Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus

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Replication strategy

After contact with the host cell membrane the adsorption of the virion happens in a matter of a few minutes. Unlike other characterized phycodnaviruses (for example the Chlorovirus described in Chapter 1) that directly inject their DNA content into the host’s plasma (Van Etten et al., 2002), the coccolithovirus virion initially maintains its integrity following entry into the cell. After passing the host’s exterior membrane the EhV capsids can be seen intact in the cytoplasm with their nucleoprotein core encapsulated by the major capsid protein. Then it takes only a few seconds for the nucleoprotein core to disassemble and release its DNA in the cell cytoplasm or directly in the nucleus. An eclipse period then takes place while the viral machinery takes over the cell metabolism and starts the assembly of new virions. The first newly produced viral capsids start to appear around 3h p.i. (Mackinder et al., 2009). According to Castberg et al. (2002) around 400 to 1000 assembled virions can be seen accumulating in the host cytoplasm before progressive release. Exit of the viral capsids occurs through a budding mechanism, in which the viruses gain a lipid envelope made of their host’s membrane (Fig. 2). Ultimately, the process leads to the disintegration of the host cell (Mackinder et al., 2009).

Phylogeny and evolution

The phylogenetic position of the coccolithoviruses is still in debate with high uncertainty regarding its evolutionary history. Several independent phylogenetic studies (Allen et al., 2006c; Larsen et al., 2008; Schroeder et al., 2002; Wilson et al., 2006) have always placed the EhV within the family Phycodnaviridae. However, the coccolithovirus do not cluster with the other Prymnesiovirus identified to date (whose hosts are phylogeneticaly close to E. huxleyi), but instead occupy a very deep position in the phycodnavirus clade (Fig. 3). This differentiation from the other members of the Phycodnaviridae led to the creation of the new genus Coccolithovirus.
The 6 RNA polymerase subunits present in the EhV genome (unique among the known phycodnaviruses) add to the singularity of the coccolithoviruses among other algal viruses. Since ancestral NCLDV contained the RNA polymerase function, it is likely that of all the phycodnaviruses sequenced so far, EhV-86 represents the virus with the lifestyle most similar to the ancestral virus (Allen et al., 2006d). The change in lifestyle represented by this loss of RNA polymerase function (i.e. from nuclear independence to nuclear dependent transcription) probably contributes to the high diversity among present day genera in the Phycodnaviridae.

Sphingolipid biosynthesis gene sequences from E. huxleyi CCMP1516

The genome sequence data of E. huxleyi CCMP1516 strain were produced by the International E. huxleyi Genome Sequencing Consortium in collaboration with the US Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/). The genome sequence data are being analyzed by the consortium members and will be published elsewhere. The amino acid sequences corresponding to the seven EhV-86 sphingolipid biosynthesis genes (Table 1) were used to identify their homologs in the E. huxleyi genome sequences, using BLASTP searches (Altschul et al., 1997) against the host’s ORFeome (the JGI reduced protein set as of April 4, 2008; E-value<10-20). For the detection of E. huxleyi 3- KSR homolog, 3-KSR homologs from green plants (Arabidopsis thaliana and Ostreococcus tauri) were used as TBLASTN queries.

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PCR-amplification and sequencing of sphingolipid biosynthesis genes from host and virus strains

Six E. huxleyi and eleven EhV strains were chosen by taking into account their distant geographical origins (Table S1) and distinct behavior regarding susceptibility to EhV infection (data not shown). To extract E. huxleyi DNA, 250 ml of late exponential growing cultures were harvested by centrifugation (14000 rpm for 2 mins). A 0.5ml pellet was recovered and initially treated with proteinase K (5 mg/ml) in a lysis buffer containing 20 mM EDTA, pH 8 and 0.5% SDS (w/v) at 65 ºC for 1 h. Major cell debris was removed by adding 600 μl of phenol to each sample and centrifuging at maximum speed for 10 min. The top layer was recovered and the DNA was extracted using an equal volume of chloroform:isoamyl alcohol (24:1). The DNA was precipitated with the addition of 0.5 × volume 7.5 M ammonium acetate, pH 7.5, and 2.5 × volume absolute ethanol. The pellet was washed 3 times in 300 μl of ice-cold 70% ethanol, after which it was dried and re-suspended in 30 μl of DNase free water. The virus isolates were directly used as DNA template for PCR without prior DNA purification. The on-line application Primer3 (Rozen and Skaletsky, 2000) was used to design primers that target homologous regions in both host and viral genes (Table 62 S2). The PCR reaction was set up as follows: 1 μl of DNA template (extracted DNA in case of the hosts, viral isolate in the case of the virus) was added to a 25 μl reaction mixture which contained: 1 U Taq DNA polymerase (Promega), 1 × PCR reaction buffer (Promega), BSA, 0.25 mM dNTPs, 2.5 mM MgCl2, 10 pmol of each primer. The PCR was conducted in a PTC-
100™ cycler (MJ Research) with an initial denaturing step of 95 ºC (5 min), followed by 35 cycles of denaturing at 95 ºC (60 s), annealing at 56 ºC (60 s), and extension at 74 ºC (60 s). A SequiTherm EXCEL II DNA Sequencing Kit-LC (EpicentreTechnologies) with a LI-COR Automated DNA Sequencer was used to sequence the PCR products. Obtained sequence data were deposited in GenBank (accession numbers from FJ531546 to FJ531633).

Table of contents :

Acknowledgements
Abstract
Résumé (in French)
Table of contents
Avant propos
Chapter 1. Introduction 
1. Virus – life’s lubricant
2. Phycodnaviridae
3. Coccolithophores
4. Emiliania huxleyi
5. Thesis Objectives
Chapter 2. Coccolithovirus – a review 
Chapter 3. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus
Supplementary data
Chapter 4. Host-virus shift of the sphingolipid pathway along an Emiliania huxleyi bloom: survival of the fattest 
Supplementary data
Chapter 5. Novel transcription features unveiled during natural coccolithovirus infection
Supplementary data
Chapter 6. Short report on attempts to isolate new coccolithophore viruses 
Chapter 7. Final discussion and perspectives 
Perspectives for future research
Annexe A. Horizontal gene transfer between Emiliania huxleyi and viruses
Annexe B. Uncoupling of Emiliania huxleyi photosynthesis: virus infection versus nutrient stress
REFERENCES 
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