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Hyaluronan Synthase of Chlorella Virus PBCV-1

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Science  05 Dec 1997:
Vol. 278, Issue 5344, pp. 1800-1803
DOI: 10.1126/science.278.5344.1800

Abstract

Sequence analysis of the 330-kilobase genome of the virus PBCV-1 that infects a chlorella-like green algae revealed an open reading frame, A98R, with similarity to several hyaluronan synthases. Hyaluronan is an essential polysaccharide found in higher animals as well as in a few pathogenic bacteria. Expression of the A98Rgene product in Escherichia coli indicated that the recombinant protein is an authentic hyaluronan synthase.A98R is expressed early in PBCV-1 infection and hyaluronan is produced in infected algae. These results demonstrate that a virus can encode an enzyme capable of synthesizing a carbohydrate polymer and that hyaluronan exists outside of animals and their pathogens.

Hyaluronan or hyaluronic acid (HA), a member of the glycosaminoglycan family that also includes heparin and chondroitan, is a linear polysaccharide composed of alternating β1,4-glucuronic acid (β1,4-GlcA) and β1,3-N-acetylglucosamine (β1,3-GlcNAc) groups. Typically the full-length polymer chains are composed of 103 to 104 monosaccharides (106 to 107daltons). HA is an important structural element in the vitreous humor of eye, synovial fluid, and skin of vertebrates (1). Furthermore, HA interacts with proteins such as CD44, RHAMM, and fibrinogen, thereby influencing many natural processes such as angiogenesis, cancer, cell motility, wound healing, and cell adhesion (2). HA also constitutes the extracellular capsules of certain bacterial pathogens such as group A and CStreptococcus and Pasteurella multocida type A (3, 4). These capsules act as virulence factors that protect the microbes from phagocytosis and complement during infection (5, 6). Because HA, a component of the host tissues, is not normally immunogenic, the capsule serves as molecular camouflage (7).

HA synthases (HASs) are integral membrane proteins that polymerize the HA molecule using activated uridine diphosphate (UDP)–sugar nucleotides as substrates. Amino acid sequences for some HASs have been deduced from gene sequencing (8); their sizes range from 419 to 588 residues. The vertebrate enzymes (DG42, HAS1, HAS2, and HAS3) and streptococcal HasA have several regions of sequence similarity. Recently, while sequencing the double-stranded DNA genome of virus PBCV-1 (Paramecium bursaria chlorella virus), we unexpectedly discovered an open reading frame (ORF), A98R (GenBank accession number U42580), encoding a 568-residue protein with similarity to the known HASs (28 to 33% amino acid identity in pairwise comparisons by FASTA) (Fig. 1).

Figure 1

Sequence similarity of HASs. The Multalin program (26) was used to align the amino acid sequences of HASsXenopus laevis DG42, human HAS2, PBCV-1 A98R, andStreptococcus pyogenes HasA (red, 90% consensus; green, 50% consensus, as calculated by Multalin) (8). In the consensus sequence, the symbols are: !, any one of I or V; $, any one of L or M; %, any one of F or Y; #, any one of N,D,E, or Q. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

PBCV-1 is the prototype of a family (Phycodnaviridae) of large (175 to 190 nm in diameter) polyhedral, plaque-forming viruses that replicate in certain unicellular, eukaryotic chlorella-like green algae (9). PBCV-1 virions contain at least 50 different proteins and a lipid component located inside the outer glycoprotein capsid (10). The PBCV-1 genome is a linear, nonpermuted 330-kb double-stranded DNA molecule with covalently closed hairpin ends (11).

On the basis of its deduced amino acid sequence, theA98R gene product should be an integral membrane protein. To test this hypothesis, we produced recombinant A98R protein inEscherichia coli and assayed the membrane fraction for HAS activity (12, 13). UDP-GlcA and UDP-GlcNAc were incorporated into polysaccharide by the membrane fraction derived from cells containing the A98R ORF on a plasmid, pCVHAS, (average specific activity of 2.5 pmol of GlcA transferred per minute per microgram of protein), but not by samples from control cells (<0.001 pmol of GlcA transferred per minute per microgram of protein). No activity was detected in the soluble fraction of cells transformed with pCVHAS. UDP-GlcA and UDP-GlcNAc were simultaneously required for polymerization. The activity was optimal in Hepes buffer at pH 7.2 in the presence of 15 mM MnCl2, whereas no activity was detected if the metal ion was omitted. The ions Mg2+ and Co2+ were ∼20% as effective as Mn2+ at similar concentrations. The P. multocida HAS (14) has a similar metal requirement, but other HASs prefer Mg2+.

We also tested the specificity of recombinant A98R for UDP-sugars (15). Only the two authentic HA precursors were incorporated into polysaccharide; neither UDP-galacturonic acid (UDP-GalA) nor UDP-N-acetylgalactosamine (UDP-GalNAc), the C4 epimers of UDP-GlcA or UDP-GlcNAc, respectively, were incorporated. Likewise, UDP-glucose (UDP-Glc) was not polymerized in place of either HA precursor. This strong substrate specificity for UDP-GlcA and UDP-GlcNAc is a general feature of the HASs HasA (13) and DG42 (16).

The recombinant A98R enzyme synthesized a polysaccharide with an average molecular size of 3 × 106 to 6 × 106 daltons (Fig.2), which is smaller than that of the HA synthesized by recombinant HasA or DG42 in vitro (∼107daltons and ∼5 × 106 to 8 × 106daltons, respectively) (13, 16). The polysaccharide was completely degraded by Streptomyces hyalurolyticus HA lyase, an enzyme that depolymerizes HA but not structurally related glycosaminoglycans such as heparin and chondroitan (17).

Figure 2

Size exclusion chromatography of polymer product of recombinant A98R HAS. Membranes derived from E. coli cells transformed with pCVHAS were incubated with both radiolabeled HA precursors diluted to the same specific activity (27). After deproteinization and removal of unincorporated precursors, samples were injected onto a Sephacryl S-500HR size exclusion column, and the radioactivity in the fractions was measured (3H, solid squares; 14C, solid circles). A duplicate sample was treated with HA lyase before deproteinization and chromatography (3H, open squares;14C, open circles); no polymer remains after digestion. Size standards: Vo arrow, void volume, HA derived from recombinant streptococcal HasA (17 ml; ≥2 × 107daltons) (13); crosshatched box, blue dextran (29 to 32 ml; average molecular size 2 ×106 daltons; Pharmacia); Vti arrow, totally included volume, UDP-sugars (37 ml).

We examined PBCV-1–infected chlorella cells for A98R gene expression. A ∼1700-nucleotide A98R transcript appeared about 15 min after infection and disappeared by 60 min after infection (18), indicating that A98R is an early gene. Consequently, we assayed membrane fractions from uninfected and PBCV-1–infected chlorella cells at 50 and 90 min after infection for HAS activity. Infected cells, but not uninfected cells, had activity (Table 1). Like the bacterially derived recombinant A98R enzyme, radioactive label incorporation from UDP-[14C]GlcA into polysaccharide depended on both Mn2+ and UDP-GlcNAc. This labeled product was also degraded by HA lyase. Disrupted PBCV-1 virions had no HAS activity.

Table 1

HAS activity of membranes derived fromChlorella cells infected with PBCV-1. The membrane fractions (370 μg of protein) from uninfected cells or cells at 50 and 90 min after infection (a.i.) were assayed with UDP-[14C]GlcA (60 μM, 0.02 μCi) in parallel reactions containing the following components as indicated (300 μM UDP-GlcNAc or 15 mM MnCl2 or both) for 1 hour at 30°C (28). HAS specific activity (presented as picomoles of [14C]GlcA transferred per hour per milligram of protein) was detected in the algal membranes after infection with PBCV-1, but not in uninfected cells.

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PBCV-1–infected chlorella cells were analyzed for HA polysaccharide by means of a highly specific 125I-labeled HA-binding protein (19, 20). Extracts from cells at 50 and 90 min after infection contained substantial amounts of HA (0.7 and 1400 ng per microgram of protein, respectively), but not extracts from uninfected algae (<0.04 ng per microgram of protein) or disrupted PBCV-1 virions (<0.04 ng per microgram of dry weight). The labeled HA-binding protein also interacted with intact infected cells at 50 and 90 min after infection, but not with healthy cells (21). Therefore, a considerable portion of the newly synthesized HA polysaccharide was immobilized at the outer cell surface of the infected algae. The extracellular HA does not play any obvious role in the interaction between the virus and its algal host because neither plaque size nor plaque number was altered by including either testicular hyaluronidase (465 units/ml) or free HA polysaccharide (100 μg/ml) in the top agar of the PBCV-1 plaque assay (9).

Among chlorella viruses, HA biosynthesis during infection is not limited to the PBCV-1 prototype strain. Thirty-three independently isolated and plaque-purified viruses from the United States, South America, Asia, and Australia were tested for the presence of an A98R-like gene and for the ability to direct production of HA polysaccharide in Chlorella NC64A. Dot-blot hybridization analyses of the individual viral genomes with the PBCV-1 A98R probe indicated that 19 isolates (58%) had a similar gene; the algal host DNA did not cross-react with the probe (21). Chlorella cells infected with each of these 19 viruses produced cell surface HA as measured by interaction with the 125I-HA–binding protein (21).

Surprisingly, the PBCV-1 genome also has additional genes, namedA609L and A100R, that encode for a UDP-Glc dehydrogenase (UDP-Glc DH) and a glutamine:fructose-6-phosphate amidotransferase (GFAT), respectively. UDP-Glc DH converts UDP-Glc into UDP-GlcA, a required precursor for HA biosynthesis. GFAT converts fructose-6-phosphate into glucosamine-6-phosphate, an intermediate in the UDP-GlcNAc metabolic pathway. Both of these PBCV-1 genes, like the A98R HAS, are expressed early in infection and encode enzymatically active proteins (22); however, these three genes do not function as an operon. Although two of these genes,A98R and A100R, are near one another in the viral genome (bases 50,901 to 52,607 and 52,706 to 54,493, respectively),A609L is located ∼240 kb away and is transcribed in the opposite orientation (bases 292,916 to 291,747). The presence of multiple enzymes in the HA biosynthesis pathway indicates that HA production must serve an important function in the life cycle of these chlorella viruses.

The details of the natural history of the phycodnaviruses are unknown. These viruses are ubiquitous in freshwater collected worldwide, and titers as high as 4 × 104 infectious viruses per milliliter of native water have been reported (23). The only known hosts for these viruses are chlorella-like green algae, which normally live as hereditary endosymbionts in some isolates of the ciliate, P. bursaria. In the symbiotic unit, algae are enclosed individually in perialgal vacuoles and are surrounded by a host-derived membrane (24). The endosymbiotic chlorella are resistant to virus infection and are only infected when they are outside the paramecium (9). We hypothesize that HA synthesis and its accumulation on the algal surface may block the uptake of virus-infected algae by the paramecium. Alternatively, the chlorella viruses might have another host in nature (such as an aquatic animal); perhaps the virus is transmitted because this other host is attracted to or binds to the HA polysaccharide on virus-infected algae.

As depicted in Fig. 1, HASs of Streptococcus, vertebrates, and PBCV-1 have many motifs of two to four residues that occur in the same relative order. These conserved motifs probably reflect domains crucial for HA biosynthesis. Regions of similarity between HASs and other enzymes that synthesize β-linked polysaccharides from UDP-sugar precursors are also being discovered as more glycosyltransferases are sequenced (25). The significance of these similar structural motifs will become more apparent as the three-dimensional structures of glycosyltransferases are determined.

The fact that Chlorella virus PBCV-1 encodes a functional glycosyltransferase that can synthesize HA is contrary to the general observation that viruses either (i) use host cell glycosyltransferases to create new carbohydrate structures, or (ii) accumulate host cell glycoconjugates during virion maturation. Furthermore, HA has been generally regarded as restricted to animals and a few of their virulent bacterial pathogens. Though many plant carbohydrates have been characterized, to our knowledge, neither HA nor a related analog has previously been detected in cells of plants or protists.

  • * To whom correspondence should be addressed: E-mail: paul-deangelis{at}OUHSC.edu

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