Identification of a Chlorovirus PBCV-1 Protein Involved in Degrading the Host Cell Wall during Virus Infection.

Chloroviruses are unusual among viruses infecting eukaryotic organisms in that they must, like bacteriophages, penetrate a rigid cell wall to initiate infection. Chlorovirus PBCV-1 infects its host, Chlorella variabilis NC64A by specifically binding to and degrading the cell wall of the host at the point of contact by a virus-packaged enzyme(s). However, PBCV-1 does not use any of the five previously characterized virus-encoded polysaccharide degrading enzymes to digest the Chlorella host cell wall during virus entry because none of the enzymes are packaged in the virion. A search for another PBCV-1-encoded and virion-associated protein identified protein A561L. The fourth domain of A561L is a 242 amino acid C-terminal domain, named A561LD4, with cell wall degrading activity. An A561LD4 homolog was present in all 52 genomically sequenced chloroviruses, infecting four different algal hosts. A561LD4 degraded the cell walls of all four chlorovirus hosts, as well as several non-host Chlorella spp. Thus, A561LD4 was not cell-type specific. Finally, we discovered that exposure of highly purified PBCV-1 virions to A561LD4 increased the specific infectivity of PBCV-1 from about 25-30% of the particles forming plaques to almost 50%. We attribute this increase to removal of residual host receptor that attached to newly replicated viruses in the cell lysates.

Chlorovirus PBCV-1 Multidomain Protein A111/114R Has Three Glycosyltransferase Functions Involved in the Synthesis of Atypical N-Glycans.

The structures of the four N-linked glycans from the prototype chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of genetic and structural analyses indicates that the protein coded by PBCV-1 gene a111/114r, conserved in all chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan, and, if biochemically corroborated, it would be the second three-domain protein coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the chloroviruses do not use the canonical host endoplasmic reticulum-Golgi glycosylation pathway to glycosylate their glycoproteins; instead, they perform glycosylation independent of cellular organelles using virus-encoded enzymes.

Validation of superior reference genes for qRT-PCR and Western blot analyses in marine Emiliania huxleyi-virus model system.

AIMS: To search for a set of reference genes for reliable gene expression analysis in the globally important marine coccolithophore Emiliania huxleyi-virus model system. METHODS AND RESULTS: Fifteen housekeeping genes (CDKA, CYP15, EFG3, POLAI, RPL30, RPL13, SAMS, COX1, GPB1-2, HSP90, TUA, TUB, UBA1, CAM3 and GAPDH) were evaluated for their stability as potential reference genes for qRT-PCR using ΔCt, geNorm, NormFinder, Bestkeeper and RefFinder software. CDKA, TUA and TUB genes were tested as loading controls for Western blot in the same sample panel. Additionally, target genes associated with cell apoptosis, that is metacaspase genes, were applied to validate the selection of reference genes. The analysis results demonstrated that putative housekeeping genes exhibited significant variations in both mRNA and protein content during virus infection. After a comprehensive analysis with all the algorithms, CDKA and GAPDH were recommended as the most stable reference genes for E huxleyi virus (EhV) infection treatments. For Western blot, significant variation was seen for TUA and TUB, whereas CDKA was stably expressed, consistent with the results of qRT-PCR. CONCLUSIONS: CDKA and GAPDH are the best choice for gene and protein expression analysis than the other candidate reference genes under EhV infection conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The stable internal control genes identified in this work will help to improve the accuracy and reliability of gene expression analysis and gain insight into complex E. huxleyi-EhV interaction regulatory networks.

Marine virus predation by non-host organisms.

Viruses are the most abundant biological entities in marine environments, however, despite its potential ecological implications, little is known about virus removal by ambient non-host organisms. Here, we examined the effects of a variety of non-host organisms on the removal of viruses. The marine algal virus PgV-07T (infective to Phaeocystis globosa) can be discriminated from bacteriophages using flow cytometry, facilitating its use as a representative model system. Of all the non-host organisms tested, anemones, polychaete larvae, sea squirts, crabs, cockles, oysters and sponges significantly reduced viral abundance. The latter four species reduced viral abundance the most, by 90, 43, 12 and 98% over 24 h, respectively. Breadcrumb sponges instantly removed viruses at high rates (176 mL h-1 g tissue dry wt-1) which continued over an extended period of time. The variety of non-host organisms capable of reducing viral abundance highlights that viral loss by ambient organisms is an overlooked avenue of viral ecology. Moreover, our finding that temperate sponges have the huge potential for constant and effective removal of viruses from the water column demonstrates that natural viral loss has, thus far, been underestimated.

Chloroviruses.

Chloroviruses are large dsDNA, plaque-forming viruses that infect certain chlorella-like green algae; the algae are normally mutualistic endosymbionts of protists and metazoans and are often referred to as zoochlorellae. The viruses are ubiquitous in inland aqueous environments throughout the world and occasionally single types reach titers of thousands of plaque-forming units per ml of native water. The viruses are icosahedral in shape with a spike structure located at one of the vertices. They contain an internal membrane that is required for infectivity. The viral genomes are 290 to 370 kb in size, which encode up to 16 tRNAs and 330 to ~415 proteins, including many not previously seen in viruses. Examples include genes encoding DNA restriction and modification enzymes, hyaluronan and chitin biosynthetic enzymes, polyamine biosynthetic enzymes, ion channel and transport proteins, and enzymes involved in the glycan synthesis of the virus major capsid glycoproteins. The proteins encoded by many of these viruses are often the smallest or among the smallest proteins of their class. Consequently, some of the viral proteins are the subject of intensive biochemical and structural investigation.

The Roles of Electrostatic Interactions in Capsid Assembly Mechanisms of Giant Viruses.

In the last three decades, many giant DNA viruses have been discovered. Giant viruses present a unique and essential research frontier for studies of self-assembly and regulation of supramolecular assemblies. The question on how these giant DNA viruses assemble thousands of proteins so accurately to form their protein shells, the capsids, remains largely unanswered. Revealing the mechanisms of giant virus assembly will help to discover the mysteries of many self-assembly biology problems. Paramecium bursaria Chlorella virus-1 (PBCV-1) is one of the most intensively studied giant viruses. Here, we implemented a multi-scale approach to investigate the interactions among PBCV-1 capsid building units called capsomers. Three binding modes with different strengths are found between capsomers around the relatively flat area of the virion surface at the icosahedral 2-fold axis. Furthermore, a capsomer structure manipulation package is developed to simulate the capsid assembly process. Using these tools, binding forces among capsomers were investigated and binding funnels were observed that were consistent with the final assembled capsid. In addition, total binding free energies of each binding mode were calculated. The results helped to explain previous experimental observations. Results and tools generated in this work established an initial computational approach to answer current unresolved questions regarding giant virus assembly mechanisms. Results will pave the way for studying more complicated process in other biomolecular structures.

In plaque-mass spectrometry imaging of a bloom-forming alga during viral infection reveals a metabolic shift towards odd-chain fatty acid lipids.

Tapping into the metabolic crosstalk between a host and its virus can reveal unique strategies employed during infection. Viral infection is a dynamic process that generates an evolving metabolic landscape. Gaining a continuous view into the infection process is highly challenging and is limited by current metabolomics approaches, which typically measure the average of the entire population at various stages of infection. Here, we took an innovative approach to study the metabolic basis of host-virus interactions between the bloom-forming alga Emiliania huxleyi and its specific virus. We combined a classical method in virology, the plaque assay, with advanced mass spectrometry imaging (MSI), an approach we termed 'in plaque-MSI'. Taking advantage of the spatial characteristics of the plaque, we mapped the metabolic landscape induced during infection in a high spatiotemporal resolution, unfolding the infection process in a continuous manner. Further unsupervised spatially aware clustering, combined with known lipid biomarkers, revealed a systematic metabolic shift during infection towards lipids containing the odd-chain fatty acid pentadecanoic acid (C15:0). Applying 'in plaque-MSI' may facilitate the discovery of bioactive compounds that mediate the chemical arms race of host-virus interactions in diverse model systems.

Nitric oxide production and antioxidant function during viral infection of the coccolithophore Emiliania huxleyi.

Emiliania huxleyi is a globally important marine phytoplankton that is routinely infected by viruses. Understanding the controls on the growth and demise of E. huxleyi blooms is essential for predicting the biogeochemical fate of their organic carbon and nutrients. In this study, we show that the production of nitric oxide (NO), a gaseous, membrane-permeable free radical, is a hallmark of early-stage lytic infection in E. huxleyi by Coccolithoviruses, both in culture and in natural populations in the North Atlantic. Enhanced NO production was detected both intra- and extra-cellularly in laboratory cultures, and treatment of cells with an NO scavenger significantly reduced viral production. Pre-treatment of exponentially growing E. huxleyi cultures with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) prior to challenge with hydrogen peroxide (H2O2) led to greater cell survival, suggesting that NO may have a cellular antioxidant function. Indeed, cell lysates generated from cultures treated with SNAP and undergoing infection displayed enhanced ability to detoxify H2O2. Lastly, we show that fluorescent indicators of cellular ROS, NO, and death, in combination with classic DNA- and lipid-based biomarkers of infection, can function as real-time diagnostic tools to identify and contextualize viral infection in natural E. huxleyi blooms.

The mutual interplay between calcification and coccolithovirus infection.

Two prominent characteristics of marine coccolithophores are their secretion of coccoliths and their susceptibility to infection by coccolithoviruses (EhVs), both of which display variation among cells in culture and in natural populations. We examined the impact of calcification on infection by challenging a variety of Emiliania huxleyi strains at different calcification states with EhVs of different virulence. Reduced cellular calcification was associated with increased infection and EhV production, even though calcified cells and associated coccoliths had significantly higher adsorption coefficients than non-calcified (naked) cells. Sialic acid glycosphingolipids, molecules thought to mediate EhV infection, were generally more abundant in calcified cells and enriched in purified, sorted coccoliths, suggesting a biochemical link between calcification and adsorption rates. In turn, viable EhVs impacted cellular calcification absent of lysis by inducing dramatic shifts in optical side scatter signals and a massive release of detached coccoliths in a subpopulation of cells, which could be triggered by resuspension of healthy, calcified host cells in an EhV-free, 'induced media'. Our findings show that calcification is a key component of the E. huxleyi-EhV arms race and an aspect that is critical both to the modelling of these host-virus interactions in the ocean and interpreting their impact on the global carbon cycle.

Light regulation of coccolithophore host-virus interactions.

Viruses that infect photoautotrophs have a fundamental relationship with light, given the need for host resources. We investigated the role of light on Coccolithovirus (EhV) infection of the globally distributed coccolithophore, Emiliania huxleyi. Light was required for EhV adsorption, and viral production was highest when host cultures were maintained in continuous light or at irradiance levels of 150-300 µmol m-2  s-1 . During the early stages of infection, photosynthetic electron transport remained high, while RuBisCO expression decreased concomitant with an induction of the pentose phosphate pathway, the primary source of de novo nucleotides. A mathematical model developed and fitted to the laboratory data supported the hypothesis that EhV replication was controlled by a trade-off between host nucleotide recycling and de novo synthesis, and that photoperiod and photon flux could toggle this switch. Laboratory results supported field observations that light was the most robust driver of EhV replication within E. huxleyi populations collected across a 2000 nautical mile transect in the North Atlantic. Collectively, these findings demonstrate that light can drive host-virus interactions through a mechanistic interplay between host metabolic processes, which serve to structure infection and phytoplankton mortality in the upper ocean.

Algal Viruses: The (Atomic) Shape of Things to Come.

Visualization of algal viruses has been paramount to their study and understanding. The direct observation of the morphological dynamics of infection is a highly desired capability and the focus of instrument development across a variety of microscopy technologies. However, the high temporal (ms) and spatial resolution (nm) required, combined with the need to operate in physiologically relevant conditions presents a significant challenge. Here we present a short history of virus structure study and its relation to algal viruses and highlight current work, concentrating on electron microscopy and atomic force microscopy, towards the direct observation of individual algae⁻virus interactions. Finally, we make predictions towards future algal virus study direction with particular focus on the exciting opportunities offered by modern high-speed atomic force microscopy methods and instrumentation.

Dynamics of transparent exopolymer particle production and aggregation during viral infection of the coccolithophore, Emiliania huxleyi.

Emiliania huxleyi produces calcium carbonate (CaCO3 ) coccoliths and transparent exopolymer particles (TEP), sticky, acidic carbohydrates that facilitate aggregation. E. huxleyi's extensive oceanic blooms are often terminated by coccolithoviruses (EhVs) with the transport of cellular debris and associated particulate organic carbon (POC) to depth being facilitated by TEP-bound 'marine snow' aggregates. The dynamics of TEP production and particle aggregation in response to EhV infection are poorly understood. Using flow cytometry, spectrophotometry and FlowCam visualization of alcian blue (AB)-stained aggregates, we assessed TEP production and the size spectrum of aggregates for E. huxleyi possessing different degrees of calcification and cellular CaCO3 :POC mass ratios, when challenged with two EhVs (EhV207 and EhV99B1). FlowCam imaging also qualitatively assessed the relative amount of AB-stainable TEP (i.e., blue:red ratio of each particle). We show significant increases in TEP during early phase EhV207-infection (∼ 24 h) of calcifying strains and a shift towards large aggregates following EhV99B1-infection. We also observed the formation of large aggregates with low blue:red ratios, suggesting that other exopolymer substances contribute towards aggregation. Our findings show the potential for virus infection and the associated response of their hosts to impact carbon flux dynamics and provide incentive to explore these dynamics in natural populations.

Transcriptional responses of the marine green alga Micromonas pusilla and an infecting prasinovirus under different phosphate conditions.

Prasinophytes are widespread marine algae for which responses to nutrient limitation and viral infection are not well understood. We studied the picoprasinophyte, Micromonas pusilla, grown under phosphate-replete (0.65 ± 0.07 d-1 ) and 10-fold lower (low)-phosphate (0.11 ± 0.04 d-1 ) conditions, and infected by the phycodnavirus MpV-SP1. Expression of 17% of Micromonas genes in uninfected cells differed by >1.5-fold (q < 0.01) between nutrient conditions, with genes for P-metabolism and the uniquely-enriched Sel1-like repeat (SLR) family having higher relative transcript abundances, while phospholipid-synthesis genes were lower in low-P than P-replete. Approximately 70% (P-replete) and 30% (low-P) of cells were lysed 24 h post-infection, and expression of ≤5.8% of host genes changed relative to uninfected treatments. Host genes for CAZymes and glycolysis were activated by infection, supporting importance in viral production, which was significantly lower in slower growing (low-P) hosts. All MpV-SP1 genes were expressed, and our analyses suggest responses to differing host-phosphate backgrounds involve few viral genes, while the temporal program of infection involves many more, and is largely independent of host-phosphate background. Our study (i) identifies genes previously unassociated with nutrient acclimation or viral infection, (ii) provides insights into the temporal program of prasinovirus gene expression by hosts and (iii) establishes cell biological aspects of an ecologically important host-prasinovirus system that differ from other marine algal-virus systems.

Population size changes and selection drive patterns of parallel evolution in a host-virus system.

Predicting the repeatability of evolution remains elusive. Theory and empirical studies suggest that strong selection and large population sizes increase the probability for parallel evolution at the phenotypic and genotypic levels. However, selection and population sizes are not constant, but rather change continuously and directly affect each other even on short time scales. Here, we examine the degree of parallel evolution shaped through eco-evolutionary dynamics in an algal host population coevolving with a virus. We find high degrees of parallelism at the level of population size changes (ecology) and at the phenotypic level between replicated populations. At the genomic level, we find evidence for parallelism, as the same large genomic region was duplicated in all replicated populations, but also substantial novel sequence divergence between replicates. These patterns of genome evolution can be explained by considering population size changes as an important driver of rapid evolution.

Coccolithovirus facilitation of carbon export in the North Atlantic.

Marine phytoplankton account for approximately half of global primary productivity 1 , making their fate an important driver of the marine carbon cycle. Viruses are thought to recycle more than one-quarter of oceanic photosynthetically fixed organic carbon 2 , which can stimulate nutrient regeneration, primary production and upper ocean respiration 2 via lytic infection and the 'virus shunt'. Ultimately, this limits the trophic transfer of carbon and energy to both higher food webs and the deep ocean 2 . Using imagery taken by the Moderate Resolution Imaging Spectroradiometer (MODIS) onboard the Aqua satellite, along with a suite of diagnostic lipid- and gene-based molecular biomarkers, in situ optical sensors and sediment traps, we show that Coccolithovirus infections of mesoscale (~100 km) Emiliania huxleyi blooms in the North Atlantic are coupled with particle aggregation, high zooplankton grazing and greater downward vertical fluxes of both particulate organic and particulate inorganic carbon from the upper mixed layer. Our analyses captured blooms in different phases of infection (early, late and post) and revealed the highest export flux in 'early-infected blooms' with sinking particles being disproportionately enriched with infected cells and subsequently remineralized at depth in the mesopelagic. Our findings reveal viral infection as a previously unrecognized ecosystem process enhancing biological pump efficiency.

Size-dependent Catalysis of Chlorovirus Population Growth by A Messy Feeding Predator.

Many chloroviruses replicate in endosymbiotic zoochlorellae that are protected from infection by their symbiotic host. To reach the high virus concentrations that often occur in natural systems, a mechanism is needed to release zoochlorellae from their hosts. We demonstrate that the ciliate predator Didinium nasutum foraging on zoochlorellae-bearing Paramecium bursaria can release live zoochlorellae from the ruptured prey cell that can then be infected by chloroviruses. The catalysis process is very effective, yielding roughly 95% of the theoretical infectious virus yield as determined by sonication of P. bursaria. Chlorovirus activation is more effective with smaller Didinia, as larger Didinia typically consume entire P. bursaria cells without rupturing them, precluding the release of zoochlorellae. We also show that the timing of Chlorovirus growth is tightly linked to the predator-prey cycle between Didinium and Paramecium, with the most rapid increase in chloroviruses temporally linked to the peak foraging rate of Didinium, supporting the idea that predator-prey cycles can drive cycles of Chlorovirus abundance.

Chitin synthesis by Chlorella cells infected by chloroviruses: Enhancement by adopting a slow-growing virus and treatment with aphidicolin.

Chlorella viruses or chloroviruses contain a gene that encodes an enzyme that catalyzes chitin synthesis. This gene is expressed early in viral infections to produce chitin on the outside of the Chlorella cell wall. Interestingly, chitin synthesis by microalgal Chlorella cells in combination with chloroviruses represents a unique eco-friendly process for converting solar energy and CO2 into useful materials. However, during the final viral infection stage, the host cells are completely lysed, so chitin should be harvested before cells lyse. To increase chitin yields, slow-growing chlorovirus isolates were adopted and the viral replication process was modified with an inhibitor of DNA synthesis. The accumulation of chitin on the surface of Chlorella cells infected with one of nine chlorovirus isolates carrying the chitin synthase gene was compared with that of CVK2 (a standard virus)-infected cells. Chlorella cells infected with CVNF-1 (a slow-growing virus) accumulated chitin over the entire cell surface within 15 min post-infection (p.i.), and chitin continued to accumulate for up to 8 h p.i. before cells lysed. This was 2-fold longer than the chitin-accumulation period for cells infected with CVK2. The addition of aphidicolin delayed the progression of the virus replication cycle and extended the chitin-accumulation period of CVNF-1-infected cells to 12 h p.i. before cells lysed. Additionally, chitin production in the aphidicolin-treated CVNF-1-infected cells was approximately 6-fold higher than in CVK2-infected cells not treated with aphidicolin. Thus, chitin synthesis in a Chlorella-virus system may be prolonged by using slow-growing viral isolates treated with aphidicolin.

Structural studies demonstrating a bacteriophage-like replication cycle of the eukaryote-infecting Paramecium bursaria chlorella virus-1.

A fundamental stage in viral infection is the internalization of viral genomes in host cells. Although extensively studied, the mechanisms and factors responsible for the genome internalization process remain poorly understood. Here we report our observations, derived from diverse imaging methods on genome internalization of the large dsDNA Paramecium bursaria chlorella virus-1 (PBCV-1). Our studies reveal that early infection stages of this eukaryotic-infecting virus occurs by a bacteriophage-like pathway, whereby PBCV-1 generates a hole in the host cell wall and ejects its dsDNA genome in a linear, base-pair-by-base-pair process, through a membrane tunnel generated by the fusion of the virus internal membrane with the host membrane. Furthermore, our results imply that PBCV-1 DNA condensation that occurs shortly after infection probably plays a role in genome internalization, as hypothesized for the infection of some bacteriophages. The subsequent perforation of the host photosynthetic membranes presumably enables trafficking of viral genomes towards host nuclei. Previous studies established that at late infection stages PBCV-1 generates cytoplasmic organelles, termed viral factories, where viral assembly takes place, a feature characteristic of many large dsDNA viruses that infect eukaryotic organisms. PBCV-1 thus appears to combine a bacteriophage-like mechanism during early infection stages with a eukaryotic-like infection pathway in its late replication cycle.