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Complete Genome Sequence and Lytic Phase Transcription Profile of a Coccolithovirus

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Science  12 Aug 2005:
Vol. 309, Issue 5737, pp. 1090-1092
DOI: 10.1126/science.1113109

Abstract

The genus Coccolithovirus is a recently discovered group of viruses that infect the globally important marine calcifying microalga Emiliania huxleyi. Among the 472 predicted genes of the 407,339–base pair genome are a variety of unexpected genes, most notably those involved in biosynthesis of ceramide, a sphingolipid known to induce apoptosis. Uniquely for algal viruses, it also contains six RNA polymerase subunits and a novel promoter, suggesting this virus encodes its own transcription machinery. Microarray transcriptomic analysis reveals that 65% of the predicted virus-encoded genes are expressed during lytic infection of E. huxleyi.

Large icosahedral viruses from the family Phycodnaviridae infect marine or freshwater eukaryotic algae, and all contain dsDNA genomes ranging from 180 to 560 kbase pair (kbp) (1, 2). Phycodnaviruses belong to a group of viruses that replicate, completely or partly, in the cytoplasm of eukaryotic cells and are termed nucleocytoplasmic large DNA viruses (NCLDVs) (3). Two other members of the Phycodnaviridae have been sequenced before this study: the 335,593–base pair (bp) genome of a virus that infects a marine filamentous brown alga, Ectocarpus siliculosus (EsV-1) (4), and the 330,744-bp genome of a virus that infects a unicellular chlorellalike green algal symbiont of the freshwater protozoa Paramecium bursaria (PBCV-1). These two viruses, which are from the same family, only have 33 genes in common (1). EhV-86, a lytic virus about 170 to 175 nm in diameter, was originally isolated by plaque assay from a seawater sample collected from a dying Emiliania huxleyi bloom in the English Channel (5). Phylogenetic analysis of its DNA polymerase gene places it in a new genus (Coccolithovirus) within the family Phycodnaviridae (6). The EhV-86 host, Emiliania huxleyi (Haptophyta), is a unicellular alga known for its elegant calcium carbonate scales, which it produces intracellularly and sequesters over its cell surface (7). It is perhaps best known for its immense coastal and open ocean blooms at temperate latitudes and is a key species for current studies on global biogeochemical cycles and climate modelling (810).

Sequence analysis (11) of EhV-86 revealed a circular genome with a length of 407,339 bp (Fig. 1) (12), making this the largest Phycodnaviridae genome sequenced to date. 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 about 560 kbp (13). Recently, Mimivirus, the largest NCLDV [previously isolated from amoeba growing in the water of a cooling tower (14)] was sequenced, revealing a 1,181,404-bp genome (15). General features of the EhV-86 genome sequence include a nucleotide composition of 40.2% G+C, a total of 472 predicted genes (coding sequences, CDSs) (Fig. 1) with an average gene length of 786 bp, a coding density of 91%, five tRNA genes [encoding amino acids Leu (containing an intron), Ile, Gln, Asn, and Arg] and two further introns (ehv064 and ehv459). In addition, we identified the location and orientation of three distinct families of repeats (designated A, B, and C) throughout the genome (Fig. 1 and fig. S1). Family A repeats are noncoding but are found immediately upstream of 86 predicted CDSs and are possible promoter elements essential for transcription of associated CDSs. Family B repeats are characterized by GC-rich regions found in CDSs, which encode proline-rich domains. Family C repeats are AT-rich, noncoding, and characteristic of virus genome origins of replication (16).

Fig. 1.

Circular representation of the 407,339-bp EhV-86 genome. The outside scale is numbered clockwise in kbp. Circles 1 and 2 (from outside in) are CDSs (forward and reverse strands, respectively), starting with CDS ehv001 at position 276 bp (color-coded gray and just below the 0-kb marker on the reverse strand). CDSs are color-coded by putative function: light green, no known function; dark green, no known function but contains transmembrane helices; gray, miscellaneous; sky blue, degradation of large molecules; red, information transfer; yellow, central or intermediary metabolism; pink, virus specific; and light blue, kinases. Circles 3 and 4 are microarray expression results (forward and reverse strands, respectively) color-coded as follows: orange, expression observed; blue, no expression observed; and gray, not determined because oligonucleotides were not designed for that particular CDS. Circles 5 to 7 are the positions of the three families of repeats: green, putative promoter family A; blue, proline-rich family B; and orange, putative origin of replication. Circle 8, G+C content. Initial sequence analysis of EhV-86 suggested a linear genome, because the ends in the sequence alignment always stopped at the same bases. However, polymerase chain reaction amplification across the termini generated a product indicating the genome had a circular stage during virus replication (30). Sequence analysis of this product revealed either an A or T (at equal ratio) single base pair overhang at each of the termini, indicating a potential method for circularization of the genome. Consequently, bases T and A were annotated to the “start” of the genome (bases 1 and 2).

Of the 472 CDSs, only 66 (14%) have been annotated with functional product predictions on the basis of sequence similarity or protein domain matches (Table 1). A large proportion of CDSs exhibited no similarity to proteins in the public databases; only 21% of the CDSs contain protein-protein basic local alignment search tool (BLASTP) results that matched with an E value lower than 0.01 (for details of sequence similarity and protein domain database matches, see table S2). Future analysis of marginal similarities may increase this proportion further. Most of the genes with functional predictions are at the “ends” of the genome; surprisingly, there are only 8 such genes within 230 CDSs in the 176-kbp central region between 156 kbp and 332 kbp. The role of this central region is unclear.

Table 1.

Genes with functional predictions identified in EhV-86. CDS, systematic gene identifier; CG, CDSs that are known NCLDV core genes (✓) (3); Ex, CDSs that are expressed (✓) or not expressed (x) at 33 hours postinfection as determined by microarray transcriptomic analysis.

CDS Position Putative function/features CG Ex
Start End
ehv014 8433 9299 Longevity-assurance (LAG1) family protein
ehv018 12303 13379 Endonuclease
ehv020 15170 15949 Putative proliferating cell nuclear antigen
ehv021 15998 17212 Serine protease
ehv022 17197 18486 Phosphoglycerate mutase family protein
ehv023 18732 19253 Deoxycytidylate deaminase
ehv026 22027 23004 Ribonucleoside-diphosphate reductase small chain
ehv028 24121 24912 Lipase
ehv030 25675 28713 DNA polymerase delta catalytic subunit
ehv031 28718 29704 Sterol desaturase
ehv041 38172 39305 Endonuclease
ehv050 45226 47838 Serine palmitoyltransferase
ehv060 55942 61926 Lectin protein x
ehv061 61979 62941 Fatty acid desaturase
ehv064 64018 68501 DNA-dependent RNA polymerase II largest subunit
ehv072 74904 75785 DNA-binding protein
ehv077 78289 79257 Transmembrane fatty acid elongation protein
ehv079 80165 80896 Lipid Phosphate phosphatase
ehv085 86013 87614 Major capsid protein
ehv093 93036 93416 HNH endonuclease family protein x
ehv101 99368 100174 Hydrolase x
ehv103 100800 101153 Vesicle-associated membrane protein
ehv104 101158 102741 Putative helicase
ehv105 102837 103331 Transcription factor S-II (TFIIS) family protein
ehv108 106901 107545 DNA-directed RNA polymerase subunit
ehv109 107546 108073 OTU-like cysteine protease
ehv110 108106 108942 RING finger protein
ehv113 110604 112046 Bifunctional dihydrofolate reductase—thymidylate synthase
ehv117 113956 115560 Phosphate permease x
ehv128 121517 122026 ERV1/ALR family protein x
ehv133 125567 126283 ATP-dependent protease proteolytic subunit
ehv136 126964 127572 Nucleic acid—binding protein
ehv141 131083 132345 Hypothetical protein
ehv151 139720 140628 Serine protease
ehv152 140747 141610 DNA binding protein
ehv158 145226 147094 DNA ligase
ehv160 147460 148464 Serine protease
ehv166 154991 155719 RING finger protein x
ehv167 155756 156016 DNA-directed RNA polymerase subunit
ehv179 167551 169176 Major facilitator superfamily protein
ehv184 171775 173091 DNA binding protein
ehv230 219949 220335 Endonuclease
ehv346 228542 229306 Lectin protein
ehv349 289563 290267 Protease
ehv358 300433 300909 Thioredoxin x
ehv361 302158 303507 Serine protease x
ehv363 303778 304569 Esterase x
ehv393 333013 334023 DnaJ domain-containing protein x
ehv397 335877 336323 Deoxyuridine 5′-triphosphate nucleotidohydrolase
ehv399 337274 337963 DNA-directed RNA polymerase subunit
ehv401 338688 339317 Ribonuclease
ehv402 339307 340335 Protein kinase x
ehv403 340415 341551 Hypothetical protein
ehv415 348487 349263 Putative fatty acid desaturase x
ehv428 362962 365202 Ribonucleoside-diphosphate reductase protein
ehv430 365923 368631 Helicase
ehv431 368760 369743 Thymidylate kinase
ehv434 370859 374329 DNA-directed RNA polymerase II subunit
ehv440 378178 379044 Proliferating cell nuclear antigen protein
ehv444 384374 387685 DNA topoisomerase
ehv447 388779 389684 Serine protease
ehv451 391491 392306 Protein kinase
ehv453 393351 394478 MRNA capping enzyme
ehv455 396954 398075 Sialidase
ehv459 398903 401004 Nucleic acid—independent nucleoside triphosphatase
ehv465 404611 405201 Putative thioredoxin protein x

Microarray analysis was used to obtain a transcriptome profile during lytic infection and to confirm whether putative CDSs were transcriptionally active. The microarray was constructed from 75-mer oligonucleotides corresponding to the sense strand of each putative CDS on the EhV-86 genome. EhV-86 gene expression (RNA transcription) was determined by hybridization to the microarray with fluorescently labeled cDNAs prepared from an infected E. huxleyi culture 33 hours postinfection. This time was chosen because the majority of cells in the culture were infected at a wide range of infection stages (fig. S3); the majority of kinetic classes of transcripts should be expressed at this point, and we detected expression of 308 EhV-86 CDSs (65%) (Fig. 1 and database S5) (17). Analysis of the expression data allowed us to confirm annotation of some overlapping CDSs. Analyses of the transcriptome revealed that many of the unknown CDSs were expressed and are therefore likely to be functional. Up to 35% of EhV-86 CDSs were either not expressed or were beyond the limit of detection at 33 hours postinfection. Interestingly, expression was not observed in a putative phosphate permease (ehv117), a protein predicted to be involved in high affinity phosphate transport (18, 19). Because this study was conducted in nutrient excess, it is possible the permease is only expressed during phosphate-limiting conditions.

Analysis of EhV-86 homologs of known genes revealed several that have never been identified in a virus before. There are at least four genes involved in sphingolipid biosynthesis, encoding sterol desaturase (ehv031), serine palmitoyltransferase (ehv050), transmembrane fatty acid elongation protein (ehv077), lipid phosphate phosphatase (ehv079), and a further two genes encoding desaturases (ehv061 and ehv415). Transcriptomic analysis revealed that all these genes except ehv415 are expressed during infection (Fig. 1). Sphingolipids are membrane lipids present in all eukaryotes and some prokaryotes and also play a key role in several processes, particularly signal transduction (20). A sphingolipid biosynthesis pathway has not previously been discovered on a virus genome. Sphingolipid biosynthesis leads to the formation of ceramide (21), which is known to suppress cell growth and is an intracellular signal for apoptosis (22, 23). Several viruses are known to induce apoptosis (24). Uniquely, EhV-86 appears to encode key components of the ceramide pathway in its genome; furthermore, it is known that there is a connection between protease activation and ceramide-induced apoptosis (25), and intriguingly EhV-86 contains eight proteases (including five serine proteases) throughout the genome (ehv021, ehv109, ehv133, ehv151, ehv160, ehv349, ehv361, and ehv447). All proteases except ehv361 are expressed during infection (Fig. 1). One theory is that this algal virus encodes a mechanism for inducing apoptosis as a strategy for killing the host cell and disseminating progeny virions during the infection cycle. Apoptosis, or programmed cell death, has been observed previously in marine phytoplankton (26), although usually as a response to nutrient or other physiological stressors (27, 28) rather than virus infection.

Further analysis of the genome reveals that EhV-86 contains 25 of the core set of 40 to 50 conserved virus genes for NCLDVs (3) that encode some of the principal features of virion structure and genome replication and expression (Table 1 and table S2). Uniquely for the Phycodnaviridae, EhV-86 contains six RNA polymerase subunits (ehv064, ehv105, ehv108, ehv167, ehv399, and ehv434), all of which are expressed. The presence of a putative virus RNA polymerase holoenzyme together with the family A repeats, which we suggest are previously unknown promoters (Fig. 1), would indicate that EhV-86 encodes its own transcription machinery. Hence, expression of some EhV-86 transcripts may occur in the cytoplasm rather than the nucleus (29). Phycodnaviridae lack RNA polymerases; thus, further phylogenetic analysis may point to coccolithoviruses being reclassified into a distinct subfamily.

EhV-86 is the largest algal virus genome sequenced to date, and it expresses a range of virus genes, revealing functions more commonly observed in animal and plant cells. Viral ceramide production raises questions about cell death in phytoplankton and may have implications for new sources of lipids in the marine food chain. Ongoing definition of the EhV-86 genome, coupled with transcriptomic analysis, will reveal the function of expressed genes that have no homologs in current databases. As more giant viruses are discovered and their genomes sequenced, there is sure to be an explosion of exciting gene discoveries and novel functions.

Supporting Online Material

www.sciencemag.org/cgi/content/full/309/5737/1090/DC1

Materials and Methods

Figs. S1, S3, S4

Table S2

Database S5

References and Notes

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