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Review

Chloroviruses Have a Sweet Tooth

by
James L. Van Etten
1,*,
Irina Agarkova
1,
David D. Dunigan
1,
Michela Tonetti
2,
Christina De Castro
3 and
Garry A. Duncan
4
1
Department of Plant Pathology and Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583-0900, USA
2
Department of Experimental Medicine and Center of Excellence for Biomedical Research, University of Genova Viale Benedetto XV/1, 16132 Genova, Italy
3
Department of Agricultural Sciences, University of Napoli, Via Università 100, 80055 Portici, NA, Italy
4
Department of Biology, Nebraska Wesleyan University, Lincoln, NE 68504-2796, USA
*
Author to whom correspondence should be addressed.
Viruses 2017, 9(4), 88; https://doi.org/10.3390/v9040088
Submission received: 17 March 2017 / Revised: 13 April 2017 / Accepted: 14 April 2017 / Published: 22 April 2017
(This article belongs to the Special Issue Viruses of Microbes)
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Abstract

:
Chloroviruses are large double-stranded DNA (dsDNA) viruses that infect certain isolates of chlorella-like green algae. They contain up to approximately 400 protein-encoding genes and 16 transfer RNA (tRNA) genes. This review summarizes the unexpected finding that many of the chlorovirus genes encode proteins involved in manipulating carbohydrates. These include enzymes involved in making extracellular polysaccharides, such as hyaluronan and chitin, enzymes that make nucleotide sugars, such as GDP-l-fucose and GDP-d-rhamnose and enzymes involved in the synthesis of glycans attached to the virus major capsid proteins. This latter process differs from that of all other glycoprotein containing viruses that traditionally use the host endoplasmic reticulum and Golgi machinery to synthesize and transfer the glycans.

1. Introduction

In discussing enzymes involved in manipulating carbohydrates, one usually does not consider viruses to play a role in this important subject. However, as described in this review, chloroviruses (family Phycodnaviridae) that infect certain isolates of single-celled, eukaryotic chlorella-like green algae are an exception to this process because they encode enzymes involved in making extracellular polysaccharides, nucleotide sugars and the synthesis of glycans attached to their major capsid glycoproteins.
The plaque-forming chloroviruses are large icosahedral (190 nm in diameter), double-stranded DNA (dsDNA)-containing viruses (genomes of 290 to 370 kb) with an internal lipid membrane. They exist in inland waters throughout the world with titers occasionally reaching thousands of plaque-forming units (PFU) per mL of indigenous water. Known chlorovirus hosts, which are normally endosymbionts and are often referred to as zoochlorellae [1,2], are associated either with the protozoan Paramecium bursaria, the coelenterate Hydra viridis or the heliozoan Acanthocystis turfacea [3,4,5,6]. Four such zoochlorellae and their viruses are Chlorella variabilis NC64A and its viruses (referred to as NC64A viruses), Chlorella variabilis Syngen 2–3 and its viruses (referred to as Osy viruses), Chlorella heliozoae SAG 3.83 and its viruses (referred to as SAG viruses) and Micractinium conductrix Pbi and its viruses (referred to as Pbi viruses). The zoochlorellae are resistant to virus infection when they are in their symbiotic relationship, because the viruses have no way of reaching their hosts.
The genomes of 43 chloroviruses infecting these four hosts have been sequenced, assembled and annotated [6,7,8,9,10,11]. Collectively, the viruses encode genes from 643 predicted protein families; however, any given chlorovirus only has 330 to 416 protein-encoding genes (PEGs). Thus, the genetic diversity among these viruses is large, and many of the proteins are unexpected for a virus. With the exception of homologs solely in other chlorovirus members, about 50% of their PEGs do not match anything in the databases.
The prototype chlorovirus Paramecium bursaria chlorella virus type 1 (PBCV-1) is an NC64A virus [12]. PBCV-1 is an icosahedron (190 nm in diameter) with a spike-like structure at one vertex and a few external fibers that extend from some of the viral capsomeres [5,13]. The outer capsid layer covers a single lipid bilayered membrane, which is essential for infection. The PBCV-1 major capsid protein (named Vp54) is a glycoprotein, and three Vp54s form a trimeric capsomere, which has pseudo-six-fold symmetry. A proteomic analysis of PBCV-1 virions revealed that the virus contains 148 virus-encoded proteins and at least one host-encoded protein [10]. The PBCV-1 genome is a linear ~331-kb, non-permuted dsDNA molecule with covalently-closed hairpin termini. Identical ~2.2-kb inverted repeats flank each 35-nucleotide-long, incompletely base-paired, covalently closed hairpin loop [14,15]. The remainder of the PBCV-1 genome contains primarily single-copy DNA that encodes ~416 putative proteins and 11 transfer RNAs (tRNAs) [5]. The G + C content of the PBCV-1 genome is 40%; in contrast, its host nuclear genome is 67% G + C. PBCV-1 and other chlorovirus genomes contain methylated bases, which occur in specific DNA sequences. The methylated bases are part of chlorovirus-encoded DNA restriction and modification systems [16].
As the title of this review indicates, many of the chlorovirus genes encode enzymes involved in various aspects of carbohydrate metabolism. We have listed putative chlorovirus genes involved in carbohydrate metabolism, which are encoded by the 43 chloroviruses whose genomes have been sequenced, in Table 1, Table 2 and Table 4. Recombinant proteins have been produced from some of these genes, and the proteins have been characterized (indicated in bold in the tables). When some of the genes were initially cloned and the recombinant proteins characterized, the genes were hybridized to many other chlorovirus genomes by dot blots to determine the distribution of the genes. Because of the large number of viruses, these experimental results are not included in the tables, unless the virus genome was subsequently sequenced.

2. Chlorovirus Encoded Polysaccharide Synthesizing Enzymes

Three PBCV-1 encoded enzymes are involved in the synthesis of the extracellular matrix polysaccharide hyaluronan (also referred to as hyaluronic acid), including glycosyltransferase Class I hyaluronan synthase (HAS; Table 1) [17,18]. Until the has gene (a098r) was discovered in PBCV-1, hyaluronan was only thought to occur in vertebrates and a few pathogenic bacteria, where it forms an extracellular capsule, presumably to avoid the immune system [19,20]. Hyaluronan is an essential constituent of the extracellular matrix in vertebrates and consists of ~10,000 or more alternating β-1,4-glucuronic acid (GlcA) and β-1,3-N-acetylglucosamine (GlcNAc) residues. Typically, the HAS enzyme is located on the inner surface of the plasma membrane. The newly-synthesized hyaluronan then moves through the membrane and cell wall to the extracellular matrix.
PBCV-1 also encodes two enzymes involved in the biosynthesis of hyaluronan precursors, glutamine:fructose-6-phosphate amidotransferase (GFAT, gene a100r) and UDP-glucose dehydrogenase (UDP-GlcDH, gene a609l; Table 2) [21]. All three PBCV-1 genes involved in hyaluronan synthesis are expressed early during virus infection, and all three transcripts decrease significantly by 60 min post-infection (PI) [18,21]. However, these three genes do not function like an operon, although two of the genes, a98r and a100r, are adjacent to one another and are co-linear in the PBCV-1 genome. In contrast, a609l is located ~240 kb away and is transcribed in the opposite orientation [17]. The identification of these three genes led to the discovery that hyaluronan lyase-sensitive hair-like fibers begin to accumulate on the surface of PBCV-1-infected host cells by 15 min PI. By 4 h PI, the infected cells are covered with a dense fibrous hyaluronan network (Figure 1) [18].
Three additional enzymes are needed to convert glucosamine-6-phosphate (GlcN-6P) to UDP-N-acetylglucosamine (UDP-GlcNAc), and these enzymes (EC2.3.1.4, EC5.4.2.3, EC2.7.7.23) are encoded by the host [22]. This is not surprising because the host NC64A cell wall is predicted to contain chitin, which is a polymer of GlcNAc residues, and so, the alga must encode these enzymes.
The has gene that encodes hyaluronan synthase is present in 12 of the 43 chloroviruses isolated from diverse geographical regions, including 5 NC64A viruses, 6 Pbi viruses and 1 Osy virus (Table 1). In contrast, the udp-glcdh gene is present in 40 of the 43 viruses, 14 of which have two copies of the gene, while the gfat gene is present in 27 chloroviruses, including 11 of the 14 NC64A viruses, all 14 Pbi viruses, one of the 13 SAG viruses and the only Osy virus that has been sequenced (Table 2). Both of these latter two genes are present in all of the 12 viruses that have a has gene, except for the one Osy virus that lacks a udp-glcdh gene.
Surprisingly, 19 of the 31 chloroviruses that lack a has gene have a gene encoding a chitin synthase (CHS). Chitin, an insoluble linear homopolymer of β-1,4-linked-GlcNAc residues, is a common component of insect exoskeletons, shells of crustaceans and fungal cell walls [23]. Chitin is rare in algal cell walls, although it has been reported to exist in some green algae [24]. Like the has gene, the chs gene is expressed as early as 10 min PI and peaks at 20–40 min PI, and the transcript disappears at 120–180 min PI. Furthermore, cells infected with chs-containing viruses produced chitin fibers on the external surface of their hosts [25]. As discussed below, many of the chloroviruses also encode chitinases and chitosanases.
At least one chlorovirus, CVK2, has replaced the PBCV-1 has gene with a 5-kb region containing chs, udp-gdh2 (a gene encoding a second UDP-GlcDH) and two other ORFs [26]. Therefore, at least some chloroviruses have changed from HAS viruses to CHS viruses or vice versa, by swapping genes.
Two NC64A chloroviruses have both has and chs genes, and at least one of them forms both hyaluronan and chitin on the surface of their infected cells [25,27]. Finally, 12 chloroviruses lack both genes, and no extracellular polysaccharides are formed on the surface of cells infected with at least one of these viruses [18]. The fact that many chloroviruses encode enzymes involved in extracellular polysaccharide biosynthesis suggests that the polysaccharides, which require a large expenditure of ATP for their synthesis, are important in the virus life cycles. However, the extracellular hyaluronan does not play an obvious role in the interaction between PBCV-1 and its algal host because neither plaque size nor plaque numbers were altered by including either hyaluronidase or free hyaluronan in the top agar of the PBCV-1 plaque assay [17].
The three genes involved in synthesizing hyaluronan have probably been obtained rather recently in evolutionary terms because the coding portions of the PBCV-1 gfat and udp-glcnc genes are 44% G + C, while the has gene is 46.7% G + C. In contrast, PBCV-1, as well as all the NC64A viruses, have a 40% G + C content [11,21].
Currently, it is not known how or why the chloroviruses acquired these polysaccharide-synthesizing genes. We have considered the following possible evolutionary advantages for acquiring these genes: (1) the polysaccharides prevent infection by a second chlorovirus; (2) they cause the infected cells to clump with uninfected host cells, thus increasing the probability that progeny viruses can infect healthy host cells; (3) they prevent paramecia from taking up infected algal cells, (4) the chloroviruses have another host in nature, and this other host is attracted to or binds to hyaluronan or chitin on virus-infected algae, which would facilitate progeny-virus infections; or (5) polysaccharides increase the functional diameter of the infected cell, which might facilitate consumption by a predator. This could aid virus movement in the water column. In regards to the first possibility, it is known that attachment of other viruses to PBCV-1-infected cells at 4 h PI is inhibited when the external surface of the host is covered with hyaluronan fibers [18]. However, this is unlikely to be the explanation for the presence of hyaluronan because normally the host, C. variabilis NC64A, is only infected by one virus, and this restriction occurs in the first few min PI [28,29]. In regards to the second possibility, host cells often clump shortly after infection, and this phenomenon, which does not always occur, could be due to hyaluronan production. The last three possibilities have not been explored experimentally.
We have experimentally tried to address the question: does the presence of hyaluronan and/or chitin on the exterior surface of the host cell wall confer an evolutionary advantage to a virus that has one or both of these genes? To answer the question, chlorella cells were co-infected with combinations of chloroviruses that: (1) have both genes; (2) only have the has gene; (3) only have the chs gene; and (4) lack both genes. The resulting lysates were then added to fresh cells and allowed to replicate and lyse. After five passages, progeny viruses were plaqued, and 20 plaques were randomly picked to determine if one virus type dominated. However, after repeating these experiments several times, no consistent pattern was obtained [30].
To ideally conduct this experiment, one would like to either add the chs gene to the PBCV-1 genome so that both genes are present, replace the has gene with the chs gene or remove the has gene so that PBCV-1 lacked both genes. Unfortunately, this experimental protocol is currently not possible because procedures are not available for reverse genetic manipulation of chlorovirus genomes. Therefore, in the experiments described above, viruses were selected that had the desired properties and also had similar growth kinetics as PBCV-1.
In addition to not knowing why the chloroviruses acquired the has and chs genes, another question is: how are the newly-forming hyaluronan and/or chitin fibers moved through the membrane and the complex cell wall to the exterior of the algal host from the plasma membrane? This phenomenon would appear to be equivalent to pushing a thread through a furnace filter. One would expect the polysaccharide fibers to bunch up underneath the cell wall. In fact, this happened when the viral has gene was expressed in cultured tobacco cells [31]. Could a pilot protein(s) that is attached to the leading end of the polymer guide the hyaluronan chain through the wall?

3. Chlorovirus Encoded Nucleotide Sugar Metabolism Enzymes

Many chloroviruses also encode enzymes involved in nucleotide sugar metabolism, as well as other sugar metabolic enzymes (Table 2). Two enzymes encoded by all of the NC64A, SAG and Syn chloroviruses, GDP-D-mannose 4,6 dehydratase (GMD) and GDP-4-keto-6-deoxy-d-mannose epimerase reductase (GMER) (Table 2), comprise a highly-conserved pathway in bacteria, plants and animals that converts GDP-d-mannose to GDP-l-fucose (Figure 2) [32]. Fucose is found in glycoconjugates of many organisms, where it often plays a fundamental role in cell-cell adhesion and recognition [33]. The Pbi chloroviruses lack both gmd and gmer genes (Table 2) even though the glycans attached to the major capsid protein from the three evaluated Pbi viruses have fucose [34,35].
In vitro reconstruction of the pathway using recombinant PBCV-1 GMD and GMER proteins resulted in the synthesis of GDP-l-fucose as expected. Unexpectedly, however, the PBCV-1 GMD also catalyzed the NADPH-dependent reduction of the intermediate GDP-4-keto-6-deoxy-d-mannose, to form GDP-d-rhamnose. That is, the enzyme has two activities, and both sugars are produced in the infected cell [32]. The PBCV-1 recombinant GMD has another property that is unusual. Unlike recombinant GMDs from many other organisms, the viral encoded enzyme is very stable when stored at either 4 °C or −20 °C [32]. The PBCV-1 GMD enzyme was crystalized, and the structure resembles other GMDs [36].
A recombinant GMD protein encoded by another chlorovirus, Acanthocystis turfacea chlorella virus 1 (ATCV-1), which has 53% amino acid identity with the PBCV-1 GMD, was also characterized because the amino acid differences between the two enzymes suggested they might have slightly different properties. In fact, the ATCV-1 GMD does not form GDP-d-rhamnose, and so, it lacks the second enzyme activity [37]. Both GMD enzymes bound NADPH tightly, and this association was essential for the stabilization and function of both enzymes, even though NADP+ is the co-enzyme required to initiate the GMD catalytic cycle. Phylogenetic analyses established that the PBCV-1 GMD is the most evolutionarily diverged of all the GMDs, whereas the ATCV-1 GMD was in a clade of bacterial GMDs [37].
The GMER enzymes from PBCV-1 and ATCV-1 have 63% amino acid identity to each other and phylogenetically are more similar to one another and to other GMERs than are the two GMDs. The possible evolutionary consequences of these differences have been discussed previously [37]. Both fucose and rhamnose are constituents of the glycans attached to the PBCV-1 and ATCV-1 major capsid proteins (see below). However, the PBCV-1 glycan contains three rhamnose residues, with one in the D-configuration, whereas only one with L-configuration is present in the ATCV-1 glycan. Perhaps there was enough natural selection pressure on the PBCV-1 GMD gene to evolve to synthesize GDP-d-rhamnose, whereas the ATCV-1 GMD did not face this pressure.
ATCV-1 and all of the SAG viruses, however, encode another enzyme, UDP-d-glucose 4,6-dehydratase (UGD), that is one of two enzymes involved in the synthesis of L-rhamnose [38], and this enzyme may contribute to rhamnose synthesis. The PBCV-1 host chlorella, which is closely related to the ATCV-1 host chlorella, encodes the second enzyme in the rhamnose pathway [22], and so the host is predicted to be able to synthesize the rhamnose required for ATCV-1 glycan synthesis.

4. Unusual Attachment of Glycans to the Chlorovirus Major Capsid Proteins

Structural proteins of many viruses, such as rhabdoviruses, herpesviruses, poxviruses and paramyxoviruses, are glycosylated. Glycans contribute to the protease resistance and the antigenicity of these viruses. Most virus glycans are linked to Asn in the protein via N-acetylglucosamine, although some viruses also have O-linked glycans attached to either Ser or Thr residues via an amino sugar, usually N-acetylglucosamine or N-acetylgalactosamine. Typically, viruses use host-encoded glycosyltransferases and glycosidases located in the endoplasmic reticulum (ER) and Golgi apparatus to add and remove N-linked sugar residues from virus glycoproteins either co-translationally or shortly after translation of the protein. This post-translational processing aids in protein folding, progression in the secretory pathway and in the regulation of host-virus interactions [39,40,41,42]. After folding the protein, virus glycoproteins are transported by host-sorting and membrane-transport functions to virus-specified regions in host membranes where they displace host glycoproteins. Progeny viruses then bud through these virus-specific target membranes, which is usually the final step in the assembly of infectious viruses. Thus, nascent viruses only become infectious after budding through the membrane, usually the plasma membrane, as they exit the cell. Consequently, the glycan portion of virus glycoproteins is host-specific. The theme that emerges from these viruses is that virus glycoproteins are synthesized and glycosylated by the same processes as host glycoproteins. Therefore, the only way to alter virus protein glycosylation is to either grow the virus in a different host or have a mutation that alters the virus protein glycosylation site.
Unlike the process described above, glycosylation of the chlorovirus major capsid proteins differs from that scenario because the viruses encode most, if not all, of the machinery for the process. In addition, the process occurs in the cytoplasm. The conclusion that the chlorovirus PBCV-1 major capsid protein (Vp54, gene a430l) is glycosylated by a different mechanism than that used by other characterized viruses originally arose from antibody studies [43]. Rabbit polyclonal antiserum prepared against intact PBCV-1 particles inhibited virus plaque formation by agglutinating the virions. However, spontaneously-derived, antiserum-resistant, plaque-forming variants of PBCV-1 occurred at a frequency of 10−5–10−6. At the time of the 1993 publication, these antiserum-resistant variants fell into four serologically-distinct classes; two additional antigenic variants have subsequently been isolated for a total of six variants (Table 3). Polyclonal antisera prepared against members of each of these antigenic classes react predominately with the Vp54 equivalents from the viruses in the class used for the immunization. Each of the Vp54 proteins from the antigenic variants migrated faster on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) than those of the strains from which they were derived, indicating a lower molecular weight. However, all of the de-glycosylated Vp54 proteins migrated at the same rate on SDS-PAGE, indicating that the differences resided in the size of the attached glycans. In addition, the nucleotide sequence of the a430l gene in each of the variants was identical to the wild-type gene, which verified that the polypeptide portion of Vp54 was not altered in the mutants. Western blot analyses of Vp54 proteins isolated from the variants, before and after removing the glycans with trifluoromethane-sulfonic acid or altering the glycan with periodic acid, also supported the notion that the antigenic variants reflected differences in the Vp54 glycans, not the Vp54 polypeptide [43].
All of the glycan antigenic variants form plaques on their C. variabilis NC64A host, so one can infer that the glycans are not directly involved in virus infection and virus replication. However, anecdotal evidence suggests that the glycans are important in virus stability because the variants with the smallest glycans do not remain infectious in storage as long as wild-type virus.
Additional observations supported the concept that PBCV-1 Vp54 glycosylation was unusual: (1) unlike viruses that acquire their glycoproteins(s) by budding through a plasma membrane, which results in infectious particles, plaque-forming PBCV-1 particles accumulate inside the host 30–40 min before virus release [47]; (2) all of the antigenic variants were grown in the same host so the glycan differences are not due to the host; (3) polyclonal antibodies to Vp54, the major capsid protein, do not react with host glycoproteins; (4) the Vp54 protein lacks an ER and Golgi signal peptide; (5) unlike most glycoproteins that exhibit size micro-heterogeneity, PBCV-1 Vp54 appears homogeneous on SDS-PAGE; in addition, mass spectrometry analysis only revealed one satellite peak that differed from the main peak by 140 Da, the approximate weight of either one arabinose or xylose residue [46]; and (6) the ability to easily crystallize Vp54 as a homotrimer provided additional evidence that the protein is essentially homogeneous [48,49].
Evidence that the N-linked Vp54 glycans are not attached to the Vp54 protein by a traditional N-linkage was initially obtained from the X-ray crystal structure of the protein. The structure revealed that the protein had four N-linked glycans at Asn positions 280, 302, 399 and 406 [48]. None of these Asn were located in an Asn-X-(Thr/Ser) sequon sequence commonly recognized by ER located glycosyltransferases [50,51,52]. This finding also explained why prior attempts to remove Vp54 glycans with enzymes that cleave traditional N-linked glycans were unsuccessful [53] Nandhagopal et al. [48] also reported that Vp54 contained two O-linked glycans. However, re-examination of the X-ray crystal data (Figure 3) indicates that no O-linked glycans are present in the protein [49], which agrees with our unsuccessful attempts to detect them by chemical procedures.

5. Glycan Structures Attached to Chlorovirus Major Capsid Proteins

The structures of the PBCV-1 Vp54 N-linked glycans were reported recently, and they consist of 8–10 neutral monosaccharide residues, producing a total of four glycoforms (Figure 4) [54]. These structures do not resemble any structure previously reported in the three Domains of Life. Among their most distinctive features are: (1) the four glycoforms share a common core structure, and the four glycoforms are related to the non-stoichiometric presence of two monosaccharides, L-arabinose and D-mannose; the most abundant glycoform consists of nine neutral monosaccharide residues organized in a highly-branched fashion; (2) the glycans are attached to the protein by a β-glucose linkage, which is rare in nature and has only been reported in glycoproteins from a few organisms [55,56,57,58]; and (3) the glycoform contains a dimethylated rhamnose as the capping residue of the main chain, a hyper-branched fucose residue and two rhamnose residues with opposite absolute configurations.
Attempts to fit the Vp54 glycan structures into the original Vp54 X-ray crystal structure [48] were unsuccessful and led to a re-examination of the original structure. This re-examination produced a structure that was compatible with the four N-linked glycan structures (Figure 3) [49]. As mentioned above, the revised structure lacks the two O-linked glycans reported originally.
The PBCV-1 Vp54 has a molecular weight of 53,790 Da. The a430l gene encodes Vp54 with a predicted molecular weight of 48,165 Da so the combined sugars have a molecular weight of 5625 Da, which is about the weight of the four glycans. Vp54 was also reported to have a myristic acid attached to the carboxyl portion of the protein [59]. However, myristic acid has not been observed in any of the recent Vp54 structural experiments, and so, its status is currently unknown. The structures of the Vp54 glycans from the PBCV-1 antigenic variants, referred to above, are currently being determined, and as expected, the structures are truncated forms of the wild-type PBCV-1 glycans [44]. PBCV-1 particles were reported to have two additional glycoproteins in addition to Vp54 [59]. Both of these glycoproteins react with the PBCV-1 antibody, and so, the glycan structures are predicted to be similar or identical to the glycans associated with Vp54. The gene encoding one of these proteins (Vp260) was identified (gene a122r). Gene a122r homologs are common in the chloroviruses, and some of the viruses have as many as five copies of the gene [60]. The role that Vp260 plays in the PBCV-1 virion is unknown.
The glycan structures of the major capsid proteins from seven more chloroviruses, which represent all four chlorovirus types, were recently reported (Figure 5) [6,34,35]; collectively, all of the glycans have a common core region (outlined in Figure 4). The common core region consists of a pentasaccharide with a β-glucose linked to an Asn residue, which is not located in the typical sequon Asn-X-(Thr/Ser). The glucose has a terminal xylose unit and a hyperbranched fucose, which is in turn substituted with a terminal galactose and a second xylose residue. The third position of the fucose unit is always linked to a rhamnose, which is a semi-conserved element because its configuration is virus dependent. Additional decorations occur on this core N-glycan and represent a molecular signature for each chlorovirus.

6. Chlorovirus PBCV-1 Encoded Glycosyltransferases

In addition to the two glycosyltransferases, hyaluronan synthase and chitin synthase previously described, the 43 chloroviruses collectively encode eight putative glycosyltransferases (Table 4). Six of these eight glycosyltransferase-encoding genes are in PBCV-1; they are scattered throughout the PBCV-1 genome. None of these six PBCV-1 encoded glycosyltransferases have an identifiable signal peptide that would target them to the ER. Furthermore, with the exception of PBCV-1 glycosyltransferases A473L (six transmembrane domains; CESA CelA-like) and A219/222/226R (nine transmembrane domains; CXCX-2), none of the four remaining PBCV-1 encoded glycosyltransferases are predicted to have transmembrane domains. Therefore, these enzymes are expected to be soluble proteins. The genes for the six PBCV-1 encoded glycosyltransferases are expressed early during PBCV-1 infection [61]. Thus, assuming the enzymes are stable, they would be available for adding sugars to the Vp54 glycans during virus replication.
The PBCV-1 a064r gene encodes a 638-amino acid protein with three predicted domains. The N-terminal 211 amino acid domain resembles a “fringe-class” of glycosyltransferases (GT-GTA) and contains the last four of the five conserved motifs characteristic of this group of glycosyltransferases [62,63], including the proposed catalytic amino acids, the Asp-X-Asp sequence in motif 3 and the first Asp residue in motif 5. However, spacing between some of the four motifs differs from that of the fringe-glycosyltransferases. As mentioned above, the A064R protein, which is only present in five NC64A viruses, lacks both an identifiable signal peptide that would target the protein to the ER and a membrane-spanning motif, in contrast to “fringe” glycosyltransferases.
The 211-amino acid A064R glycosyltransferase domain was cloned, and the recombinant protein was crystallized [64]. The 1.6 Å crystal structure of the peptide has a mixed α/β fold containing a central, six-stranded β sheet flanked by α helices. The overall fold is similar to the catalytic domains in retaining glycosyltransferases in the GT-A group, family 34, although the amino acid similarity between them is low. Zhang et al. [64] suggested that the A064R glycosyltransferase bound to UDP-glucose better than to UDP-galactose or UDP-N-acetyl glucosamine. However, these binding experiments were conducted prior to knowing the Vp54 glycan structures. Now, there is evidence that the glycosyltransferase domain adds L-rhamnose to the distal xylose residue in the core structure [45].
Analysis of the six PBCV-1 antigenic variants revealed mutations in a064r that correlated with a specific antigenic class, B (EPA-1) (Table 3). The a064r gene in all six of these antigenic variants was sequenced to determine if mutations in a064r correlated with the EPA-1 antigenic variation [46]. The a064r sequences from three of the mutants had single nucleotide substitutions, which produced a single amino acid substitution in the glycosyltransferase portion of the A064R protein. Two of the amino acid substitutions occurred in the Asp-X-Asp motif (domain 3), and the other one was in domain 4. A fourth variant had an extra base in the coding sequence, which created a frame shift mutation in the gene. Finally, the entire gene was deleted in the other two antigenic variants.
Dual infection experiments with some of the different antigenic variants established that viruses containing wild-type a064r complemented and recombined with viruses that contained variant a064r to form wild-type virus. Therefore, it was concluded that a064r encodes a glycosyltransferase involved in the synthesis of the Vp54 glycan [46].
As noted above, the protein product of the a064r gene contains three domains with domain 1 being the glycosyltransferase. Domain 2 does not match anything in GenBank, but the C-terminal domain 3 is predicted to be a methyltransferase. We suspect that this C-terminal domain of approximately 200 amino acids is involved in methylating the terminal L-rhamnose in the Vp54 glycan [45].
A homolog of PBCV-1 glycosyltransferase, A546L (GT-GT4), has also been produced and crystallized [65]. The a546l gene homolog was from another NC64A chlorovirus NY-2A (gene b736l), and the 396-amino acid protein resembles members in the GT4 family of glycosyltransferases in the CAZy classification [66,67]. However, its biochemical function remains to be elucidated.
Of the eight glycosyltransferases encoded by the 43 chloroviruses, only two of them, homologs of PBCV-1 A111/114R and A075L, are present in all of the viruses, and so, they are predicted to be involved in the synthesis of the core glycan structure. A111/114R is especially interesting because it is predicted to have at least two glycosyltransferase catalytic domains.
Now that structures of the glycans from the chlorovirus major capsid proteins are becoming available, one can begin to characterize the viral encoded glycosyltransferases biochemically. One question that needs to be addressed is: Are the sugars added sequentially to the Vp54 protein backbone or are the glycans initially synthesized independently of Vp54, possibly on a lipid carrier and then attached to the protein in a single step? A slight variation of these two possibilities is that the core glycan is synthesized independently of the protein and then attached to Vp54. Additional sugars could then be added sequentially to these core glycans [68]. We suspect that this viral encoded glycosylation pathway represents a previously undescribed pathway, possible even a pathway that existed in eukaryotes prior to the ER and Golgi glycosylation pathway [18].

7. Additional Chlorovirus Encoded Sugar Metabolism Enzymes

Besides the chlorovirus-encoded enzymes described above, the viruses have four additional genes predicted to encode enzymes involved in sugar metabolism (Table 2). Recombinant proteins have not been produced from any of these genes, and so, it is unknown if they encode functional enzymes. These putative enzymes include an acetyltransferase (AT) encoded by all 43 chloroviruses, a D-lactate dehydrogenase (D-LD) encoded by 32 chloroviruses, fumarate reductase (FRD) encoded by five chloroviruses and ADP-ribosyl glycohydrolase (ADP-RGH) encoded by nine chloroviruses, all but two of which are Pbi viruses. The roles these putative enzymes play in the viral life cycles are unknown.

8. Chlorovirus-Encoded Polysaccharide Degrading Enzymes

In addition to the polysaccharide synthesizing enzymes described above, the chloroviruses also encode polysaccharide-degrading enzymes (Table 5). The chloroviruses are unique among viruses infecting eukaryotic organisms in that they, like bacteriophages, need to penetrate a rigid algal cell wall to initiate infection. The icosahedral shaped chlorovirus PBCV-1 has a spike-like structure at one vertex [13], which appears to make the initial contact with the cell wall of its host, C. variabilis NC64A [69]. Attachment is immediately followed by host cell wall degradation at the point of contact by a virus-packaged enzyme(s) [70]. After wall degradation, the viral internal membrane fuses with the host membrane to produce a narrow (~5 nm in inner diameter), membrane-lined tunnel, which allows entry of the viral DNA and some viral proteins [71]. This membrane fusion results in immediate host membrane depolarization [72] and potassium ion efflux [73]. This process results in an empty capsid remaining on the host cell surface.
In addition to virus entry into the host cells, nascent infectious PBCV-1 viruses exit the cells at 6–8 h PI by lysis of the plasma membrane and the cell wall. Therefore, it is not surprising that the chloroviruses encode polysaccharide-degrading enzymes in order to enter and exit the host cell. In fact, PBCV-1 encodes five such enzymes (Table 5), including two chitinases [74,75], a chitosanase [74,76], a β-1,3 glucanase [77] and an alkaline alginate lyase [78] or a polysaccharide lyase, cleaving chains of β- or α-1,4-linked glucuronic acids [79,80]. Recombinant proteins have been produced from each of these genes and shown to have the expected activity. Interestingly, the β-1,3 glucanase gene is expressed very early and disappears by 60 min PI. The protein is also made very early and disappears by 90 min PI [77]. Therefore, this enzyme is unlikely to be involved in either viral entry or viral exit from the cell. One possible function for the enzyme is to degrade host β-1,3 glycans, which might serve as host storage polysaccharides. Gene transcripts from the other four polysaccharide-degrading enzymes are present throughout the viral life cycles [74,81].
Experiments conducted about 30 years ago established that a crude enzyme preparation made from PBCV-1 lysates, named lysin, had good wall degrading activity and could be used to produce C. variabilis NC64A protoplasts [82,83]. Therefore, it was assumed that one or more of the five PBCV-1 encoded enzymes would be packaged in the PBCV-1 virion and be responsible for degrading the host cell wall at the point of infection. In fact, Yamada et al. [76] reported that a chitosanase activity was packaged in a closely-related chlorovirus, CVK2. However, an ensuing report [75] indicated that the CVK2 chitosanase activity was due to incomplete purification of the virion. Subsequently, a PBCV-1 proteome study identified 148 virus-encoded proteins and one host-encoded protein in highly purified virions [10]. Surprisingly, none of the five polysaccharide-degrading enzymes were packaged in the PBCV-1 virions.
Consequently, the 148 virus-encoded proteins packaged in the PBCV-1 particles were re-examined for possible polysaccharide or cell wall degrading activity. This effort revealed that one of the PBCV-1-encoded proteins packaged in the virion, A561L, has a putative glycosyl hydrolase domain. A recombinant protein produced from this domain has cell wall degrading activity, and the protein is under active investigation [84]. Homologs of the A561L domain (named A561L lysin) are present in most of the chloroviruses (Table 5), but not all. For example, viruses NYs-1 and CVR-1 appear to lack an a561l gene homolog encoding this domain, and the similarity between the predicted A561L homolog from viruses NY-2B and WI0606 is not very high. The apparent absence of the protein from these viruses deserves to be investigated further because one would expect the enzyme(s) that degrades the host cell wall during virus infection would be highly conserved.
Twenty-four of the 43 chloroviruses encode a protein that has a polysaccharide deacetylase domain (Table 5). Viruses in three of the four types (Osy being the exception) have the gene, but it is also missing in some viruses in each of the three types, so the gene is clearly not required for the success of the viruses. Its role might be to remove the acetyl group from chitin during host cell wall degradation.
In addition to these glycolytic enzymes, 42 of the 43 chloroviruses, encode a functional glycosylase protein that initiates pyrimidine photodimer excision [85,86]. The enzyme is part of a DNA repair pathway.

9. Conservation of the Chlorovirus Encoded Sugar Enzymes

Only three of the 21 chlorovirus encoded proteins listed in Table 1, Table 2 and Table 4 are present in all 43 chloroviruses, and these would be considered to be core proteins. The three are an acetyltransferase (AT), an exostosin glycosyltransferase (EXT) and a family A glycosyltransferase (GT-A). As noted above, we predict that the two glycosyltransferases are involved in the synthesis of the glycan core attached to the major capsid protein. The predicted function of the acetyltransferase is unknown.
Two of the seven viral encoded proteins involved in polysaccharide degrading activity are conserved in all of the viruses, the chitosanase (CHIS) and a putative bifunctional chitinase/lysozyme (BCHIL) (Table 4). Presumably these two enzymes play a role in the release of the nascent viruses from the cell. As indicated above, they are not packaged in the PBCV-1 virion, and so, they are not involved in the immediate early virus infection process.
The presence or absence of some of the chlorovirus sugar encoding enzymes displays some interesting patterns. For example, the GMD and GMER encoding genes are present in all of the NC64A, SAG and Osy viruses and absent in all of the Pbi viruses. This observation would suggest that the three virus types that have these genes would be more closely related to each other than to the Pbi viruses. However, a phylogenetic tree that shows the evolutionary relationship between the 43 viruses based on 29 concatenated core proteins [6] indicates that SAG and Pbi viruses are in the same branch and that the NC64A and Osy viruses are in a separate branch. Therefore, these results would imply that the SAG and NC64A/Osy viruses either acquired the genes separately after the four virus types had separated from a common ancestor or that the chlorovirus ancestor had both genes and for some reason, they were lost in the Pbi lineage.
Most of the other protein patterns are even more difficult to explain. For example, the GFAT encoding gene is present in all of the Pbi viruses and present in 11 of the 14 NC64A viruses and one SAG virus. Several of the other genes have similar complicated patterns and await explanations.

10. Sugar Enzymes Coded by Other Large DNA Viruses

This review has focused on carbohydrate enzymes encoded by the chloroviruses, primarily because these enzymes have been the most intensively studied. However, as new giant viruses are being discovered and their genomes sequenced, it is clear that some of them encode putative enzymes involved in carbohydrate manipulations. The most extensively studied of these other large DNA viruses is Acanthamoeba polyphaga mimivirus, which has genes encoding both glycosyltransferases and nucleotide sugars (see the recent review by Piacente et al. [87]). Other large DNA viruses encoding putative sugar manipulating enzymes include prasinoviruses (family Phycodnaviridae like the chloroviruses) that infect small marine green algae, including Ostreococcus, Bathycoccus and Micromonas species; these viruses have clusters of putative genes for enzymes involved in nucleotide-sugar metabolism and glycosyltransferases [88]. Similar genes are present in other members of the Mimiviridae family, including Phaeocystis globosa virus [89] and Cafeteria roenbergensis virus [90]. Putative glycosyltransferase-encoding genes have also been reported in the genomes of pandoraviruses [91], Pithovirus sibericus [92] and Mollivirus sibericus [93].
In conclusion, it is becoming clear that virus-encoded sugar-manipulating enzymes and glycosylation systems can no longer be considered a hallmark solely of cellular organisms, but that some viruses also encode unique and complex glycan systems, which are still largely unknown. One encourages young glycobiologists to consider working on some of these systems.

Acknowledgments

The authors thank the many undergraduate students, graduate students, postdocs and collaborators who have worked on various aspects of the chlorovirus encoded sugar enzymes over the past 25 years. This research has been supported by grants from NIH, NSF, DOE and the Stanley Medical Research Institute over the years.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Location of hyaluronan on the surface of infected Chlorella variabilis NC64A cells and ultrastructural changes in the algal cell wall after chlorovirus Paramecium bursaria chlorella virus type 1 (PBCV-1) infection. The figure shows the cross-sections of (A) the surface of the uninfected cells; (B) cells at 4 h post-infection (PI); and (C) cells at 4 h PI that were treated with hyaluronan lyase. Note that after treatment with hyaluronan lyase, the cell surface resembles the surface of uninfected cells. C is the cell wall, and Cyto is cytoplasm. Micrographs were taken from Graves et al. [18] with permission.
Figure 1. Location of hyaluronan on the surface of infected Chlorella variabilis NC64A cells and ultrastructural changes in the algal cell wall after chlorovirus Paramecium bursaria chlorella virus type 1 (PBCV-1) infection. The figure shows the cross-sections of (A) the surface of the uninfected cells; (B) cells at 4 h post-infection (PI); and (C) cells at 4 h PI that were treated with hyaluronan lyase. Note that after treatment with hyaluronan lyase, the cell surface resembles the surface of uninfected cells. C is the cell wall, and Cyto is cytoplasm. Micrographs were taken from Graves et al. [18] with permission.
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Figure 2. Scheme of the biosynthesis of GDP-l-fucose and GDP-d-rhamnose. PBCV-1 GDP-d-mannose 4,6 dehydratase (GMD) catalyzes both the dehydration of GDP-d-mannose to the intermediate GDP-4-keto-6-deoxy-d-mannose and the NADPH-dependent reduction of this latter compound to GDP-d-rhamnose. NADP+ serves as the cofactor for GMD during the internal oxidoreduction reaction involved in the dehydration process. The epimerization and the NADPH-dependent reduction of the 4-keto group leading to GDP-l-fucose are carried out by PBCV-1 GDP-4-keto-6-deoxy-D-mannose epimerase reductase (GMER). Figure was taken from Tonetti et al. [32] with permission.
Figure 2. Scheme of the biosynthesis of GDP-l-fucose and GDP-d-rhamnose. PBCV-1 GDP-d-mannose 4,6 dehydratase (GMD) catalyzes both the dehydration of GDP-d-mannose to the intermediate GDP-4-keto-6-deoxy-d-mannose and the NADPH-dependent reduction of this latter compound to GDP-d-rhamnose. NADP+ serves as the cofactor for GMD during the internal oxidoreduction reaction involved in the dehydration process. The epimerization and the NADPH-dependent reduction of the 4-keto group leading to GDP-l-fucose are carried out by PBCV-1 GDP-4-keto-6-deoxy-D-mannose epimerase reductase (GMER). Figure was taken from Tonetti et al. [32] with permission.
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Figure 3. Structure of the revised PBCV-1 Vp54 monomer. The two jelly-roll domains are colored in green and red, respectively. The glycans located on the surface are shown as a space-filling representation of their atoms and are colored according to the residue they are attached to (Asn-280: green, Asn-302: black, Asn-399: red, Asn-406: blue). Taken from De Castro et al. [49] with permission.
Figure 3. Structure of the revised PBCV-1 Vp54 monomer. The two jelly-roll domains are colored in green and red, respectively. The glycans located on the surface are shown as a space-filling representation of their atoms and are colored according to the residue they are attached to (Asn-280: green, Asn-302: black, Asn-399: red, Asn-406: blue). Taken from De Castro et al. [49] with permission.
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Figure 4. Structures of PBCV-1 Vp54 N-glycans. Arabinose and mannose are not stoichiometric substituents and create four different glycoforms. The two on the left are the most abundant, and both have mannose. The structure at the bottom represents the conserved core oligosaccharide that is present in all of the chloroviruses studied to date. Residues within the box are those strictly conserved, while rhamnose (outside the box) is a semi-conserved element because its absolute configuration is virus dependent. The figure was modified from De Castro et al. [34,54] with permission.
Figure 4. Structures of PBCV-1 Vp54 N-glycans. Arabinose and mannose are not stoichiometric substituents and create four different glycoforms. The two on the left are the most abundant, and both have mannose. The structure at the bottom represents the conserved core oligosaccharide that is present in all of the chloroviruses studied to date. Residues within the box are those strictly conserved, while rhamnose (outside the box) is a semi-conserved element because its absolute configuration is virus dependent. The figure was modified from De Castro et al. [34,54] with permission.
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Figure 5. Structures of N-glycans from seven chloroviruses representing all four chlorovirus types. Substituents in brackets are not stoichiometric. All sugars are in the pyranose form, except where specified. Virus NY-2A is an NC64A virus, virus Osy-NE5 an Osy virus, viruses ATCV-1 and TN603 SAG viruses and MT325, CVM-1 and NE-JV-1 Pbi viruses. This figure was modified from [6,34,35] with permission.
Figure 5. Structures of N-glycans from seven chloroviruses representing all four chlorovirus types. Substituents in brackets are not stoichiometric. All sugars are in the pyranose form, except where specified. Virus NY-2A is an NC64A virus, virus Osy-NE5 an Osy virus, viruses ATCV-1 and TN603 SAG viruses and MT325, CVM-1 and NE-JV-1 Pbi viruses. This figure was modified from [6,34,35] with permission.
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Table
Table 1. Chlorovirus encoded enzymes involved in the synthesis of polysaccharides.
Table 1. Chlorovirus encoded enzymes involved in the synthesis of polysaccharides.
HostVirusesHAS 1CHS 2CBP 3
NC64AAN69C 390R395L, 438L
AR158 C418RC423L, C475L
CviK1102R365R370L, 414L
CvsA1 375R380L, 427L
IL-3A 386L, 432L
IL-5-2s1134L 503L, 562L
KS-1B 314L, 360L
MA-1D485L362R367L, 472L
MA-1E 113L407L, 451L
NE-JV-4 390L
NY-2B 542L, 484L
NY2A B139R, B472RB480L
NYs-1137R 360R, 495L, 555L
PBCV-1a A098R A333L, A348Dl, A383R
SYNOSY-NE-5038R 167L, 184L
PbiAP110A 152L, 175R828R
CVA-1 150L, 169R834R
CVB-1 177L791R
CVG-1146R 792R
CVM-1 165L, 186R832R
CVR-1 838R
CZ-2 798R
Can18-4163R 839R
FR483N124R N690R
FR5L 151L797R
MT325M128R M701R
NE-JV-1 278R, 282R734L
NW665133R 821R
OR0704.2.2116R 804R
SAGATCV-1 Z734R
Br0604L 834R431R
Can0610SP 438R, 442R
Canal-1 746R405R
GM0701 852R436R
MN0810 087R, 900R466R, 468R, 531L
MO0605SPH 435R
NE-JV-2 462R
NE-jv-3 431R
NTS-1 893R461R, 463R, 529L
OR0704.3 431R
TN603 869R425R
W10606 457R
1 Hyaluronan synthase; 2 chitin synthase; 3 chitin binding proteins, except for the chitinase proteins reported in Table 5; a the recombinant protein has the predicted activity. The numbers refer to the protein names, and the R and L refer to the strand orientation.
Table
Table 2. Chlorovirus encoded enzymes involved in sugar metabolism.
Table 2. Chlorovirus encoded enzymes involved in sugar metabolism.
HostVirusesGFAT 1UDP-GlcDH 2GMD 3GMER 4UGD 5AT 6D-LD 7ADP-RGH 8FRD 9
NC64AAN69C109R384R, 487R129R334L 739L055R
AR158C132RC413R, C729LC155RC344L C767L
CviK1105R359R, 662L122R312L 742L055R
CvsA1064R368R083R321L 716L131R
IL-3A104R375R, 685L126R326L 726L051R
IL-5-2s1130R492R, 858L106L417L 896L
KS-1B 066R261L 643L
MA-1D481L355R, 838L456L284L 872L
MA-1E116R396R134R355L 806L045R
NE-JV-4 131R340L 740L064R
NY-2B 473R, 836L185R408L 881L
NY2AB143RB465RB163RB395L B853L
NYs-1143R483R, 846L167R404L 879L
PBCV-1a A100Ra A609La A118Ra A295L A654LA053R
SYNOSY-NE-5039R 045R139L 340L015R308R
PbiAP110A071R146L 893R053L
CVA-1056R144L 900R040L205R
CVB-1071R172L 856R056L221R
CVG-1050R812L 857R
CVM-1069R159L 893R052L222R
CVR-1062R151L 906R046L210R
CZ-2059R718L 865R048L 917L
Can18-4061R859L 908R048L212R
FR483N035RN712L N747R N170R
FR5L087R145L 863R076L
MT325M036R, M037RM719L M758RM026L
NE-JV-1081R291R 861R810L
NW665046R846L 889R 189R
OR0704.2.2062R722L 862R051L
SAGATCV-1 Z571La Z804LZ282LZ544RZ147LZ295L
Br0604L 667L, 839R934L332L631R173L350L
Can0610SP 687L965L338L658R170L355L
Canal-1 605L, 751R847L329L576R188L343L898L886R
GM0701 664L, 856R954L337L629R180L354L
MN0810887L720L, 904R992L365L689R204L379L
MO0605SPH 656L897L341L625R181L359L 943R
NE-JV-2 708L981L367L672R186L383L
NE-JV-3 679L935L332L648R175L347L 981R
NTS-1 714L, 898R1012L378L681R188L391L
OR0704.3 676L960L335L639R179L354L
TN603 659L, 873R966L326L625R179L342L
W10606 679L916L360L651R185L375L 962R
1 Glutamine-fructose-6-phosphate aminotransferase; 2 UDP-glucose-6-dehydrogenase; 3 GDP-d-mannose dehydratase; 4 GDP-4-keto-6-deoxy-d-mannose epimerase/reductase (=GDP-l-fucose synthase 2); 5 UDP-d-glucose 4,6-dehydratase, 6 acetyltransferase; 7 d-lactate dehydrogenase; 8 ADP-ribosylglycohydrolase; 9 fumarate reductase; a the recombinant proteins have the predicted activities. The numbers refer to the protein names, and the R and L refer to the strand orientation.
Table
Table 3. PBCV-1 antigenic variants that affect the molecular weight of the major capsid glycoprotein.
Table 3. PBCV-1 antigenic variants that affect the molecular weight of the major capsid glycoprotein.
Antisera ClassesPredicted MW (kDa) cSDS-PAGE Estimates (kDa) d
Class aLabel b
+Wild-type54.154
FCME654not determined
AP9152.853
EEPA-1552.8not determined
BEPA-251.652
CE1L351.151
DP1L650.550.5
a Listed in order of predicted molecular weight based on nuclear magnetic resonance (NMR) analysis (De Castro et al., [44,45]); b representative mutant strain label; c the gene encoding the PBCV-1 major capsid protein (a430l) is wild-type in sequence and does not vary among antisera classes; d Graves et al. [46]. MW, molecular weight.
Table
Table 4. Chlorovirus encoded enzymes involved in synthesizing glycans attached to virus major capsid proteins.
Table 4. Chlorovirus encoded enzymes involved in synthesizing glycans attached to virus major capsid proteins.
HostVirusesEXT 1GT-A 2GT-GT4 3GT-GTA 4CESA CelA-Like 5CSCS-2 6GT 7GT17 8
NC64AAN69C078L123R559R065R104R255R
AR158C093LC150RC661L C265R C559R
CviK1080L117R594L 518L242R
CvsA1039L077R611L 535L247R
IL-3A071L120R606L060R099R240R
IL-5-2s1175R109L773L 313R 649R
KS-1B024L060R528L009R046L170R
MA-1D531R4549L753L 194R 637R
MA-1E533R128R702L 626L281R, 210R
NE-JV-4085L124R631L074R108L250R
NY-2B116L180R754L 160R323R 633R
NY2A107LB159RB736L B618R
NYs-1098L162R760L 187R 641R
PBCV-1A075LA111/114RA546LA064RA473LA219/222/226R
SYNOSY-NE-5025L044R283L 097L
PbiAP110A013L548R 226R 970R
CVA-1016L532R 220R 971R
CVB-1025L538R 232R811L918R
CVG-1019L520R 217R815L920R
CVM-1022L550R 237R 953R
CVR-1020L545R 225R 977R
CZ-2012L532R380L 822L932R
Can18-4020L557R 229R862L971R
FR483N012LN472R N191RN715LN805R
FR5L046L537R 819L926R
MT325M009LM467R M186RM721LM813R
NE-JV-1079L464L 801R930R
NW665015L532R 849L955R
OR0704.2.2017L549R382L 823L923R
SAGATCV-1Z830RZ120RZ667L Z178L, Z823R, Z417LZ425RZ347R
Br0604L959R137R 225L, 952R, 483L489R399R
Can0610SP1007R140R789L 210L, 1002R, 978L, 487L495R407R
Canal-1874R141R164L 871R447R380R
GM0701977R141R752L 228L, 975R, 486L493R405R
MN08101009R165R 244L 424R
MO0605SPH932R138R164L 230L, 926R, 479L488R412R
NE-JV-21020R150R804L 1015R, 992L, 516L523R438R
NE-JV-3970R145R778L 215L, 210L, 964R, 484L493R407R
NTS-11044R156R 1016L, 516L 441R
OR0704.31006R146R944L 1001R, 972L, 485L, 219L493R404R
TN603991R141R 226L, 986R, 475L483R400R
WI0606951R141R 233L, 945R, 506L514R430R
1 Exotosin glycosyltransferase; 2 glycosyltransferase family A; 3 glycosyltransferase GT4-type super family; 4 glycosyltransferase GTA-type super family; 5 CESA CelA-like cellulose synthase catalytic subunit (UDP-glucose as substrate); 6 cellulose synthase catalytic subunit; 7 glycosyltransferase; 8 glycosyltransferase family 17.
Table
Table 5. Chlorovirus encoded enzymes involved in degrading polysaccharides.
Table 5. Chlorovirus encoded enzymes involved in degrading polysaccharides.
HostVirusesCHI 1CHIS 2GUN 3BCHIL 4Lysin 5GH 6CD 7ALGL 8
NC64AAN69C297R331L102L204R540R 387L250L
AR158 C342LC126LC220RC681L C415LC263L
CviK1 309L99L194R610L 362L237L
CvsA1 318L058L200R627L 371L, 373L243L
IL-3A285R323L097L196R624L 378L235L
IL-5-2s1373R415L140R262R796L 495L310L
KS-1B211R257L048R132R546L 166L
MA-1D241R, 242R283L491R147R777L 359L191L
MA-1E 352L114R194R, 229R717L 399L277L
NE-JV-4298R337L110R205R648L 246L
NY-2B367R406L158L270R777L 476L319L
NY2A B393LB137LB239RB756L B469LB288L
NYs-1360R403L134L251R 487L
PBCV-1a A260Ra A292La A094La A181/182Ra A561L a A215L
SYNOSY-NE-5119R138L037L117L290L 099R
PbiAP110A106R122R174L942R306R 158L338R
CVA-1102R114R167L942R297R 156L327R
CVB-1129R143R 890R310R 183L342R
CVG-1105R113R142L896R303R 329R
CVM-1122R133R184L927R330R 171L360R
CVR-1109R121R174L948R 163L335R
CZ-2070R079R114L902R263R 293R
Can18-4113R121R160L944R316R 349R
FR483 N087RN119LN779RN262R N293R
FR5L098R106R 897R300R 157L336R
MT325M085RM091RM124LM791RM258R M289R
NE-JV-1328L218L275L088R592L050L269L472L
NW665 088R128L921R281R 315R
OR0704.2.2074R082R113L895R257R 292R
SAGATCV-1Z780LZ204RZ819LZ814LZ511L Z771L
Br0604L 253R, 254R950L902R, 942L518L 832L895L
Can0610SP 244R985R, 1001L936R, 996R613L 919L
Canal-1815L242R866L138L, 855L546L 743L806L
GM0701 251R972L917R, 963L526L 846L910L
MN0810 271R082L162L, 1003L659L 896L959L
MO0605SPH871L257R920L911L588L 862L
NE-JV-2950L258R1000R, 1013L1008R634L 932L
NE-JV-3907L244R957L948L603L 897L
NTS-1973L276R1024R1039R556L 886L961L
OR0704.3 251R979R, 999L918R, 994R604L 910L
TN603 253R983L939R, 977L520L 867L931L
WI0606889L262R938L929L612L 878L
1 Chitinase; 2 chitosanase; 3 1-3-beta glucanase; 4 bifunctional chitinase/lysozyme; 5 lysin homologs from encoded by PBCV-1 CDS A561L; 6 glycosyl hydrolase; 7 chitin deacetylase; 8 alanine alginate lyase; a the recombinant proteins have the predicted activity.

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Van Etten, J.L.; Agarkova, I.; Dunigan, D.D.; Tonetti, M.; De Castro, C.; Duncan, G.A. Chloroviruses Have a Sweet Tooth. Viruses 2017, 9, 88. https://doi.org/10.3390/v9040088

AMA Style

Van Etten JL, Agarkova I, Dunigan DD, Tonetti M, De Castro C, Duncan GA. Chloroviruses Have a Sweet Tooth. Viruses. 2017; 9(4):88. https://doi.org/10.3390/v9040088

Chicago/Turabian Style

Van Etten, James L., Irina Agarkova, David D. Dunigan, Michela Tonetti, Christina De Castro, and Garry A. Duncan. 2017. "Chloroviruses Have a Sweet Tooth" Viruses 9, no. 4: 88. https://doi.org/10.3390/v9040088

APA Style

Van Etten, J. L., Agarkova, I., Dunigan, D. D., Tonetti, M., De Castro, C., & Duncan, G. A. (2017). Chloroviruses Have a Sweet Tooth. Viruses, 9(4), 88. https://doi.org/10.3390/v9040088

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