Infection by a Giant Virus Induces Widespread Physiological Reprogramming in Aureococcus Anophagefferens – A Harmful Bloom Algae

While viruses with distinct phylogenetic origins and different nucleic acid types can infect and lyse eukaryotic phytoplankton, “giant” dsDNA viruses have been found to be associated with important ecological processes, including the collapse of algal blooms. However, the molecular aspects of giant virus – host interactions remain largely unknown. AaV, a giant virus in the Mimiviridae clade, is known to play a critical role in regulating the fate of brown tide blooms caused by the pelagophyte Aureococcus anophagefferens. To understand the physiological response of A. anophagefferens CCMP1984 upon AaV infection, we studied the transcriptomic landscape of this host-virus pair over an entire infection cycle using a RNA-sequencing approach. A massive transcriptional reprogramming of the host was evident as early as 5 min post-infection, with modulation of specific processes likely related to both host defense mechanism(s) and viral takeover of the cell. Infected Aureococcus showed a relative suppression of host-cell transcripts associated with photosynthesis, cytoskeleton formation, fatty acid and carbohydrate biosynthesis. In contrast, host cell processes related to protein synthesis, polyamine biosynthesis, cellular respiration, transcription and RNA processing were overrepresented compared to the healthy cultures at different stages of the infection cycle. A large number of redox active host-selenoproteins were overexpressed, which suggested that viral replication and assembly progresses in a highly oxidative environment. The majority (99.2%) of annotated AaV genes were expressed at some point during the infection cycle and demonstrated a clear temporal-expression pattern and an increasing relative expression for the majority of the genes through the time course. We detected a putative early promoter motif for AaV, which was highly similar to the early promoter elements of two other Mimiviridae members, indicating some degree of evolutionary conservation of gene regulation within this clade. This large-scale transcriptome study provides the insight into the Aureococcus ‘virocell’, and establishes a foundation to test hypotheses regarding metabolic and regulatory processes critical for AaV and other Mimiviridae members.


Introduction:
Viruses are thought to lyse cells and release cellular organic and inorganic nutrients that either become available for microbial growth or are exported to the deep ocean (Wilhelm and Suttle, 1999). With an estimated 10 31 virus particles in the sea (Angly et al., 2005), the geographical scale and impact of these processes are enormous -viral activity can turn over an estimate of 150 gigatons of carbon per year (Suttle, 2007). To accomplish this, it has been historically thought that viruses encode a minimal amount of genomic information that instructs host cells to produce new virus particles. Using almost entirely the host machineries, hundreds of virus particles can be produced from one host cell. As an example, Hepatitis B virus encodes only four overlapping genes in a 3.2 kb genome (Liang, 2009), whereas several Picornavirales members, which are widespread in the ocean, only code for one or two proteins (Lang et al., 2009). This paradigm has been challenged by discovery of 'giant' eukaryotic viruses -viruses that rival bacterial cells in terms of their physical size and genomic content (Raoult et al., 2004;Moniruzzaman et al., 2014;Wilhelm et al., 2016;Wilhelm et al., 2017). Phylogenetic analyses of members of this group (known collectively as nucleocytoplasmic large DNA viruses, NCLDVs) (Iyer et al., 2001) have revealed that a major portion of the genomic content of these giant viruses has been acquired from the eukaryotic hosts and other sources through horizontal gene transfer (HGT), some of which are passed vertically through the course of viral evolution (Filee et al., 2007;Koonin and Yutin, 2010;Moniruzzaman et al., 2014). This genomic complement renders these viruses more autonomous from the host cell, empowering them to control individual processes in the complex eukaryotic cells and produce virus-specific macromolecules (Wilson et al., 2005;Claverie and Abergel, 2010).
Giant viruses are thought to play important roles in constraining photosynthetic protists in both marine and freshwater ecosystems (Short, 2012;Moniruzzaman et al., 2017). And while there is growing information regarding large virus diversity, seasonality and roles in host dynamics, there is a dearth of information regarding the molecular underpinnings of conversion of a healthy host cell into a virus producing 'machine' (aka the "virocell") (Forterre, 2011). Both protists and their large viruses are complex compared to their prokaryotic counterparts -microbes and phages. Yet molecular information is necessary to understand how viral infection can select for resistance in hosts, or shape the macromolecules released during cell lysis into surrounding waters. Indeed, given that infection is an ongoing and prevalent process in the oceans, a significant amount of the particulate chemistry that oceanographers measure may be due to infection phenotypes. Understanding the molecular aspects of giant virus infections can also reveal important markers of infection that can be used to track and differentiate infected cells from healthy cells in situ.
Giant viruses infecting eukaryotic algae are functionally diverse, although they do share a few core proteins (Yutin et al., 2009). As a consequence, significant differences in the molecular basis of interactions can be expected between different eukaryotic host-virus pairs, although not much is known in this regard. High throughput techniques, like transcriptomics and/or metabolomics, have only been focused on a few ecologically relevant host-virus systems. From the work that exists we know that giant viruses genes, like those from Mimivirus and PBCV-1, are expressed quickly upon infection: PBCV-1 gene transcripts have been detected within 7 min of infection (Blanc et al., 2014). These viruses are also known to capture genes through HGT from their hosts and diverse sources, although function of these genes (and even if they are transcribed) remain largely unknown. Critical insights have been obtained regarding the modulation of cellular processes of Emiliania huxleyi -the most abundant coccolithophore alga in the world's ocean -upon infection by a large virus, EhV (Vardi et al., 2012). This includes virus-mediated regulation of the host's lipid biosynthesis resulting in programmed cell death and modulation of the host redox state during infection. (Vardi et al., 2009;Rosenwasser et al., 2016).
In Mimivirus, elaborate virus factories -cytoplasmic sites for virus replication and assemblyhave been detected. Despite these important discoveries, a significant knowledge gap exists regarding the physiological response of a host to giant virus infection.
Aureococcus anophagefferens is a bloom forming pelagophyte which causes recurrent brown tides along the east coast (Gobler et al., 2005). A giant virus (AaV), it was isolated during a brown tide event and shown to infect and lyse Aureococcus in culture (Rowe et al., 2008). A subsequent genomic study revealed the 'chimeric' nature of AaV; which picked up large number of genes from diverse cellular sources (Moniruzzaman et al., 2014), while statistical analysis of metatranscriptomic data from a brown tide bloom demonstrated active infection of Aureococcus by AaV during the peak of the bloom (Moniruzzaman et al., 2017). In addition, AaV is one of the few algae infecting viruses in the Mimiviridae, a clade of giant viruses that infect both photosynthetic and heterotrophic protists (Moniruzzaman et al., 2014). The availability of genome sequences for both AaV and its host (Gobler et al., 2011;Moniruzzaman et al., 2014) and recurrent brown tide blooms (Gastrich et al., 2004;Gobler et al., 2007) makes this host-virus pair an interesting model system. No information is available on the progressive changes in the molecular processes of Aureococcus cells upon infection, which might provide critical insights on the metabolic pathways and cellular components that can impact virus production. Moreover, the possible roles and activity of the large number of xenologs that AaV has acquired from its host, other organisms, and its NCLDV ancestor remain to be elucidated.
In this study, we employed transcriptomics to resolve the molecular response of A. anophagefferens to infection by AaV. Our experimental design examined the transcriptomic landscape throughout the AaV infection cycle (with an emphasis on the early phase) to capture the host cellular response and viral transcriptional landscape. This study also provides insight into the molecular interaction between a giant algal virus in the Mimiviridae clade and its host.

Experimental setup
Aureococcus anophagefferens CCMP 1984 was maintained in modified L-1 medium (Hallegraeff et al, 2003) at an irradiation level of 100 µmol photons m −2 s −1 and a temperature of 19 o C for a 14:10 (h) light-dark cycle. Prior to the experiment, Aureococcus cultures were grown to a mid-log phase concentration of ~1.95 x 10 6 cells/ml. Five biological replicates (2.0 L) of Aureococcus cultures at a concentration of 7.5 x 10 5 cells/ml were started within two hours of the onset of the light cycle. The cultures were inoculated with AaV at a particle multiplicity of infection (pMOI) of ~18. pMOI of 18 was chosen because it led to ~98% reduction of cell numbers 48 hr post-infection in assays conducted in-house. Due to the absence of a plaque assay to determine infectious units for AaV, we defined pMOI as total virus particles (not plaque forming units) for our experiment. For each biological replicate, control cultures were inoculated with the same volume of a heat-killed viral lysate. The heat killed-lysate was generated by exposure to microwaves (Keller et al., 1988) for 5 min. Samples for sequencing were collected at 5 min, 30 min, 1 h, 6 h, 12 h and 21 h after inoculation to capture the range of infection states seen before lysis, which starts at ~ 24 h (Rowe et al., 2008). For RNA extraction, 250 ml subsamples were filtered through 0.8-µM pore-size ATTP filters (EMD Millipore, Darmstadt, Germany) and immediately flash frozen in liquid nitrogen prior to storage at -80° C. Unfiltered samples (for cell enumeration) and samples passed through 0.45-µM polyvinylidene fluoride syringe filters (Merck, Darmstadt, Germany) for free virus count were preserved in 0.5% glutaraldehyde at -80° C from each sample at each time point.

Cell and free virus density estimates
Aureococcus cells were enumerated using a GUAVA-HT6 flow cytometer (EMD Millipore, Darmstadt, Germany) gated on the red chlorophyll fluorescence. Free virus particle densities from each time point was determined following Ortmann and Suttle, 2009. Samples were thawed at room temperature and diluted 100 times using L-1 medium prior to counting. The diluted samples were collected on 25-mm diameter Whatman Anodisc (Sigma-Aldrich, St. Louis, MO, USA) inorganic membrane filters having a nominal pore-size of 0.02 µM. The filters were allowed to air-dry for 15 min following incubation with 15 µL of 4,000X diluted Syber Green (Lonza, Rockland, ME, USA). The filters were then fixed using an anti-fade solution (50:50 PBS/glycerol and 0.1% p-Phenylenediamine) (Noble and Fuhrman, 1998). Slides were observed through a Leica DM5500 B microscope at 1000X magnification with a L5 filter cube (excitation filter: 480/40, suppression filter: BP 527/30) (Leica Microsystems CMS GmbH, Hesse, Germany). For each sample, 20 random fields (1 µM by 1 µM) were enumerated and averaged. The following formula was used to estimate the VLPs/ml in each sample:

‫כ‬ ‫ܦ‬
Where, V f = average virus count/field, A a = total filterable area of Anodisc (excluding the Oring), A g = Area of eyepiece grid, V f = volume filtered (mL), D = dilution factor.

Bioinformatics analysis
Sequencing reads were initially trimmed in CLC Genomics Workbench 9.0 (Qiagen, Hilden, Germany). Reads with a quality score cut-off of ≤ 0.3, or with ambiguous bases ('N's), were discarded. Reads passing quality control were mapped to the Aureococcus (NCBI. Accession no ACJI00000000) and AaV genome sequence (NCBI. Accession no NC_024697) with stringent mapping criteria (95% similarity, 70% length matching). Differential expression of genes in the virus-treated samples compared to the controls was determined at each time point using edgeR (Robinson et al., 2010) program implemented in the CLC Genomics Workbench 9.0. P-values were adjusted for False Discovery Rate (FDR) using Benjamini-Hochberg (BH) procedure (Benjamini and Hochberg, 1995). The number of reads mapped to each AaV gene was rarefied by library size. Values from biological replicates at each time point were averaged prior to hierarchical clustering of the viral gene expression.
Functional enrichment within the framework of Gene Ontology (GO) terms (positive or negative fold changes) was determined using BiNGO (Maere et al., 2005). GO enrichment for differentially expressed genes is complicated by at an arbitrary fold-change cut-off imposed prior to the enrichment analysis, which excludes the genes with fold-change values even marginally similar to that cut-off. To partially alleviate this problem, we ran the enrichment analysis on gene sets selected using two absolute fold change cut-offs: >1.5 and >1.3. Using both these cut-offs recovered mostly same GO processes, however some of the processes were missed by each of the individual approaches. Since our analysis is largely exploratory, results obtained from both cut-off were investigated for interesting GO processes. We report all the GO terms recovered by this approach in Supplementary Table 1. The up-or down-regulation of KEGG pathways were determined using z-test as implemented in 'GAGE' R package (Luo et al., 2009). This analysis employed input from all the genes, irrespective of fold-change level or statistical significance, and looked for coordinated expression changes within a particular pathway. The resulting Pvalues for both the analyses were corrected for false discovery rate (FDR) using Benjamini-Hochberg (BH) procedure (Benjamini and Hochberg, 1995). We considered a FDR corrected pvalue ≤ 0.1 to be significant for both GO and KEGG pathway enrichment.

Cell growth dynamics and RNA-seq output
Cultures inoculated with heat-killed lysates displayed growth patterns similar to a healthy . About ~20% of the reads from all the samples could not be aligned to the host or viral genomes, which likely originated from incomplete parts of the host genome.

Gene expression dynamics of AaV
Transcripts from 116 viral genes were present in the infected culture as early as 5 min postinfection (Supplementary table 1 expression of major capsid protein was found to be dramatically high at 21 h -encompassing more than 50% of the virus specific reads and ~6% of the entire libraries at that time point. There are 137 genes from AaV which have nucleocytoplasmic large DNA virus orthologous groups (NCVOG) (Yutin et al., 2009) and/or cluster of orthologous groups (COG) (Tatusov et al., 2000) assignments, giving insights into their potential function (Supplementary table 1).
Based on the cluster analysis, we found 7 of 9 annotated viral-methyltransferases were expressed

Global transcriptional remodeling of the virus infected host
Viral infection induced a dramatic and rapid reprogramming of host cell gene expression, which was reflected in the number of Aureococcus genes that were differentially expressed compared to the uninfected culture ( Figure 3). Even at 5-min post infection, we observed 13.4% of the 11,570 genes of Aureococcus were differentially expressed, with 412 genes having fold changes of > 1.5, and 588 genes with a fold change < -1.5 (FDR corrected p < 0.05) (Figure 3). With exception of the 1-h time point, the number of genes over or underrepresented compared to control showed a tendency to increase over time, with the highest number of genes observed to be differentially expressed occurring at the 12 h time point (42.9%).
The number of differentially expressed host genes was dramatically reduced at 1-h compared to other time points: only 82 genes were found to be differentially expressed (

DNA mismatch repair, "immune response" and polyamine biosynthesis
Surprisingly, we detected a GO term "immune response" that was down-regulated around 6-h and 12-h post-infection ( Figure 4). Upon closer inspection, the genes annotated by this function were found to be guanylate-binding proteins (GBP). GBPs belong to interferon-gamma inducible GTPase superfamily that are widely known to promote defense against viruses and other intracellular pathogens in humans, mice and other mammals (Vestal and Jeyaratnam, 2011). We determined that a number of GBP proteins were down-regulated 30-min post-infection and onwards ( Table 2). The phagosome pathway was also down-regulated immediately after infection, while autophagy pathway was down-regulated 6-h post-infection ( Figure 4).
Interestingly, GBPs are known to transport antimicrobial peptides, NADPH oxidase components and the machinery of autophagy in the phagosomal compartments (Dupont and Hunter, 2012).
These observations indicate that GBPs might be part of a defense network in Aureococcus against infection. Circumvention of this network is probably crucial in establishing successful infection by AaV.
DNA mismatch repair and damaged DNA-binding related GO processes were found to be overrepresented around 6-h post-infection ( Figure 4). All of the genes annotated with these GO processes were found to encode proteins having MutS and MutL domains, which are important players in detecting and recruiting repair machineries to mismatched bases on DNA (Modrich, 2006). Viral infection induced significant over-expression of a number of these proteins within 5-min of infection (Supplementary figure 6). These include a MutS homolog and a small MutS related protein (smr). By 30 min, more of these genes showed significant increased expression, which include a MutS2, MutL and another smr homolog (Supplementary figure 6). Around 6-h, all these genes remained highly expressed compared to control. Several genes were found to be down-regulated at 12 h but up-regulated at 21 h, indicating varying degree of regulatory controls acting upon them over the course of infection (Supplementary figure 6).
Data exploration using GO analysis also revealed an overrepresentation of spermidine synthase activity at 6 h post infection. Ornithine decarboxylase converts L-ornithine to putrescine, which is further converted to spermidine by spermidine synthase. Aureococcus homologs of ornithine decarboxylase and spermidine synthase were found to be up-regulated in the virus-infected culture from 6 h onwards (Supplementary figure 7). Additionally, a homolog of Ncarbamoylputrescine amydase (Aurandraft_59241) was also found to be up-regulated during this time. This suggests that cellular spermidine and putrescine pools increased during the intermediate and late phase of infection.

Alteration of photosynthesis and photoprotection related processes upon virus infection
We  Table 3). Taken together, the data indicate that photosynthetic capacity of the

virus infected cells decreased during the early and intermediate stage of infection -throughout
the light cycle. During the 12 and 21-h, photosynthesis and thylakoid related GO processes were underrepresented in the infected cells compared to the non-infected ones (Figure 4). This could indicate that photosynthesis related genes peaked in expression during mid-night and pre-dawn in the healthy cultures, a phenomenon that has been observed in other algae such as Ostreococcus tauri (Monnier et al., 2010). In addition, isoprenoid biosynthesis was downregulated around the same time. Isoprenoids are an important functional and structural part of the photosynthetic apparatus and photosynthetic electron careers, with roles in regulating the fluidity of photosynthetic membranes. (Havaux, 1998). Also, isoprenoid compounds like zeaxanthin and β -carotene are involved in photoprotection -they dissipate excessive light energy through heat (Peñuelas and Munné-Bosch, 2005  The heme biosynthesis pathway, which leads to the production of photosynthetic pigments including chlorophyll a and other tetrapyrrolic pigments, is a crucial metabolic pathway in photosynthetic organisms (Obornik and Green, 2005). While genes involved in light harvesting, photosystem structure and isoprenoid biosynthesis were generally under-expressed, we found porphyrin biosynthesis genes in this pathway to be overexpressed immediately after infection ( Figure 5). All the genes involved in synthesis of the precursor of protoporphyrin-X were upregulated immediately after infection, but were down-regulated by 30-mins post-infection ( Figure 5). In contrast, genes involved in chlorophyll a biosynthesis from protoporphyrin-X did not show significant up or down-regulation at this time ( Figure 5). Collectively, this data indicates that porphyrin derivatives accumulated early in the infected cell, possibly leading to photooxidative damage of different cellular components, including chloroplasts (Reinbothe et al., 1996). Additionally, host DNA photolyase activity was found to be down-regulated 5-min postinfection ( Figure 4) -a process critical in repairing UV-mediated formation of pyrimidine dimers in DNA (Thiagarajan et al., 2011).

Changes in expression associated with the selenoproteome
Aureococcus has 59 predicted selenoproteins -the highest reported amongst all eukaryotes (Gobler et al., 2011). Out of these, 35 showed significant (FDR p < 0.1) fold-changes at least at one time point (Figure 6). While several selenoproteins were found to be under-expressed immediately post-infection, at 12 and 21-h a large number of selenoproteins showed increased expression relative to controls ( Figure 6). We found O-phosphoseryl-tRNA(Sec) selenium transferase, a gene involved in producing selenocysteinyl-tRNA from L-seryl-tRNA(Sec) to be overexpressed (Supplementary figure 9). Cystathionine beta-lyase and Selenocysteine (Sec) lyase, two genes involved in conversion of Sec into methionine and alanine, also showed increased expression compared to control (Supplementary figure 9). Although no known selenium transporter has been characterized in Aureococcus, it is known that opportunistic transport of selenium using phosphate transporters might be common in plants, fungi and algae (Lazard et al., 2010). Aureococcus has six annotated phosphate transporters. Among these, either 3 or 4 of the transporters showed significant positive differential expression at 12-h and 21-h, respectively, during infection (Supplementary Figure 10). However, this could also imply that AaV proliferation may require elevated concentration of phosphate in cell. Phosphate depletion has been shown to massively reduce the burst size of PpV in Phaeocystis pouchetii (Carreira et al., 2013), PBCV in Chlorella sp. (Carreira et al., 2013) and MpV in Micromonas pusilla (Maat et al., 2014). Five of the overexpressed selenoprotein genes were methionine sulfoxide reductases ( Figure 6), genes involved in reversing the oxidation of methionine by reactive oxygen species (ROS), thereby repairing the oxidative damage in proteins (Moskovitz, 2005).
Three copies of glutathione peroxidases (GPx) were also over-expressed compared to control, which are crucial in reducing H 2 O 2 or organic hydroperoxides, thereby minimizing oxidative damage to cellular components (Ursini et al., 1995). Glutathione S-transferase (GST), a selenoprotein, was over-expressed during the last three time points. GSTs have diverse functions in the cell, including detoxification of electrophilic metabolites of xenobiotics into less reactive compounds by catalyzing their conjugation with glutathione (GSH) (Veal et al., 2002). GSTs are also known to participate in oxidative stress protection by conjugating GSH with secondary ROS molecules that are produced upon ROS reacting with cellular constituents (Danielson et al., 1987). In addition, some GSTs show GPx activity (Tan and Board, 1996). Sel U and Sel H, two selenoproteins involved in redox functions and Sep15, a transcript whose product is involved in protein folding in endoplasmic reticulum (Labunskyy et al., 2009) were overexpressed in the late stage of virus infection (Figure 6).
A number of redox active proteins not incorporating selenium were also overexpressed during the last two time points. This includes a Cu-Zn superoxide dismutase (Aurandraft_59136), which dismutates superoxide anion (O 2 -), leading to production of H 2 O 2 . We detected overexpression of a dehydroascorbate reductase homolog (Aurandraft_67072), which is involved in recycling of ascorbate, during the last three time points. Ascorbate acts as a key antioxidant in the cell by directly neutralizing superoxide radicals, singlet oxygen or hydroxyl radical (Noctor and Foyer, 1998). Collectively, this data indicate that virus infected cells were responding to the oxidative stress induced by AaV invasion and proliferation, and a large number of selenoproteins participated in this process.

Transcriptional landscape of AaV infection
This study provides initial insight into the gene expression dynamics of an algal virus in the Mimiviridae clade (Santini et al., 2013). AaV has a 21-30 h infection cycle, with free virus production observable by 21 hours post-infection, which steadily increases over time . The almost immediate transcription of viral genes indicated a rapid modulation of host cellular processes directed towards transcribing the viral mRNAs (Figure 2 Table 1).
The expression of carbohydrate metabolism genes (putatively acquired by HGT) in AaV also leads to some intriguing possibilities. It is known that unsaturated glucuronyl hydrolases remove the terminal unsaturated sugar from the oligosaccharide products released by polysaccharide lyases (Jongkees and Withers, 2011). Healthy Aureococcus cells are surrounded by a fibrous glycocalyx, which is absent from the virus infected cells (Gastrich et al., 1998): it is thus compelling to speculate that the role of a polysaccharide lyase and glucuronyl hydrolase during infection is to make the cell membrane accessible for virus attachment.

The Aureococcus 'virocell' -transcriptional remodeling upon virus infection
One observation within this study was a rapid transcriptional response of the host after virus treatment ( Figure 3). In part the differentially expressed gene pool might reflect host defense response to virus attack. However, this response also potentially includes genes that are rapidly manipulated by the virus to transform the cellular environment in favor of virus propagation. This rapid change at transcription level perhaps represents how the transformation of a healthy cell into a 'virocell' (Forterre, 2011) is initiated.
Relative to competing plankton, Aureococcus has a larger number of nuclear-encoded LHC proteins, which augment the photosynthetic reaction center in collecting light energy (Gobler et al., 2011). It is known that lower light level can delay the virus-mediated lysis of Aureococcus, and photosynthetic efficiency is not significantly different between infected and non-infected cultures within 24-h of infection (Gobler et al., 2007). However, the molecular basis of how AaV infection can influence the host photosynthetic capacity is largely unknown. Immediately after infection, transcripts for photosynthesis related processes proportionally decreased relative to control, with increasing number of LHC proteins being down-regulated as infection progressed (Supplementary figure 8). AaV propagation was found to be adversely affected at low lightwith cultures incubated in low light (~3 µmol quanta m -2 s -1 ) taking more than 7 days to be reduced to <10 4 cells/mL compared to high light (~110 µmol quanta m -2 s -1 ) incubated culture, which took 3 days to be reduced to similar concentration (Gobler et al., 2007). Down-regulation of photosynthesis was also observed in Chlorella upon infection with PBCV-1 (Seaton et al., 1995) and Heterosigma akashiwo infected by either RNA or DNA viruses (Philippe et al., 2003).
Photosynthesis was found to be down-regulated in a wide range of plants in response to pathogen invasion (e.g., virus and bacteria) (Bilgin et al., 2010) and was suggested to be an adaptive response to biotic attack. It is important to note that down-regulation of gene expression doesn't necessarily mean immediate loss of function -specifically, the proteins involved in light reaction might have a long functional lifetime (Bilgin et al., 2010). Thus, the actual effect of immediate down-regulation of photosynthesis gene expression on viral attack remains to be elucidated. It was proposed that slow turnover of many photosynthesis related proteins allows the host to redirect resources for immediate defense mechanisms without dramatically reducing its photosynthetic capacity (Bilgin et al., 2010). High light requirement of the virus and the capacity of Aureococcus to grow in a low light environment might itself act as a natural defense mechanism at the community level, where delayed virus production can eventually lead to fewer host-virus contacts and infection.
In photosynthetic organisms, different chlorophyll precursors are formed as part of its biosynthetic pathway. However, accumulation of such precursors, especially protoporphyrin IX, can lead to photosensitivity (Inagaki et al., 2015). It has been demonstrated that various porphyrin derivatives might have broad antiviral activity -however, the activity is mostly extracellular. For example, an alkylated porphyrin (chlorophillide) was found to cause damage to the hepatitis B-virus capsid (Guo et al., 2011), leading to loss of virion DNA. Thus, increased porphyrin concentrations in AaV infected cells might increase the oxidative stress, making the cellular environment hostile for the invading viral components (Mock et al., 1998). Intriguingly, AaV encodes a Phaeophorbide a oxygenase gene (AaV_372) which is a key regulator in heme/chlorophyll breakdown (Hörtensteiner, 2013). Transcripts for this gene were initially reported to be part of the CroV proteome (Fischer et al., 2014). It was also found that majority of the packaged proteins, including photolyase, were late proteins (Fischer et al., 2014). A photolyase is also encoded into AaV genome, which was found to be expressed late ( The majority of the selenoproteins characterized to date have redox-active functions, however, they can also have a wide range of biological roles (Labunskyy et al., 2014). Some viruses can encode Sec containing proteins, with bioinformatics evidence provided for several mammalian viruses (Taylor et al., 1997): indeed, a Sec-containing glutathione peroxidase experimentally characterized in HIV-1 (Zhao et al., 2000). Given the unrivaled compendium of Aureococcus selenoproteins within the eukaryotic domain, we were interested in how the expressions of these proteins would be modulated by AaV infection and their possible role in virus propagation. Our study indicates that a number of up-regulated selenoproteins are possibly involved in viral protein synthesis and preventing oxidation of these viral proteins, especially during the late phase of infection ( Figure 6). It has been demonstrated that Sec-containing methionine sulfoxide reductases (MSR) are efficient, showing 10-50 fold higher enzymatic activity compared to the Cys-containing MSRs (Kim et al., 2006). Additionally, selenoproteins deemed crucial in regulating the redox state of the cells (e.g., glutathione peroxidase and dehydroascorbate reductase) were also overrepresented during infection. The cellular pro-/antioxidant balance is a highly complex process, involving a cascade of enzymatic activity and interconnected pathways.
As was aptly put by Schwarz (1996), "it is difficult to distinguish between association and causation as well as between primary and secondary effects of a given virus on ROS mediated

Conclusion:
Host-virus interactions at nanoscale eventually shape ecosystem processes at geographical scales (Brussaard et al., 2008). Resolving the molecular aspects of ecologically relevant host-virus interactions is critical to understand the role of viruses in the biogeochemical processes, as well as the factors that drive the co-evolution of virus-host systems . In this study, we gleaned insights into the transcriptomic response of a harmful alga upon infection by a giant virus. The ultimate fate of a cell going through lytic infection is to produce progeny viruses, which is accomplished through a different transcriptomic and metabolic trajectory relative to a healthy cell. The most likely outcome of this massive transcriptional response is a reprogrammed metabolic profile -specific metabolites might regulate viral replication and might be incorporated in the virion particles. The altered virocell metabolism might even influence large scale ecological processes; for example, differential uptake or release of specific compounds by virocells might alter the nutrient dynamics, thereby affecting the coexisting microbial communities (Ankrah et al., 2014). This study will provide an important foundation to generate and test new hypotheses regarding individual metabolic or regulatory processes that can have important biogeochemical consequences, and perhaps more importantly, place the "virocell" into a better ecological context.