered on the genome by either temporal orfunctional class, suggesting that transcription regulation mustoccur via  cis - and possible  trans -acting regulatory elements.To understand the dynamics of PBCV-1 global gene expres-sion during virus replication, we constructed a microarray con-taining 50-mer probes to each of the 365 PBCV-1 CDSs. * Corresponding author. Mailing address: Nebraska Center for Vi-rology, University of Nebraska, Lincoln, NE 68583-0900. Phone: (402)472-3168. Fax: (402) 472-3323. E-mail: jvanetten@unlnotes.unl.edu.† Supplemental material for this article may be found at http://jvi.asm.org/.  Published ahead of print on 14 October 2009.532  cDNAs from poly(A)-containing RNAs isolated from cells atseven times after PBCV-1 infection were competitively hybrid-ized against a reference sample on the microarray. To furtherdelineate early and late gene expression, cells were treated with the DNA replication inhibitor, aphidicolin, prior to infec-tion. The results provide the first comprehensive transcrip-tional map of the virus genome, conferring insights about thecharacterization of each PBCV-1 CDS, the majority of whichhave unknown functions. In addition, the microarray data sug-gest that viral DNA replication plays a significant role in thetemporal regulation of gene expression. MATERIALS AND METHODSRNA isolation and drug treatment.  Chlorella  strain NC64A cells (10 8 cells/ml) were infected with PBCV-1 at a multiplicity of infection of 5 to ensure synchro-nous infection. Uninfected cells and cells at 20, 40, 60, 90, 120, 240, and 360 minp.i. were harvested by centrifugation (4,000 rpm) for 5 min at 4°C and disrupted with glass beads (0.25 to 0.30 mm in diameter) by using a bead beater (DisruptorGenie; Scientific Industries, Bohemia, NY) in the presence of TRIzol (Invitro-gen, Carlsbad, CA). RNAs were isolated by using the Absolutely RNA miniprepkit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. RNA integrity was verified in denaturing 1% agarose gels by monitoring host cytoplas-mic and chloroplast rRNAs. Total RNA was quantified with a NanoDrop spec-trophotometer (NanoDrop Technologies, Wilmington, DE).To determine the effect of virus DNA synthesis on virus gene expression,aphidicolin (20   g/ml) was added to the cells 15 min prior to infection, andsamples were collected at the same times after infection as described above.Control samples were obtained from infected nontreated cells at the same times.Preliminary experiments to determine optimal drug dosage and time of applica-tion indicated that 20   g of aphidicolin/ml completely inhibits DNA synthesis in15 min. Microarray construction and hybridization.  The PBCV-1 genome is predictedto have 365 CDSs and 11 tRNA encoding genes. The tRNAs sequences were notincluded in the microarrays. Fifty-mer probes representing each CDS in thePBCV-1 genome were designed and synthesized by MWG Biotech (Ebersberg,Germany) (20 to 80% GC, with a melting temperature of 60 to 80°C). A table with the probes’ sequences is in Supplement S1 in the supplemental material.Probes were spotted onto CMT-GAPS silane-coated slides (Corning, Lowell,MA) by using Omnigrid 100 (Genomic Solutions, Ann Arbor, MI) according tothe manufacturer’s instructions. Probes were printed in quadruplicates on everyslide. For each time point, 20   g of total RNA was reverse transcribed by usingoligo(dT) as a primer, and cDNAs were labeled with either Cy3- or Cy5-dUTP(GE Healthcare, Piscataway, NJ) with the SuperScript indirect cDNA labelingsystem (Invitrogen) according to the supplier’s directions. The reference sample,for the time course experiments, consisted of a pool of transcripts obtained bymixing equal amounts of total RNA from each time point. Competitivehybridization experiments were conducted for each sample against the ref-erence sample (15, 18, 41). For the aphidicolin experiments, a direct com-parison was carried out with each treated sample versus the correspondinguntreated infected control.Labeled cDNAs were resuspended in 40   l of preheated (68°C) Ambionhybridization buffer 2 (Ambion, Austin, TX). Arrays were hybridized (42°C) for16 h in a Corning hybridization chamber (Corning, Lowell, MA). The slides were washed twice (42°C) in 2  SSC (1  SSC is 0.15 M sodium chloride plus 0.015 Msodium citrate)–0.5% sodium dodecyl sulfate (SDS) for 15 min, followed by two FIG. 1. Timeline representing the PBCV-1 life cycle in  Chlorella  strain NC64A. Numbers represent minutes after infection. CDSs expressedbefore viral DNA synthesis begins were classified as early (black arrow), CDSs expressed after DNA synthesis begins were classified as late (whitearrow), and CDSs expressed before and after DNA synthesis begins were classified as early/late (arrow with diagonal lines). Electron micrographs A and B were reproduced with permission from Meints et al. (22), and micrographs C and D were reproduced with permission from Meints etal. (21).V OL  . 84, 2010 CHLORELLA VIRUS PBCV-1 TRANSCRIPTION 533   washes in 0.5   SSC–0.5% SDS for 15 min. Slides were then dried by low-speedcentrifugation and subjected to fluorescence detection with an Axon 4000Bscanner (Molecular Devices, Sunnyvale, CA). Microarray analysis.  Results from three independent experiments were ana-lyzed by using the GenePix Pro v.6.0 software (Molecular Devices) and TIGRmicroarray software suite (TM4) (28). Several transformations were performedto eliminate low-quality data, to normalize the measured intensities using theLowess algorithm, and to regulate the standard deviation of the intensity of theCy5/Cy3 ratio across blocks. CDSs that displayed statistically significant modu-lation were identified by a one-way analysis of variance, using  P   values of    0.01as a cutoff. For the aphidicolin experiments, significant analysis of microarray(33) was used to identify CDSs with statistically significant changes in expressioncompared to an untreated infected sample (false discovery rate,   5%). CDSs with similar expression profiles were grouped into different clusters with a K-means algorithm by using Euclidean distance and 50 maximum iterations.PBCV-1 microarray data sets were deposited at NCBI’s Gene Omnibus Express(GEO) under the accession number GSE18421. RESULTS AND DISCUSSIONMicroarray quality check.  To evaluate our probes, equalamounts of PBCV-1 genomic DNA (2  g) were labeled in twoindependent reactions with either Cy3-dCTP or Cy5-dCTP(GE Healthcare) by using random primers (Invitrogen). Thetwo reactions were then competitively hybridized with theprobes in the microarray. No difference was detected in hy-bridization of the two DNA samples on all spots (results notshown), indicating the probes were specific and also excludingany preferential hybridization to one of the dyes. In addition,to check for host cross-hybridization, PBCV-1 DNA (2  g) waslabeled with Cy3-dCTP and host  Chlorella  strain NC64A DNA (2   g) with Cy5-dCTP. Only two PBCV-1 CDSs (A260R and A625R) hybridized with host DNA; however, these two CDSare false positives since recently available  Chlorella  strainNC64A genome sequence did not have detectable homologoussequences. PBCV-1 transcription program.  RNAs were isolated frominfected cells at 20, 40, 60, 90, 120, 240, and 360 min p.i.Competitive hybridization results from each time point againstthe RNA reference pool revealed that transcripts of 360 (99%)of the 365 PBCV-1 CDSs display statistically significant varia-tion in at least one of the experimental time points (Fig. 2).CDSs A60L, A328L, A482R, and A646L did not pass statisticaltests in the time course experiment. CDS A689L was not spot-ted onto the array because it was accidentally omitted duringthe probe synthesis. The gene expression analysis was based onrelative levels, rather than absolute levels of expression. Map-ping the PBCV-1 transcription pattern to the genome revealedno large regions that were biased as to time of expression,indicating that gene expression in PBCV-1 is mostly controlledby multiple initiation sites (Fig. 3). However, a few early or lateCDSs clustered in small regions of the genome, as can be seenin shaded areas in Fig. 3. Interestingly, these small regions arealso clustered and conserved in another sequenced chlorovirus(NY-2A) that infects the same host chlorella (results notshown). Unlike the phage T4 genome, where most late CDSsare located in contiguous regions on the same DNA strand(18), PBCV-1 expression did not show a strong strand-specificbias. In addition, there is no relationship between time of expression and G  C content of the genome (Fig. 3).Classification of PBCV-1 CDSs was based on when the tran-script was detected by the microarray. Globally, the 360 statis-tically significant CDSs were grouped into three classes (Fig.4): (i) 227 (62%) of the CDSs were expressed before viralDNA synthesis begins at 60 to 90 min p.i.; (ii) these 227 CDSs were divided into two classes: transcripts of 127 CDSs disap-peared prior to initiation of virus DNA synthesis (consideredearly), while transcripts of 100 CDSs were still detected after virus DNA synthesis begins (considered early/late); (iii) tran-scripts of 133 (37%) CDSs were detected after virus DNA synthesis begins (considered late). Functional categorization of PBCV-1 CDSs was reported elsewhere (8), and the functionaldistribution compared to each transcriptional category is sum-marized in Fig. 4 and 5. Forty-four of the PBCV-1 encodedproteins have been expressed, and recombinant proteins wereshown to be functional enzymes. These are indicated with anasterisk in Fig. 5. The functions of the remaining CDSs areeither putative or unknown. A previously described putative promoter sequence (AATGACA) and a similar sequence (ATGACAA) (7, 14) weredetected in 50 early or early/late PBCV-1 CDSs. However,promoter sequences for most early, early/late, and late CDSsremain unidentified. Early CDSs.  A total of 127 (35%) of the 360 PBCV-1 CDSs were expressed early, 20 to 60 min p.i. (see Supplement S2 inthe supplemental material). Sixty-one percent of the earlyCDSs have no known function. Many of the early CDSs arepredicted to encode the machinery for the virus to begin DNA replication. In fact, PBCV-1 encodes seven proteins involvedin DNA replication, recombination and repair that were ex-pressed early including:    DNA polymerase (A185R), super-family III helicase (A456L), DNA topoisomerase II (A583L),RNase H (A399R), and PCNA (A574L). A pyrimidine dimer-specific glycosylase (A50L), a well-characterized DNA repairenzyme involved in pyrimidine photodimer excision (20), wasalso expressed early. Additional PBCV-1 encoded proteinsinvolved in virus DNA synthesis and DNA recombination werein the early/late class including, DNA primase (A468R), a 5  -3  exonuclease (A166R) and a second PCNA (A193L). ThePBCV-1 genome contains methylated nucleotides, both N 6 -methyl adenine and 5-methylcytosine (35). Therefore, it is notsurprising that the virus encodes three functional DNA meth- yltransferases that were transcribed early: two enzymes thatform N6-methyladenine (A251R and A581R) and one thatforms 5-methylcytosine (A517L).PBCV-1 DNA synthesis also requires large quantities of de-oxynucleotide triphosphates (dNTPs) that cannot be accountedfor simply by recycling deoxynucleotides from host DNA. By4 h p.i., the total amount of DNA in the cell increases fourfolddue to viral DNA synthesis (37). To guarantee a supply of dNTPs in nonproliferating host cells, large DNA viruses, in-cluding PBCV-1, encode proteins involved in dNTP biosynthe-sis: dUTP pyrophosphatase (A551L), thioredoxin (A427L),thymidylate synthase X (A674R), and cytosine deaminase(A200R) CDSs were transcribed early. Additional dNTP syn-thesizing CDSs in the early/late class included aspartate tran-scarbamylase (A169R), both subunits of ribonucleotide reduc-tase (A476R and A629R), glutaredoxin (A438L), and dCMPdeaminase (A596R).Several PBCV-1 CDSs predicted to encode proteins in- volved in transcription were also expressed early. These pro-teins included three putative transcription factors (TFIIB[A107L], TFIID [A552R], and TFIIS [A125L]), two helicases 534 YANAI-BALSER ET AL. J. V IROL  .  FIG. 2. Heat map illustrating the expression of 360 PBCV-1 CDSs during the infection cycle. cDNAs from each time point were labeled withCy5, and the reference control was labeled with Cy3. Color code represents the log 2  (Cy5/Cy3) ratio for each time point. CDSs with similarexpression profiles were grouped into three classes representing early, early/late, and late by using a K-means algorithm. Each column correspondsto the time point when total RNA was collected (numbers represent minutes after infection). Each row represents a different CDS in PBCV-1. A list of all of the CDSs is available in Supplement S2 in the supplemental material.535  (SWI/SNF helicase [A548L] and superfamily II helicase[A241R]), and RNase III (A464R). The genes for two enzymesinvolved in mRNA capping, an RNA triphosphatase (A449R)and a guanylyltransferase (A103R), were also transcribedearly. The products of at least some of these early CDSs areundoubtedly involved in the switching of virus early gene tran-scription to late gene transcription. A few PBCV-1 enzymes involved in protein synthesis andprotein degradation were transcribed early, including transla-tion elongation factor-3 (A666L), ubiquitin C-terminal hydro-lase (A105L), Skp1 protein (A39L), SCF-E3 ubiquitin ligase(A481L), a zinc metallopeptidase (A604L), and an ATPase(AAA     class) (A44L).Unlikeotherviruses,PBCV-1encodesatleastpart,ifnotall,of the machinery required to glycosylate its major capsid protein(19), including five glycosyltransferases (11, 38, 43). Further- FIG. 3. Mapping of the PBCV-1 transcriptome. Blue arrows, early CDSs; green arrows, early/late CDSs; red arrows, late CDSs. Arrow pointsindicate the transcription direction. Shaded areas indicate small transcription CDS clusters that are also conserved in another chlorovirus (NY-2A),except for the areas marked with asterisks. The middle circle shows the G  C content of the genome. Note that the PBCV-1 genome is linear, anda circular map was generated for illustration purposes only.536 YANAI-BALSER ET AL. J. V IROL  .
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