The consumption of viruses returns energy to food chains.

Viruses impact host cells and have indirect effects on ecosystem processes. Plankton such as ciliates can reduce the abundance of virions in water, but whether virus consumption translates into demographic consequences for the grazers is unknown. Here, we show that small protists not only can consume viruses they also can grow and divide given only viruses to eat. Moreover, the ciliate Halteria sp. foraging on chloroviruses displays dynamics and interaction parameters that are similar to other microbial trophic interactions. These results suggest that the effect of viruses on ecosystems extends beyond (and in contrast to) the viral shunt by redirecting energy up food chains.

Combination of hydrophobicity and codon usage bias determines sorting of model K+ channel protein to either mitochondria or endoplasmic reticulum.

When the K+ channel-like protein Kesv from Ectocarpus siliculosus virus 1 is heterologously expressed in mammalian cells, it is sorted to the mitochondria. This targeting can be redirected to the endoplasmic reticulum (ER) by altering the codon usage in distinct regions of the gene or by inserting a triplet of hydrophobic amino acids (AAs) into the protein's C-terminal transmembrane domain (ct-TMD). Systematic variations in the flavor of the inserted AAs and/or its codon usage show that a positive charge in the inserted AA triplet alone serves as strong signal for mitochondria sorting. In cases of neutral AA triplets, mitochondria sorting are favored by a combination of hydrophilic AAs and rarely used codons; sorting to the ER exhibits the inverse dependency. This propensity for ER sorting is particularly high when a common codon follows a rarer one in the AA triplet; mitochondria sorting in contrast is supported by codon uniformity. Since parameters like positive charge, hydrophobic AAs, and common codons are known to facilitate elongation of nascent proteins in the ribosome the data suggest a mechanism in which local changes in elongation velocity and co-translational folding in the ct-TMD influence intracellular protein sorting.

Lessons from Chloroviruses: the Complex and Diverse Roles of Viruses in Food Webs.

Viruses can have large effects on the ecological communities in which they occur. Much of this impact comes from the mortality of host cells, which simultaneously alters microbial community composition and causes the release of matter that can be used by other organisms. However, recent studies indicate that viruses may be even more deeply integrated into the functioning of ecological communities than their effect on nutrient cycling suggests. In particular, chloroviruses, which infect chlorella-like green algae that typically occur as endosymbionts, participate in three types of interactions with other species. Chlororviruses (i) can lure ciliates from a distance, using them as a vector; (ii) depend on predators for access to their hosts; and (iii) get consumed as a food source by, at least, a variety of protists. Therefore, chloroviruses both depend on and influence the spatial structures of communities as well as the flows of energy through those communities, driven by predator-prey interactions. The emergence of these interactions are an eco-evolutionary puzzle, given the interdependence of these species and the many costs and benefits that these interactions generate.

The Astounding World of Glycans from Giant Viruses.

Viruses are a heterogeneous ensemble of entities, all sharing the need for a suitable host to replicate. They are extremely diverse, varying in morphology, size, nature, and complexity of their genomic content. Typically, viruses use host-encoded glycosyltransferases and glycosidases to add and remove sugar residues from their glycoproteins. Thus, the structure of the glycans on the viral proteins have, to date, typically been considered to mimick those of the host. However, the more recently discovered large and giant viruses differ from this paradigm. At least some of these viruses code for an (almost) autonomous glycosylation pathway. These viral genes include those that encode the production of activated sugars, glycosyltransferases, and other enzymes able to manipulate sugars at various levels. This review focuses on large and giant viruses that produce carbohydrate-processing enzymes. A brief description of those harboring these features at the genomic level will be discussed, followed by the achievements reached with regard to the elucidation of the glycan structures, the activity of the proteins able to manipulate sugars, and the organic synthesis of some of these virus-encoded glycans. During this progression, we will also comment on many of the challenging questions on this subject that remain to be addressed.

Functional Genomic Analyses Reveal an Open Pan-genome for the Chloroviruses and a Potential for Genetic Innovation in New Isolates.

Chloroviruses (family Phycodnaviridae) are large double-stranded DNA (dsDNA) viruses that infect unicellular green algae present in inland waters. These viruses have been isolated using three main chlorella-like green algal host cells, traditionally called NC64A, SAG, and Pbi, revealing extensive genetic diversity. In this study, we performed a functional genomic analysis on 36 chloroviruses that infected the three different hosts. Phylogenetic reconstruction based on the DNA polymerase B family gene clustered the chloroviruses into three distinct clades. The viral pan-genome consists of 1,345 clusters of orthologous groups of genes (COGs), with 126 COGs conserved in all viruses. Totals of 368, 268, and 265 COGs are found exclusively in viruses that infect NC64A, SAG, and Pbi algal hosts, respectively. Two-thirds of the COGs have no known function, constituting the "dark pan-genome" of chloroviruses, and further studies focusing on these genes may identify important novelties. The proportions of functionally characterized COGs composing the pan-genome and the core-genome are similar, but those related to transcription and RNA processing, protein metabolism, and virion morphogenesis are at least 4-fold more represented in the core genome. Bipartite network construction evidencing the COG sharing among host-specific viruses identified 270 COGs shared by at least one virus from each of the different host groups. Finally, our results reveal an open pan-genome for chloroviruses and a well-established core genome, indicating that the isolation of new chloroviruses can be a valuable source of genetic discovery. IMPORTANCE Chloroviruses are large dsDNA viruses that infect unicellular green algae distributed worldwide in freshwater environments. They comprise a genetically diverse group of viruses; however, a comprehensive investigation of the genomic evolution of these viruses is still missing. Here, we performed a functional pan-genome analysis comprising 36 chloroviruses associated with three different algal hosts in the family Chlorellaceae, referred to as zoochlorellae because of their endosymbiotic lifestyle. We identified a set of 126 highly conserved genes, most of which are related to essential functions in the viral replicative cycle. Several genes are unique to distinct isolates, resulting in an open pan-genome for chloroviruses. This profile is associated with generalist organisms, and new insights into the evolution and ecology of chloroviruses are presented. Ultimately, our results highlight the potential for genetic diversity in new isolates.

Efficient assays to quantify the life history traits of algal viruses.

IMPORTANCE: Viruses play a crucial role in microbial ecosystems by liberating nutrients and regulating the growth of their hosts. These effects are governed by viral life history traits, i.e., by the traits determining viral reproduction and survival. Understanding these traits is essential to predicting viral effects, but measuring them is generally labor intensive. In this study, we present efficient methods to quantify the full life cycle of lytic viruses. We developed these methods for viruses infecting unicellular Chlorella algae but expect them to be applicable to other lytic viruses that can be quantified by flow cytometry. By making viral phenotypes accessible, our methods will support research into the diversity and ecological effects of microbial viruses.

Viral Chemotaxis of Paramecium Bursaria Altered by Algal Endosymbionts.

Chemotaxis is widespread across many taxa and often aids resource acquisition or predator avoidance. Species interactions can modify the degree of movement facilitated by chemotaxis. In this study, we investigated the influence of symbionts on Paramecium bursaria's chemotactic behavior toward chloroviruses. To achieve this, we performed choice experiments using chlorovirus and control candidate attractors (virus stabilization buffer and pond water). We quantified the movement of Paramecia grown with or without algal and viral symbionts toward each attractor. All Paramecia showed some chemotaxis toward viruses, but cells without algae and viruses showed the most movement toward viruses. Thus, the endosymbiotic algae (zoochlorellae) appeared to alter the movement of Paramecia toward chloroviruses, but it was not clear that ectosymbiotic viruses (chlorovirus) also had this effect. The change in behavior was consistent with a change in swimming speed, but a change in attraction remains possible. The potential costs and benefits of chemotactic movement toward chloroviruses for either the Paramecia hosts or its symbionts remain unclear.

Chlorovirus PBCV-1 protein A064R has three of the transferase activities necessary to synthesize its capsid protein N-linked glycans.

Paramecium bursaria chlorella virus-1 (PBCV-1) is a large double-stranded DNA (dsDNA) virus that infects the unicellular green alga Chlorella variabilis NC64A. Unlike many other viruses, PBCV-1 encodes most, if not all, of the enzymes involved in the synthesis of the glycans attached to its major capsid protein. Importantly, these glycans differ from those reported from the three domains of life in terms of structure and asparagine location in the sequon of the protein. Previous data collected from 20 PBCV-1 spontaneous mutants (or antigenic variants) suggested that the a064r gene encodes a glycosyltransferase (GT) with three domains, each with a different function. Here, we demonstrate that: domain 1 is a ß-l-rhamnosyltransferase; domain 2 is an α-l-rhamnosyltransferase resembling only bacterial proteins of unknown function, and domain 3 is a methyltransferase that methylates the C-2 hydroxyl group of the terminal α-l-rhamnose (Rha) unit. We also establish that methylation of the C-3 hydroxyl group of the terminal α-l-Rha is achieved by another virus-encoded protein A061L, which requires an O-2 methylated substrate. This study, thus, identifies two of the glycosyltransferase activities involved in the synthesis of the N-glycan of the viral major capsid protein in PBCV-1 and establishes that a single protein A064R possesses the three activities needed to synthetize the 2-OMe-α-l-Rha-(1→2)-ß-l-Rha fragment. Remarkably, this fragment can be attached to any xylose unit.

N-glycans from Paramecium bursaria chlorella virus MA-1D: Re-evaluation of the oligosaccharide common core structure.

Paramecium bursaria chlorella virus MA-1D is a chlorovirus that infects Chlorella variabilis strain NC64A, a symbiont of the protozoan Paramecium bursaria. MA-1D has a 339-kb genome encoding ca. 366 proteins and 11 tRNAs. Like other chloroviruses, its major capsid protein (MCP) is decorated with N-glycans, whose structures have been solved in this work by using nuclear magnetic spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry along with MS/MS experiments. This analysis identified three N-linked oligosaccharides that differ in the nonstoichiometric presence of three monosaccharides, with the largest oligosaccharide composed of eight residues organized in a highly branched fashion. The N-glycans described here share several features with those of the other chloroviruses except that they lack a distal xylose unit that was believed to be part of a conserved core region for all the chloroviruses. Examination of the MA-1D genome detected a gene with strong homology to the putative xylosyltransferase in the reference chlorovirus PBCV-1 and in virus NY-2A, albeit mutated with a premature stop codon. This discovery means that we need to reconsider the essential features of the common core glycan region in the chloroviruses.

Sterol Biosynthesis in Four Green Algae: A Bioinformatic Analysis of the Ergosterol Versus Phytosterol Decision Point.

Animals and fungi produce cholesterol and ergosterol, respectively, while plants produce the phytosterols stigmasterol, campesterol, and ß-sitosterol in various combinations. The recent sequencing of many algal genomes allows the detailed reconstruction of the sterol metabolic pathways. Here, we characterized sterol synthesis in two sequenced Chlorella spp., the free-living C. sorokiniana, and symbiotic C. variabilis NC64A. Chlamydomonas reinhardtii was included as an internal control and Coccomyxa subellipsoidea as a plant-like outlier. We found that ergosterol was the major sterol produced by Chlorella spp. and C. reinhardtii, while C. subellipsoidea produced the three phytosterols found in plants. In silico analysis of the C. variabilis NC64A, C. sorokiniana, and C. subellipsoidea genomes identified 22 homologs of sterol biosynthetic genes from Arabidopsis thaliana, Saccharomyces cerevisiae, and C. reinhardtii. The presence of CAS1, CPI1, and HYD1 in the four algal genomes suggests the higher plant cycloartenol branch for sterol biosynthesis, confirming that algae and fungi use different pathways for ergosterol synthesis. Phylogenetic analysis for 40 oxidosqualene cyclases (OSCs) showed that the nine algal OSCs clustered with the cycloartenol cyclases, rather than the lanosterol cyclases, with the OSC for C. subellipsoidea positioned in between the higher plants and the eight other algae. With regard to why C. subellipsoidea produced phytosterols instead of ergosterol, we identified 22 differentially conserved positions where C. subellipsoidea CAS and A. thaliana CAS1 have one amino acid while the three ergosterol producing algae have another. Together, these results emphasize the position of the unicellular algae as an evolutionary transition point for sterols.

Structure of the chlorovirus PBCV-1 major capsid glycoprotein determined by combining crystallographic and carbohydrate molecular modeling approaches.

The glycans of the major capsid protein (Vp54) of Paramecium bursaria chlorella virus (PBCV-1) were recently described and found to be unusual. This prompted a reexamination of the previously reported Vp54 X-ray structure. A detailed description of the complete glycoprotein was achieved by combining crystallographic data with molecular modeling. The crystallographic data identified most of the monosaccharides located close to the protein backbone, but failed to detect those further from the glycosylation sites. Molecular modeling complemented this model by adding the missing monosaccharides and examined the conformational preference of the whole molecule, alone or within the crystallographic environment. Thus, combining X-ray crystallography with carbohydrate molecular modeling resulted in determining the complete glycosylated structure of a glycoprotein. In this case, it is the chlorovirus PBCV-1 major capsid protein.

The N-glycan structures of the antigenic variants of chlorovirus PBCV-1 major capsid protein help to identify the virus-encoded glycosyltransferases.

The chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) is a large dsDNA virus that infects the microalga Chlorella variabilis NC64A. Unlike most other viruses, PBCV-1 encodes most, if not all, of the machinery required to glycosylate its major capsid protein (MCP). The structures of the four N-linked glycans from the PBCV-1 MCP consist of nonasaccharides, and similar glycans are not found elsewhere in the three domains of life. Here, we identified the roles of three virus-encoded glycosyltransferases (GTs) that have four distinct GT activities in glycan synthesis. Two of the three GTs were previously annotated as GTs, but the third GT was identified in this study. We determined the GT functions by comparing the WT glycan structures from PBCV-1 with those from a set of PBCV-1 spontaneous GT gene mutants resulting in antigenic variants having truncated glycan structures. According to our working model, the virus gene a064r encodes a GT with three domains: domain 1 has a ß-l-rhamnosyltransferase activity, domain 2 has an α-l-rhamnosyltransferase activity, and domain 3 is a methyltransferase that decorates two positions in the terminal α-l-rhamnose (Rha) unit. The a075l gene encodes a ß-xylosyltransferase that attaches the distal d-xylose (Xyl) unit to the l-fucose (Fuc) that is part of the conserved N-glycan core region. Last, gene a071r encodes a GT that is involved in the attachment of a semiconserved element, α-d-Rha, to the same l-Fuc in the core region. Our results uncover GT activities that assemble four of the nine residues of the PBCV-1 MCP N-glycans.

Chloroviruses Lure Hosts through Long-Distance Chemical Signaling.

Chloroviruses exist in aquatic systems around the planet and they infect certain eukaryotic green algae that are mutualistic endosymbionts in a variety of protists and metazoans. Natural chlorovirus populations are seasonally dynamic, but the precise temporal changes in these populations and the mechanisms that underlie them have heretofore been unclear. We recently reported the novel concept that predator/prey-mediated virus activation regulates chlorovirus population dynamics, and in the current study, we demonstrate virus-packaged chemotactic modulation of prey behavior.IMPORTANCE Viruses have not previously been reported to act as chemotactic/chemoattractive agents. Rather, viruses as extracellular entities are generally viewed as non-metabolically active spore-like agents that await further infection events upon collision with appropriate host cells. That a virus might actively contribute to its fate via chemotaxis and change the behavior of an organism independent of infection is unprecedented.

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.

Predators catalyze an increase in chloroviruses by foraging on the symbiotic hosts of zoochlorellae.

Virus population growth depends on contacts between viruses and their hosts. It is often unclear how sufficient contacts are made between viruses and their specific hosts to generate spikes in viral abundance. Here, we show that copepods, acting as predators, can bring aquatic viruses and their algal hosts into contact. Specifically, predation of the protist Paramecium bursaria by copepods resulted in a >100-fold increase in the number of chloroviruses in 1 d. Copepod predation can be seen as an ecological "catalyst" by increasing contacts between chloroviruses and their hosts, zoochlorellae (endosymbiotic algae that live within paramecia), thereby facilitating viral population growth. When feeding, copepods passed P. bursaria through their digestive tract only partially digested, releasing endosymbiotic algae that still supported viral reproduction and resulting in a virus population spike. A simple predator-prey model parameterized for copepods consuming protists generates cycle periods for viruses consistent with those observed in natural ponds. Food webs are replete with similar symbiotic organisms, and we suspect the predator catalyst mechanism is capable of generating blooms for other endosymbiont-targeting viruses.

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.

Biophysical Approaches to Solve the Structures of the Complex Glycan Shield of Chloroviruses.

The capsid of Paramecium bursaria chlorella virus (PBCV-1) contains a heavily glycosylated major capsid protein, Vp54. The capsid protein contains four glycans, each N-linked to Asn. The glycan structures are unusual in many aspects: (1) they are attached by a ß-glucose linkage, which is rare in nature; (2) they are highly branched and consist of 8-10 neutral monosaccharides; (3) all four glycoforms contain a dimethylated rhamnose as the capping residue of the main chain, a hyper-branched fucose residue and two rhamnose residues ''with opposite absolute configurations; (4) the four glycoforms differ by the nonstoichiometric presence of two monosaccharides, L-arabinose and D-mannose ; (5) the N-glycans from all of the chloroviruses have a strictly conserved core structure; and (6) these glycans do not resemble any structures previously reported in the three domains of life.The structures of these N-glycoforms remained elusive for years because initial attempts to solve their structures used tools developed for eukaryotic-like systems, which we now know are not suitable for this noncanonical glycosylation pattern. This chapter summarizes the methods used to solve the chlorovirus complex glycan structures with the hope that these methodologies can be used by scientists facing similar problems.

Identification of Intrahelical Bifurcated H-Bonds as a New Type of Gate in K+ Channels.

Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K+ channels, KcvS and KcvNTS. These small viral encoded K+ channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K+ channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in KcvNTS and 40% in KcvS. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in KcvS. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels.

Virus-host interactions: insights from the replication cycle of the large Paramecium bursaria chlorella virus.

The increasing interest in cytoplasmic factories generated by eukaryotic-infecting viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA virus Paramecium bursaria chlorella virus 1 (PBCV-1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV-1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and Mimivirus. Specifically, PBCV-1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membrane-containing sites. In sharp contrast, factories generated by Mimivirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV-1 infection, which was recently reported to result in significant pathological outcomes in humans and mice, proceeds through a bacteriophage-like infection pathway.

Structure of the N-glycans from the chlorovirus NE-JV-1.

Results from recent studies are breaking the paradigm that all viruses depend on their host machinery to glycosylate their proteins. Chloroviruses encode several genes involved in glycan biosynthesis and some of their capsid proteins are decorated with N-linked oligosaccharides with unique features. Here we describe the elucidation of the N-glycan structure of an unusual chlorovirus, NE-JV-1, that belongs to the Pbi group. The host for NE-JV-1 is the zoochlorella Micractinium conductrix. Spectroscopic analyses established that this N-glycan consists of a core region that is conserved in all of the chloroviruses. The one difference is that the residue 3OMe-L-rhamnose is acetylated at the O-2 position in a non-stoichiometric fashion.