Briefings in Functional Genomics Advance Access originally published online on October 11, 2006
Briefings in Functional Genomics 2006 5(4):273-279; doi:10.1093/bfgp/ell033
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The coccolithovirus microarray: an array of uses
Corresponding author. Michael J. Allen, Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth, PL1 3DH, UK. E-mail: mija{at}pml.ac.uk
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ABSTRACT |
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The Coccolithoviridae is a recently discovered family of giant algal viruses. Here, we review the genomic and transcriptomic characterization of this family based on the results generated from a coccolithovirus microarray. The microarray has been used to aid the annotation of the genome, to investigate the infection process at the transcriptional level and to assess the diversity in genomic content within the family.
Keywords: Coccolithovirus, Coccolithoviridae, Microarray
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BACKGROUND |
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TOPABSTRACTBACKGROUNDSUMMARYAcknowledgementsReferences |
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Emiliania huxleyi is a marine coccolithophorid with worldwide distribution capable of forming vast blooms that can cover up to 10 000 km2. Its production of calcium carbonate coccoliths (Figure 1), and its role in CO2 cycling and dimethyl sulphide (DMS) production makes E. huxleyi an important species with respect to past, present and future marine primary productivity. Our ever increasing awareness of climate change and global warming makes E. huxleyi a key species for current investigation. Despite this, the molecular biology of E. huxleyi and its recently discovered viruses has remained relatively unstudied. Viruses have been shown to be a major cause of bloom termination [1, 2]. Emiliania huxleyi virus 86, EhV-86, is a giant virus that infects the marine coccolithophorid E. huxleyi and was originally isolated from a bloom in the English Channel off the south coast of England in 1999 [1].
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EhV-86 is the type species of the genus Coccolithovirus within the family Phycodnaviridae [1, 3]. The Phycodnaviridae comprise a diverse collection of large dsDNA viruses that infect algae [4]. To date, three Phycodnaviridae have been sequenced in their entirety and have revealed incredibly diverse and predominantly unknown genomes [3, 5–7]. EhV-86 is currently the largest fully sequenced Phycodnaviridae; however, other larger algal virus genomes are known to exist, such as the virus that infects Pyramimonas orientalis, a marine microalga, which has a genome of around 560 kbp [8].
The 407 339 bp genome of EhV-86 is the second largest virus genome ever sequenced, though viruses with larger genomes are known. By far the largest is the Mimivirus which has a genome of 1 181 404 bp [9]. EhV-86 was initially predicted to encode 472 protein coding sequences (CDSs) [3]. The majority of EhV-86 CDSs exhibit no similarity with proteins in the public databases; a mere 21% of the CDSs contain protein–protein basic local alignment search tool (BLAST) results that matched an E value lower than 0.01. However, EhV-86 does contain homologues for 25 of the core set of conserved genes found in the nuclear-cytoplasmic large DNA virus (NCLDV) family [10]. EhV-86 homologues of known genes encode six RNA polymerase subunits (the presence of RNA polymerase is, so far, unique among the algal viruses), mRNA capping enzyme, DNA polymerase, DNA ligase, DNA topoisomerase, sphingolipid biosynthesis enzymes, eight proteases, major capsid protein, phosphate permease, two thioredoxins, ribonuclease and ribonucleoside-diphosphate reductase [3, 10]. Many of these are core genes found in numerous members of the NCLDV family (which includes the Phycodnaviridae, Asfariviridae, Mimiviridae, Iridoviridae and Poxviridae), however, the presence of sphingolipid biosynthesis genes is unique to this virus and their origin and role during infection is yet to be determined [10]. In addition, three distinctive repeat families are found within the genome and have been predicted to function as non-coding promoter elements, proline rich coding regions and an origin of replication [3].
Microarrays are commonly constructed for model organisms for which there is extensive sequence data. This is not the case for viral genomes which tend to be relatively small. Previously, virus-based microarrays have only been used extensively with the well-studied herpes viruses and also the shrimp white spot virus systems [11–14]. However, the large size of the EhV-86 genome and its highly unknown nature easily justified the construction of a microarray to aid in the genomic analysis. Here we review the construction of the coccolithovirus microarray and the variety of applications that it has been used for.
The EhV-86-based coccolithovirus microarray
The sequencing of EhV-86 was hindered by the highly repetitive nature of the genome, making the alignment of genome contigs particularly problematic.
Due to the delay in genome completion, the construction of the EhV-86 microarray was carried out in parallel to the sequencing effort. Indeed, the EhV-86 microarray was designed and constructed before the full genome was completely sequenced and, therefore, before full annotation had been completed. Of the 472 genes predicted initially, 425 (90%) are represented on the microarray. A single gene-specific 75-mer oligonucleotide probe was designed for virus genes using Oligo 6 (MBI, http://www.oligo.net/) for a microarray hybridization temperature of 65°C. Oligo 6 is a multifunctional program that searches for and selects oligonucleotides on the basis of factors such as hybridization temperature, secondary structure and specificity. Hairpin-free hybridization probes were designed with a 3' bias within selected stability limits. Oligonucleotide length was fixed to 75, the acceptable 3'-Dimer 
G set to –8.0 kcal/mol, the maximum length of acceptable dimers was 2 bp, the 3' stability range was set between –4.8 and –13.5 kcal/mol, GC clamp stability of –12.0 kcal/mol, minimum acceptable loop G set to –2.0 kcal/mol. Oligo Tm range was set between 40 and 50°C initially, for the design of the majority of oligonucleotides, and then the range gradually increased (from 20 to 60°C) to encompass the remaining genes that failed to have a suitably designed oligonucleotide.
Probe elements were deposited and immobilized onto amino silane treated glass slides (Corning GAP II) using a BioRobotics MicroGrid 2 printer. Each virus-specific probe was printed in triplicate along with a collection of negative and positive control probes. Probes were printed in a 4 x 4 meta-grid, each sub-grid composed of 12 rows and 13 columns. Spots from rows 1–4 were reprinted in rows 5–8 and 9–12. Rows 1, 5 and 9 contained the 10 SpotReport Alien PCR products (Stratagene) and were used as a positive control and, when relevant, to confirm consistency between cyanine-3 (Cy3) and cyanine-5 (Cy5) scanning channels. Ten exogenous alien mRNA spikes (Stratagene) can be added to the labelling reaction along with experimental RNA to allow assessment of microarray quality and orientation. Furthermore, microarray sensitivity, specificity, signal linearity, consistency and dye ratios can also be assessed using this system. Other control probes included were 3x SSC buffer, human COT-1 DNA, poly(dA) (40–60 bases in length, single stranded). Ten E. huxleyi host probes were included on the array for the genes encoding ribulose bisphosphate carboxylase (rbcL), calcium binding protein (gpa), 18S rRNA, 16S rRNA, phosphate permease, elongation factor Tu (tufA), photosystem I apoprotein A1 (psaA), photosystem II protein D1 (psbA), fucoxanthin chlorophyll binding protein and type I actin. All microarray data (including microarray design, hybridization and data analysis) was MIAME/Env compliant and was stored and curated in maxdLoad2 prior to submission in MAGE/ML format to the EBI ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) (see Table 1 for accession numbers) [15, 16]. In addition, this information is also available at EnvBase, the NERC Environmental Genomics Data Catalogue, (http://envgen.nox.ac.uk/) under the accession number egcat:000010.
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Applications
Since the construction of the EhV-86 microarray, it has been used for a wide range of purposes. Initially, the microarray was used to aid in the annotation of the genome, then to investigate the transcriptional profile during the early stages of infection, followed more recently to assess the diversity of the coccolithoviruses within the Plymouth Virus Collection (PVC).
Aiding the annotation of a genome
The majority of the EhV-86 genome is composed of genes of which the function is totally unknown. Indeed, rarely among sequenced genomes, a number of predicted genes fail to find any matches whatsoever in the public databases. The highly unknown nature of the genome makes the task of annotation difficult: without functional information it is difficult to assess whether what we predict to be a gene is actually a gene. However, by using a microarray to detect the presence of transcripts the evidence for the presence of a gene is increased: if it looks like a gene and it is expressed like a gene, the chances are, it is a gene. Of course, not all expressed transcripts are translated into functional proteins; however, the detection of transcript for a putative gene vastly increases the chances that it is a real gene. This approach was used effectively during the annotation of the EhV-86 genome [3]. We believe this was the first time that a preliminary genome annotation has been aided by transcriptional data generated on a microarray.
RNA was extracted from E. huxleyi infected cells 33 h post infection. At this stage the vast majority of E. huxleyi cells are infected and viral transcripts dominate the mRNA message. Indeed, at 6 h post infection, virus transcripts account for less than 10% of mRNA transcripts, whereas at 12 and 24 h post infection around 95% of mRNA is viral in origin (unpublished data). Viruses start being released 4 h post infection, then there is a gradual and steady release of viruses for 24–48 h until the host cell finally disintegrates (lyses) (unpublished data). Hence at 33 h post infection many rounds of re-infection would have taken place suggesting that the various transcriptional stages of the virus replication cycle should be represented. A direct labelling approach was used to label mRNA from total RNA using anchored oligo dT primers. cDNA from both uninfected and infected cultures was labelled with either Cy3 or Cy5 fluorescent dyes. Cy3/Cy5 dye labelled cDNA was hybridized to the microarray in a dual hybridization so that infected cultures could be compared directly with the uninfected state. This approach was used successfully to detect message for 65% of the original 472 predicted genes in EhV-86 (307 of the 425 that were tested) [3]. The use of statistical analysis was kept to a minimum: genes were either regarded as on or off based on each probe spot being individually assessed using a manual scoring system performed on the original microarray images. A gene was considered to be expressed if signal was detected (i.e. higher than background level) in at least two of the three replicate spots within an array, in all four replicate microarray experiments. However, differential levels of expression were observable on the microarray with some probes showing higher levels of expression than others [3]. A good example of this were genes associated with a 100 kb region identified previously as being a recent acquisition by the virus genome [10, 17]. The genes in this region fail to provide any significant matches using a BLAST analysis hence their function is a mystery [10].
Assignment of transcripts into kinetic classes
Following the lytic phase transcriptional work the microarray was used to outline the transcriptional profile generated by EhV-86 during the early stages of infection. By random amplifying the mRNA population and using the microarray, the expression profile of the EhV-86 infection of E. huxleyi was determined over the first 4 h of infection [18]. RNA samples were extracted at 0, 1, 2 and 4 h post infection. At these early stages of the infection process, virus message is at such low copy numbers that the direct labelling strategy used previously was insufficient to generate detectable signals. Therefore, mRNA message was amplified using a PCR-based amplification system (Roche). This system boasts >100 000 fold increase in message by random PCR amplification of the entire mRNA population followed by a further T7 promoter-driven amplification of signal. The cRNA produced using this method has been shown to be of higher quality than that produced by linear amplification and gives highly reproducible results [19]. The system is most commonly used when using the very low amounts of RNA isolated by laser capture microdissection [20]. A combination of two hybrid primers, oligo(dT)-T7-TAS and random hexamer-TAS primers, is used to create double stranded cDNA. The target amplification sequence (TAS) primer is a random sequence that has no significant homology to anything in the public databases, allowing the amplification of cDNA by PCR using TAS primers. The resulting PCR-amplified cDNA can then be transcribed into labelled cRNA by a linear amplification step driven from the T7 promoter.
This highly effective labelling strategy allowed the designation of EhV-86 CDSs into three groups (T1, T2 and T4) on the basis of when their transcripts are first detected (1, 2 or 4 h post infection). To produce the 33 h post infection microarray profile described in Section 1, an entire 2 l culture of exponentially growing cells (106 cells ml–1) was needed to produce enough RNA for the direct labelling method (Table 1). However, when using the amplification system much smaller samples could be taken (around 250 ml of exponentially growing cultures 106 cells ml–1) which provided more than enough RNA for labelling. The smaller sampling volume makes experimental design more manageable and, crucially, allows more frequent sampling points.
The results obtained suggested two distinct phases to the infection process: a primary phase dominated by a group of CDSs localized to a sub-region of the genome, which have no database homologues and are associated with a unique promoter element; and a secondary phase during which CDSs are transcribed from the remainder of the genome [18]. These experiments raise interesting questions on the life cycle and infection strategy of EhV-86 with particular emphasis on the role of the virally encoded RNA polymerase. It remains to be determined if the host RNA polymerase is responsible for the transcription of the primary phase transcripts or if the viral RNA polymerase is packaged into the virion in order to allow their immediate expression upon infection [18]. Since the majority of EhV-86 CDSs are unknown, the designation of CDSs into transcriptional categories is a significant step forward in determining their function.
As mentioned previously, preliminary annotation was performed whilst the EhV-86 genome sequencing was still in progress. This accounts for the lack of oligonucleotide probes on the coccolithovirus microarray for 47 putative CDSs. However, as more sequence became available the criteria for the annotation of a CDS altered as the increasingly unknown nature of the majority of the genome became apparent. As such, seven oligonucleotides probes were designed for CDSs that were not represented in the final annotation due to their failure to meet the strict criteria for CDS prediction [3]. Nevertheless these oligonucleotides probes remained as relics on the EhV-86 microarray. During the course of this in-depth transcriptional profiling, the transcript for a hitherto unrecognized CDS in the EhV-86 genome was detected. The putative CDS ehv364A is located at 311 501–311 653 bp on the EhV-86 genome and is predicted to encode a protein of 51 amino acids. Unsurprisingly (since it was not annotated originally) BLAST searches reveal no matches in the GenBank database. The lack of database homologues made functional inferences of ehv364A unfeasible, but the identification of a transcript for this previously unannotated CDS (based on microarray transcriptional data) is another step forward in genome annotation. Hence, the number of CDSs on EhV-86 was increased by 1, to 473.
In addition to the transcriptional profiling of the early stages of infection, this labelling method was also applied to an RNA extraction performed on purified EhV-86 virions. A single transcript was detected (unpublished data) suggesting that EhV-86 may package at least one transcript into its infective virions, although this is yet to be confirmed using quantitative PCR.
Assessment of genomic diversity
The PVC contains 12 virus strains which are capable of infecting E. huxleyi. These strains have been collected over the 4-year period between 1999 and 2003 from sampling sites in the English Channel and in a Norwegian Fjord [1, 2, 21]. Construction of the EhV-86 microarray has also allowed the genetic diversity (in terms of genomic content) of the coccolithoviruses in the PVC to be assessed. A disadvantage of using a specific strain/species microarray for this purpose is that the microarray can only tell you what is highly variable or not present in a particular genome. Any genomic additions would go undetected since there is no probe on the array to detect it. However, the use of the array for screening for genomic content can provide a wealth of information on genetic diversity within a family of closely related species.
By producing Cy3 labelled DNA using random primers and 500 ng of virus DNA we hybridised each virus genome in our strain collection to the EhV-86 microarray. At least 70 genes found in EhV-86 are absent or highly variable from one or more of the genomes of the coccolithoviruses found in the PVC (data submitted for publication). The distinctive pattern in genetic content displayed by each of the strains suggested a complicated series of gene loss/addition events. In particular, a high intensity of deleted or variable genes was found in the 100 kb region of unknown function suggesting this section of the genome may be under a high selection pressure. Initial analysis was limited to the 12 strains in the collection [21]; each strain required a single hybridization to determine the genetic make up. Thus, using only 12 microarray slides, the entire coccolithovirus strain collection was scrutinized. This method is a relatively cheap, efficient and quick way to glean an insight into the genetic content of a family of closely related species. This approach has commonly been used to assess bacterial diversity, but, hitherto, not virus diversity [22–27]. Genes of interest that are conserved among all strains or are lacking and/or variable in some strains can be identified rapidly for further downstream analysis.
Data from a second sequencing project focusing on EhV-163, a coccolithovirus of similar-sized genome which was isolated from a geographically distinct region from EhV-86, was also analysed in light of the microarray data [1]. More than 260 contigs were created, allowing a comparison of over 200 complete CDSs between the two strains (GenBank accession numbers DQ127552 [GenBank] -DQ127818 [GenBank] ) [28]. By analysing sequence data and the results from these genomic DNA hybridizations it was estimated that approximately 80% of the EhV-163 genome had been sequenced [28]. Among other things, the genomic DNA hybridizations confirmed the deletion of a CDS that the sequence data suggested was absent in EhV-163 [28]. Furthermore, a very rough approximation of 10 mismatching nucleotides, spread over the 75-mer microarray oligonucleotide, was found to be sufficient to cause hybridization levels to fall below significant levels.
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SUMMARY |
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TOPABSTRACTBACKGROUNDSUMMARYAcknowledgementsReferences |
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Over a relatively short period of time, a predominately uncharacterized virus has had its genome annotated, the kinetic profile of its transcripts determined and the diversity within its family assessed. The coccolithovirus microarray has been integral to these discoveries. The results have indicated a plethora of new and novel genes, a distinctive biphasic transcriptional pattern during the infection process and an unexpectedly large degree of genomic content variability within the Coccolithoviridae. The construction of the coccolithovirus microarray has rapidly increased the understanding of the virus and its infection strategy, yet clearly there is still much to learn. Future work will, without doubt, build on the techniques described here to further our knowledge of this amazing virus and its family.
Indeed, the advent of the genomic era has accelerated our understanding of genome function beyond all expectations. Microarrays are a powerful tool, but are typically used in well-studied model biological systems in which complex transcriptional cascades are mapped with ever increasing precision. However, as we show here, microarrays can prove to be just as powerful and useful a tool when dealing with less studied, non-model systems.
| Key Points The cocolithovirus microarray has been used to:
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confirm expression of predicted CDSs
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Acknowledgements |
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TOPABSTRACTBACKGROUNDSUMMARYAcknowledgementsReferences |
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This research was supported by grants awarded to WHW from the National Environment Research Council (NERC) Environmental Genomics thematic program (ref. NE/A509332/1 and NE/D001455/1) and from Marine Genomics Europe, through framework FP6 of the European Commission. We would like to acknowledge support from NERC Environmental Bioinformatics Centre, Centre for Ecology and Hydrology, Oxford for help with microarray data storage and administration. This article is dedicated to the memory of Anna Goostrey.
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FOOTNOTES |
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Mike Allen and Willie Wilson work at Plymouth Marine Biology (United Kingdom), an independent and impartial collaborative centre of the Natural Environment Research Council (NERC).
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References |
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