JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 88, No. 4, 353-361. 1999

REVIEW

Chlorella Viruses as a Source of Novel Enzymes TAKASHI YAMADA,* NITI CHUCHIRD, TAKERU KAWASAKI, KENSHO NISHIDA, AND

SHINGO HIRAMATSU

Department of Molecular Biotechnology, Graduate School of Advanced Matter, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima 739-8527, Japan

Received 2 July 1999/Accepted 31 July 1999

A special advantage has been conferred upon Chlorella cells as tools in biotechnology when viruses (Pbycodnaviridae) infecting Chlorella cells were discovered and isolated. The viruses are large icosabedral particles (150-200 nm in diameter), containing a giant, 330380 kbp long, linear dsDNA genome. Recently, the nucleotide sequence of the 330,740-bp genome of PBCV-1, the prototype virus of Pbycodnaviridae, was determined, and up to 702 open reading frames (ORFs) were identilied along the genome. The possible genes present include those encoding a variety of enzymes involved in the modification of DNA, RNA, protein and polysaccbarldes as well as those involved in the metabolism of sugars, amino acids, lipids, nucleotides and nucleosides. Many of these genes are actually expressed during viral infection, with functional enzymes detected in the host cytoplasm or incorporated into the virion. The successful utilization of these viral enzymes as various DNA restriction and modification enzymes (Cvi enzymes) that are now commercially available is well documented. Also noteworthy are virion-associated chitinase and cbitosanase activities that have potentially important applications in the recycling of natural resources. The virions of Chlorella viruses contain more than 50 different structural proteins, ranging in size from 10 to 200 kDa. Some of these proteins may be replaced with useful foreign proteins using recombinant DNA technology. The proteins of interest can be recovered easily from the viral particles, and collected by centrifugation after complete lysis of the host Chlorella cells.

[Key words: Phycodnaviridae (Chlorella viruses), dsDNA genome, complete nucleotide sequence, gene identification]

Large icosahedral, dsDNA-containing viruses (Chjorel- la virus, Phycodnaviridae (1)) that infect certain strains of the unicellular green alga Chlorella are ubiquitous in natural environments (2-4). These viruses were first found in Chlorella-like algae (zoochlorella) that are endo- symbiotic with Paramecium bursaria (5) and isolated from zoochlorella of Hydra viridis (6, 7) and P. bursaria (8). The viruses can be assayed by plaque formation and produced in large quatities with some exsymbiotic Chlo- rella strains such as NC64A (9), SAG-241-80 (10) and Pbi (11). One of the remarkable features of Chlorella viruses is their large, 330-380-kbp dsDNA genomes that potentially encode more than several hundred genes. Recently, the nucleotide sequence of the 330,740-bp genome of PBCV-1, the prototype virus of Phycodna- viridae, was completed, and 702 open reading frames (ORFs) were identified along the genome, of which 377 are predicted to encode functional proteins; 121 of these resemble proteins in databases (12-16). These proteins include a variety of enzymes involved in modification of DNA, RNA, protein and polysaccharides as well as those involved in the metabolism of sugars, nucleotides and nucleosides, amino acids and lipids (GenBank acces- sion no. U42580). The putative enzymes predicted for each PBCV-1 ORF are listed in Table 1. Some of these genes were demonstrated to be actually expressed during viral infection, and functional enzymes were detected in the host cytoplasm or were incorporated into the virion.

Biochemical or enzymological information about these gene products is at present very scarce, but a few exam-

* Corresponding author.

ples studied so far reveal that Chlorella viral enzymes have novel, interesting properties as described below. This is why Chlorella viruses have attracted attention in the fields of basic as well as applied science. In this review, attention is focused on the production of useful enzymes regarding Chlorella viruses as novel enzyme sources.

INFECTION CYCLE OF CHLORELLA VIRUSES

In the normal lytic cycle, viral particles attach to the surface of the host Chlorella cells, digest the cell wall at the point of attachment, and inject the viral core into the host cytoplasm leaving an empty capsid on the cell surface. This mode of penetration resembles that of bac- teriophage T4 which infects Escherichia co/i cells. Viral DNA synthesis begins about 1 h postinfection (pi.) and by 2-3 h p.i., parts of the viral capsid accumulate in the host cytoplasm. Mature, DNA-filled virions appear by 4 h p.i. and about 2-4 h later the host cells burst, releas- ing viral progeny into the culture medium (17). Viral gene transcription is, for convenience, divided into early and late stages (18). The junction between these stages is about l-2 h p.i., which coincides with the initiation of viral DNA synthesis. The replication cycle of Chlorella viruses is schematically shown in Fig. 1.

One-step growth experiments with PBCV- 1 revealed that the viral progeny is first released about 3 to 4 h p.i. and that the release of the virus is completed by disrup- tion of host cells within 8 to 10 h p.i. (19). Mechanical disruption of the infected cells releases infectious viruses 30 to 50min prior to spontaneous lysis. The burst size is

353

354 YAMADA ET AL. J. BIOSCI. BIOENG.,

TABLE 1. Putative genes encoded on the Chlorelia virus PBCV-la

DNA, RNA interacting proteins T4 endonuclease (ASOL), T4 intron endonuclease (A134L, A651L), R exonuclease (A166R), SPOl group I intron homing endonuclease (A422R), T4 endonuclease V (A50L), DNA polymerase III p (A493L), DNA polymerase B (A185R), Transposase (A625R), DNA ligase (A544R), DNA topoisomerase II (A583L), RNA ribozyme (A125L), RNA-binding protein (A478L, A646L), RNase III (A464R), Proliferating cell nuclear antigen (DNA polymerase a processing factor) (A193L, A574L), Reverse transcriptase (A399R), RNP-1 (A478L, A646L), Helicase (A548L), DNA-binding: Leucine zipper protein (A123R), Zn finger protein (A564L), RING finger (A481L) Helix- loop-helix DNA binding protein (A322L), Chromosomal protein (A437L), Nucleolin (A619L), Centromere-binding protein (A659R)

Restriction/modification enzymes M.CviAII (A251R), R.CviAII (A252R), M.CviJI (A517L, A530R, A683L), R.CvIAI (A579L), M.CviAI (A581R)

Transcription RNA polymerase II LS (A533R), RNA polymerase III (A357L), RNA polymerase u-factor (A470R), mRNA-capping enzyme (A103R), TFIIB (A107L), TFIIS (A125L), TFIID (A552R), TFbean (A379L), Transcription activator (A354R, A548L), Negative regulator (A373R), Regulatory protein (A140R), mRNA 3’ processing protein (A256L)

Translation tRNA L(UUG), tRNA I(AUA), tRNA L(CUC), tRNA N(AAC), tRNA K(AAG), tRNA N&AU), tRNA K(AAA), tRNA R(AGA), Elongation factor EF-3 (A666L), Aminoacyl tRNA synthetase (A3R, AllL, AlOOR, A689L), Ser-tRNA selenium transferase (A298L), Asp-tRNA synthetase (A312L), Ribosomal proteins: cpS5 (A88R), mtS5 (A261R), L29(A394R), mtProtein (A408L), cpSl1 (A577L), PO (A618L), elF3 (A590L)

Nucleotide metabolism Viral thymidine kinase (AlOR), Ribonucleoside triphosphate reductase (A129R), Cytidine/deoxycytidylate deaminase (A200R, A596R, A629R), Aspartate transcarbamylase (A169R), Thymidine kinase (A243R, A539R), Nucleoside triphosphate phosphohydrolase (A363R), Deoxycytidine kinase (383R), Ribonucleotide reductase small subunit (A476R) and large subunit (A629R), dUTP pyrophosphatase (A551L)

Amino acid metabolism Leu/Ile/Val/Thr-binding protein (A9R), Ornithine decarboxylase (A207R), Histidine decarboxylase (A598L), Serine dehydratase (A468R), Prolyl4-hydroxylase (a-subunit) (A85R), Aminotransferase (A543L)

Sugar manipulation enzymes p-1,3-Glucanase (A94L), GDP-mannose dehydratase (A118R), Chitinase (A181R, A182R), Endochitinase (A26OR), Chitosanase (A292L), Glucosamine synthetase (AlOOR), Colanic acid synthetase (A295L), Cellulose synthetase (A473L), Hyauronan synthase (A98R), UDP-Glc dehydrogenase (A609L), Glutamine:fructose-6 phosphate aminotransferase (AlOOR), Cyclomaltodextrin glucanotransferase (A326L), ,9- Glucosidase (A401R), ,%1,4-Xylanase (A424R), n-Mannosidase (A444L), Endoglucanase El (A5OOL)

Protein manipulation enzymes Thiol protease (A14R), Protein prenyltransferase a subunit (A60L), Protein kinase (A34R, A98R, A248R, A277L, A278L, A282L, A289L, A424R, A614L, A617R, A649R), Protein phosphatase (Tyr) (A305), Protein phosphatase S/T (A402R), Protein kinase inhibitor (A607R), Subtilisin-like protease (A154L), Glutamyl aminopeptidase (A297L), Zn-metallopeptidase (A521L, A604L), Papain-like protease (A328L), Pepsinogen III (A341L)

Lipid-manipulating enzymes Glycerophosphoryl diester phosphodiesterase (A49L), Diacylglycerol kinase (A87R), Lysophospholipase (A271L), Bile acid hydrolase (A284L)

Energy metabolism 2-Hydroxyacid dehydrogenase (A53R), Glyceraldehyde 3-p dehydrogenase (A127R), Aldehyde dehydrogenase (A203R), Ribulose-1,5- bisphosphate carboxylase small subunit (A257L), Phoyosystem II 10 Kd phosphoprotein (A314R), ATP synthase a (A366L) and 16-kd proteolipid subunit (AA621L), Fructose 6-p phosphotransferase (A426R), Alcohol dehydrogenase (A443R), Glycerol 3-p hydrogenase (A572R), Nucleotide sugar dehydrogenase (A609L)

Other enzymes Ubiquinol/Cyt-C reductase (A44L), Ubiquitin carboxy-terminal hydrolase (A105L), Homospermidine synthase (A237R),Cu/Zn superoxide dismutase (A245R), K+ ion channel protein (A25OR), r&S-like aminotransferase (A543L), Ubiquitin carboxyterminal hydrolase (A105L), Cyt-C heme-binding protein (A267L, A382R, A448L), ABC transporter (A445L, A666L), Cytochrome P450 (A454L), Fibronectin-binding protein (A180R), Thioredoxin (A427L), Glutaredoxin (A438L). Phophopantethenic-binding protein (A540L), transposase (A625R)

Other proteins Major capsid proteins (AlOR, AllL, A383R, A430L, A558L, A622L), ATP/GTP-binding motifs (A39L, A44L, A51L, A78R, A153R, A231L, A241R(helicase), A318R, A351L, A360R, A378L, A392R, A416L, A456L, A512R, A546L, A561L, A565R), Transmembrane domain (A51L, A77L, A85R, A94L, A98R, A122R, A130R, A135L, A157L, A162L, A163R, A168R, A175R, A196L, A199R, A201L, A213L, A219R, A229L, A230R, A263L, A265L), A273L, A320R, A352L, A401R, A405R, A407L, A413L, A473L, A494R, A497R, A503L, A602L), Prokaryotic lipoprotein attachment site (A503L, A607R, A609L, A628L, A687L), Ankyrin repeats (ASR, A7L, A8L, A247R, A330R, A607R, A672R, A682L), Collagen a-l (A205R), GAG protein (A49OL), Chemotaxis protein (A492L), Homeobox domain (A189R, A605L), Signal recognition particle (A537L), Ca-binding (EF-hand)(A208R),T4 mobD7 (A227L), Early nodulin N-75 (A594R), Neurofilament triplet H (A282L), Lectin-like protein (A321R, A627R), Integrin p-6 (A570R), Golgi complex protein (A636R), Peroxisomal protein (A662L)

a ORF designation is based on the references (12-16). Homology with a FASTA score of more than 100 in amino acid sequences is listed. Some assignments may be inappropriate.

usually 200 to 350 plaque-forming units (pfu) per cell in Chlorella cells grown in the dark or those grown under cells grown under light. Chlorella viruses replicate most light and treated with the photosynthetic inhibitor efficiently in actively growing host cells and poorly in dichlorophenyldimethyl urea (DCMU) prior to viral in- stationary-phase cells. Although they also replicate in fection, the burst size is reduced by 50% in Chlorella

CHLORELLA VIRAL ENZYMES 355

FIG. 1. Schematic representation of the replication cycle of Chlorella virus based on electron-microscopic observations. Virus particles attach to the surface of the host Chlorella cells, digest the cell wall at the point of attachment, and inject the viral core into the host cytoplasm leaving an empty capsid on the cell surface. Viral DNA accumulation and assembly of the viral capsid occur solely in the host cytoplasm. Mature, DNA-filled virions appear by 4 h pi. and host cells burst, releasing the virus progeny into the culture medium 6-8 h p.i. Enzyme activities involved in the host cell wall digestion are not yet well characterized.

cells grown in the dark (19). From a one-liter culture of host cells containing lo8 cells/ml, 3.5 X 1013 pfu of virus particles can be produced; this amount corresponds to about 2 x lo3 AX0 units and 100 mg of particles. The viral particles are easily precipitated from the Chlorellu cell lysate by centrifugation at 15,OOOrpm (Fig. 2). Purified viruses can be stored at 4°C for many years without any significant loss of infectivity.

FRACTIONATION OF VIRAL STRUCTURAL PROTEINS

Chforella viruses are large icosahedral particles 1% 200 nm in diameter. The physical properties of the viral particles as characterized for PBCV-1 are also shared by most of the other viral isolates, except for slight varia- tions. The molecular weight estimated for the PBCV-1 virion is approximately 1 x log (19, 20). PBCV-1 sedi- ments in sucrose density gradients at about 2300s (8). The virion contains about 64% protein, 21 to 25% dsDNA, and 5 to 10% lipid (19, 21). Electron microgra- phs of negatively stained viral particles indicate that the viral particles consist of electron-dense cores surrounded by multilaminate shells (Fig. 2). There is a lipid layer within the outer glycoprotein capsid (21).

Viral structural proteins can be separated by SDS-poly- acrylamide gel electrophoresis (PAGE) into more than 50 species ranging in apparent size from 10 to more than 200 kDa. Although the protein separation patterns vary among the viruses (3), the typical pattern described for PBCV-1 is as follows (2): The major capsid protein of 54 kDa (Vp54) that is glycosylated makes up about 40% of the total viral protein. It behaves as a dimer under some electrophoretic conditions. In addition to Vp54, the proteins Vp14.5, Vp71 and Vp135 (also a glycoprotein) are also exposed to the virus surface (21). Four PBCV-1 proteins (Vp27.5, Vp54, Vp55 and Vp135) are thought

FIG. 2. Electron micrograph of negatively stained Chlorella virus CVK2 particles. The virus particles were obtained by centrifugation after lysis of the host Chlorelia (NC64A) cells. Bar marker represents 200 nm.

to be myristylated, and six proteins (Vp14, Vp20, Vp29, Vp36, Vp45 and Vp60) are probably phosphoproteins. Except for these data, very little is known about the loca- tion, properties, structure and function of the individual protein constituents of Chlorellu viral particles. Recent- ly, Yamada et al. (22) have established a method to frac- tionate capsid proteins from the viral core of CVK2 (a Chlorella virus isolated in Kyoto) by treatment with 4 M urea. With this treatment, at least seven different proteins with molecular masses of 16.2, 20, 20.5, 25, 41, 45 and 52 kDa were released reproducibly from the viral particles into the soluble fraction (Fig. 3). Com- puter-aided comparison of the N-terminal amino acid sequences of these proteins with those of ORFs identified in the PBCV-1 genome resulted in a one-to-one cor- respondence, except for Vp16.2, as follows: Vp20, Vp20.5, Vp25, Vp41, Vp45 and Vp52 correspoded to A168R, A523R, A203R, A625L, A430L and A430L of PBCV-1, respectively (23). Vp16.2 may be a species- specific protein. There is no information about the bio- logical functions of these proteins except for that on Vp52, Vp45, and Vp41, all of which are related to the major capsid protein Vp54 of PBCV-1 or its homologs. Interestingly, some of the CVK2 capsid proteins were processed at their N-terminal regions by two different proteolytic activities (23).

ENZYME ACTIVITY DETECTED IN CHLORELLA VIRAL PARTICLES

Fractionation and separation of the viral proteins by gel electrophoresis made it possible to detect and assign various enzyme activities to individual protein bands using zymographic methods (24, 25). So far, several active enzymes have been detected in CVK2 viral particles, in- cluding chitosanase, chitinase, protein kinase, RNase and superoxide dismutase.

Chitinase and chitosanase When CVK2 proteins were separated into the capsid and core particle fractions as mentioned above (22, 23), and separated and assayed by SDS-PAGE with chitosan or chitin as the substrate in the gel matrix (24), several enzymatically active bands with molecular masses ranging from 35 to 70 kDa were

356 YAMADA ET AL. .1. BIOSCI. &OENti..

protease

detergent

urea

I centrifugation

DNA+proteins

FIG. 3. Fractionation of Chlorella virus CVK2 structure pro- teins. By treatment with 4 M urea, several capsid proteins are sepa- rated from the viral core (22). The major capsid protein Vp52 that is glycosylated constitutes about 40% of the total proteins (released into the soluble fraction by 4 M urea treatment). This protein behaves as a dimeric form under nondenaturing conditions. The glycosylated cap- sid protects the particle against attacks by detergents and proteases. Various enzymatic activities are found in the core fraction consisting of more than 50 different proteins.

detected in the core fraction. Of these, a 65kDa band exhibited the strongest chitosanase activity and a few bands in the 50- to 60-kDa range showed chitinase activi- ties.

Chitinase catalyzes the hydrolysis of chitin, a p-1,4- linked homopolymer of N-acetyl-D-glucosamine, while chitosanase acts on chitosan, partially or fully deacetylat- ed chitin, as the substrate. In recent years, chitin and chitosan themselves as well as the products derived from their hydrolysis have received much attention because of their many potential applications in biochemical, agri- cultural and environmental science fields (26, 27). Chitinases are ubiquitously found in a wide range of organisms including bacteria (28-30), fungi (31), insects (32), crustaceans (33), higher plants (34, 35) and some vertebrates. Chitosanases were also described in prokary- otes and fungi (36) and more recently in plants (37). However, enzymes of viral origin are rare, so that the nature and function of Chlorella viral chitinases and chitosanases are very interesting.

A few ORFs identified in the PBCV-1 genome, A181R, A182R and A260R, and A292L (16), show sig- nificant amino acid sequence homology with chitinases and chitosanases of various organisms. The chitinase gene (vchti-I) encoded by the Chlorella virus CVK2 that corresponds to ORFs A181R and A182R of PBCV-1 (16) has been cloned and characterized (38). The vChti-1 ORF consisted of 2508 bp corresponding to 836 aa residues. The predicted amino acid sequence most closely resembles chitinases from bacteria and fungi, especially

those of E. coli (33% amino acid identity), Saccharo- polyspora (Streptomyces) erythrae (29% identity), and Aeromonas sp. (29% identity). According to the clas- sification based on amino acid sequence similarities (39, 40), these chitinases belong to the family 18 of gly- cosyl hydrolases, especially to the bacterial subfamily- C (41). A peculiar structural feature of the vChti-1 chitinase is a duplication of possible catalytic domains consisting of conserved regions 1 and 2. Chitinases with two catalytic sites have also been reported for the hyper- thermophilic archeon Pyrococcus kodakaraensis KODl (42) and for a plant pathogenic bacterium Flexibacter sp. FL824A (43). In CVK2 vChti-1 chitinase, these domains are connected by repeat sequences of amino acids rich in Pro (Fig. 4). The vChti-I gene was expressed in virus- infected Chlorella cells late in infection, and its product, a 94-kDa protein, showed strong chitinase activity and remained in the cell lysate. A total of 16.4 units of chitinase activity was obtained from a 1-I culture of Chlo- rella cells.

As for chitosanase, the vChta-I gene of CVK2 that corresponds to A292L of PBCV-1 (16) encodes 328 aa with a predicted molecular mass of 36.8 kDa; the gene has been cloned and characterized (44). The predicted amino acid sequence of an N-portion (174 aa) of this gene product showed 22 to 25% identity with those of various bacterial chitosanases. The vChta-l gene was expressed in CVKZinfected cells late in infection. Wes- tern blot analysis with specific antisera raised against the vChta-1 protein identified two proteins, 37 and 65 kDa, in virus-infected cells: The larger protein, which was most likely produced by read-through into a downstream gene, was packaged in the virion, while the smaller pro- tein remained in the cell lysate. Therefore, the C’-exten- sion of the 65-kDa protein may function as a virion-tar- geting signal. This signal can be used to package a pro- tein of interest into the virion. Both large and small pro- teins showed strong chitosanase activity in zymographic assays. The specific chitosanase activity of the 65-kDa enzyme in the CVK2 virion was 10 U/mg protein, and a total of 2500 units of the enzyme activity was recovered from a 1-l Chlorella culture.

The larger 65-kDa chitosanase assembled into the virion presumably functions at the beginning of the infec- tion, while the smaller 37-kDa chitosanase as well as the vChti-1 chitinase remain in the host cytoplasm where they most likely aid in the digestion of the host cell wall prior to the release of the viruses. Since the structure and chemical composition of the Chlorella cell wall are very complicated (44, 45), the enzymological characteris- tics of these polysaccharide-degrading enzymes of Chlo- rella viruses are of great interest.

Protein kinases In protein kinase renaturation as- says, three CVK2 proteins, Vp37 (37 kDa), Vp60 (60 kDa) and Vp73 (73 kDa), that are all located in the viral core fraction, showed both protein autophosphoryla- tion and casein kinase activity (23). Several ORFs in the PBCV-1 genome showed apparent protein kinase motifs, for example, A248R, A277L, A278L, A289L and A561L (Table 1). Although Vp73 most likely corres- ponds to A561L of PBCV-1, the assignment of Vp37 and Vp60 requires additional experimental data. A 35-kDa protein corresponding to A248R exhibited protein kinase activity; however, it was not packaged into the virion (46).

The CVK2 protein kinase activity was more markedly

VOL. 88, 1999 CHLORELLA VIRAL ENZYMES 357

N’ Region 1 Region 2 Pro-rich PVDPK

region repeat Region 1 Region 2

FIG. 4. Schematic representation of the molecular arrangement of CVK2 vChti-1 chitinase protein. Regions 1 and 2 conserved in the catalytic domain of family 18 glycosyl hydrolases are indicated by hatched boxes. A proline-rich region and PVDPK-repeats are shown in the figure (39).

enhanced by Mn2+ (2-10mM) than Mg2+. Protein kinase activity enhanced by Mn2+ is also reported for African swine fever virus (47). These enzymes may have a role in some early processes of the replication cycle, including uncoating and transcriptional regulation. The properties and functions of these protein kinases remain to be characterized.

RNases Two CVK2 core proteins, Vp16 (16 kDa) and Vp45 (45 kDa), showed strong RNase activity when assayed by zymography for nucleic acid-modifying en- zymes (26). The RNase activity of Vp16 was stable over a wide range of pH, while that of Vp45 became weak under alkaline conditions. Some ORFs identified in the PBCV-1 genome showed significant amino acid sequence homology with various RNases; for example, A464R resembles RNase III of various organisms (12). For development as reagents in biotechnology, the activities of CVK2 should be characterized in greater detail.

Cu/Zn-superoxide dismutase (SOD) Zymwwhy also revealed that a CVK2 core protein band of 34 kDa possesses superoxide dismutase activity. Western blot analysis with bovine anti-Cu/Zn-SOD antibody showed that this protein is a dimer of a 20-kDa subunit (Vp20). The monomeric form did not show any SOD activity on the gel. This protein is equivalent to the ORF A245R of PBCV-1 that exhibits 50% or more amino acid identity with Cu/Zn-SOD of a variety of aerobic organisms in- cluding bovine (48), yeast (49) and rice (50). The A245R gene encodes a protein of 187 amino acids, which is about 30 amino acids larger than the usual Cu/Zn-SOD of other organisms (51); initiation of A245R translation at an internal ATG site 22 codons downstream from the first ATG codon would create a protein similar in size to other Cu/Zn-SODS. The resulting size is in good agree- ment with the observed CVK2 band on a zymogram. It would be of interest to elucidate whether this enzyme activity is involved in defence against host-generated reac- tive oxygen species.

As described above, Chlorella viral particles consist of more than 50 different proteins. Each of them has been assigned to individual ORFs identified in the PBCV-1 genome. Some proteins localized in the viral particles may prove to have important and useful applications (52).

ENZYMES EXPRESSED IN CHLORELLA VIRUS-INFECTED CELLS

Many Chlorella viral gene products that are not incor- porated into the virion exert actual enzymatic activities in virus-infected host cells during infection. Such pro- teins characterized to date include several very interest- ing enzymes as follows: site-specific endonucleases (re- striction enzymes) and their cognate DNA methyltrans- ferases (modification enzymes), DNA ligase, cis-syn pyrimidine dimer-specific glycosylase, DNA polymerase B, mRNA capping enzyme, hyaluronan synthase, and aspartate transcarbamylase.

DNA methyltransferase and DNA site-specific endo-

nuclease Chlorella viruses encode various kinds of restriction and modification enzymes. In the case of PBCV-1, the divergent ORFs A581R and A579L encode the M.CviAI N6-methyladenine (6 mA) DNA methyl- transferase (53) and its cognate R.CviAI DNA site- specific (restriction) endonuclease (54), respectively. The M.CviAI DNA methyltransferase (methylates GmATC) and R.CviAI restriction endonuclease (cleaves/GATC) are the first modification and restriction enzymes disco- vered in the Chlorella viruses. Following this, M.CviAII (methylates %JATG) and R.CvMII (cleaves C/ATG) were found in PBCV-l-infected cells (55). The cor- responding genes are A251R and A252R in the PBCV-1 genome, respectively (16). To date, various sequence- specific DNA methyltransferases and their cognate restric- tion endonucleases have been characterized in different Chlorella viruses as listed in Table 2. Remarkably, Chlo- rella virus NY-2A encodes at least 12 DNA endo- nucleases and methyltransferase genes (56). R.CviJI encoded by Chlorella virus IL-3A (57) is unique in that its activity can be modulated to recognize either a two- or three-base sequence. In the presence of ATP, R.CviJI* cleaves RGCN and YGCY sites, but not YGCR sites. Some of these enzymes are now commercial- ly available. Since there are a large number of Chlorella viruses in natural environments, some of them may con- tain novel enzymes with interesting properties.

Both DNA methyltransferase and its cognate DNA restriction endonuclease activity first appeared in virus- infected host cells between 30 and 60min p.i., coinciding with the onset of host DNA degradation (53, 54). The site-specific endonucleases might help degrade host DNA so that the resulting deoxynucleotides are recycled into viral DNA. Methylation of newly synthesized viral DNA by the cognate methyltransferase would protect it from self-digestion (53, 54).

It is also noteworthy that there is an ORF (A422R) in the PBCV-1 genome that shows significant amino acid sequence homology with group I-intron homing endo- nuclease. Group I introns occur frequently in Chlorella virus genes (58, 59), and may function in the horizontal transmission of these introns.

DNA ligase Chlorella viruses also encode a DNA ligase gene (A544R in PBCVl) that is expressed in virus- infected host cells early in infection. DNA ligase charac- terized from PBCV-1 (60) is the smallest member of the covalent nucleotidyl transferase super family, which includes the ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes. The enzyme catalyzes efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP. PBCV-1 ligase was unable to ligate across a 2-nucleotide gap and ligat- ed poorly across a 1-nucleotide gap. The enzyme seals nicked duplex DNA substrates consisting of a 5’-phos- phate-terminated strand and a 3’-hydroxyl-terminated strand annealed to a template strand, but cannot ligate a nicked duplex composed of two DNAs annealed on an RNA template (61). Although PBCV-1 DNA ligase

358 YAMADAETAL. J. BIOSCI. BIOENG.,

Virus

PBCV-1

TABLE 2. DNA restriction and modification enzymes from virus-infected Chlorella cells

DNA methyltransferase Recognition sequencea DNA restriction endonuclease Recognition sequenceb Reference

M. CviAI G*ATC CviAI /GATC 53, 54 - M.CviAII *CATG CviAII C/ATG 55

NC-IA M.CviBI G*ANTC CviBII G/ANTC 87;88 M.CviBII G*ATC M.CvrBIII TCG*A

NY-M M.CviQI GT*AC CviQI G/TAC 56 M.CviQII R*AR M.CviQIII TCG*A M.CviQIV G*ATC M.CviQV TGC*A M.CviQVI G*ANTC M.CviQVII C*ATG M.CviQVIII RG*C(T/C/G) M. CviQIX *cc M.CviQX *CGR M.CviJI RG*C(T/C/G) 57 M.CviSI TGC*A 89 M.CviSII C*ATG M.CviSIII TCG*A M.CviSIV G*ATC M.CviSV RC*CG M.CvBVI RG*C(T/C/G)

XZ-6E M.CvrRI TGC*A CviRI TGKA 90, 91 CvrRII G/TAC

IL-3A SC-IA

a Asterisk indicates a methylated base. b Slash indicates a cleavage site.

efficiently joins a 3’-OH RNA to a S-phosphate DNA, it cannot join a 3’-OH DNA to a Y-phosphate RNA. The enzyme tolerates mismatches involving 5’-A:G and 5’- G:A mispairs, which reduce ligase activity by two orders of magnitude. Chlorella virus DNA ligase has the poten- tial to affect genomic integrity by embedding ribonucleo- tides in viral DNA and by sealing nicked molecules with mispaired ends, thereby generating missense mutations (6%

Wendonuclease V-homolog: the cis-syn pyrimidine dimer-specific glycosylase The ORF A50L of PBCV-1 encodes a homolog of endonuclease V of bacteriophage T4 (63), which is a cis-syn pyrimidine dimer-specific glycosylase (PDG) involved in pyrimidine photodimer ex- cision (64). Chlorella virus PDG (cv-PDG) is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer (65). DNAs containing both types of pyrimidine dimers are cleaved by the en- zyme with similar catalytic efficiencies. This novel en- zyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endo- nuclease V-like enzymes specific toward other DNA photoproducts.

DNA polymerase B A PBCV-1 ORF, A185R, en- codes the DNA polymerase of the eukaryotic family B. The amino acid sequence of its nucleotide polymerizing domain (motifs II-VI-III-I-V) is most similar to that of human alpha DNA polymerase, yeast DNA polymerase I, and eukaryotic viral DNA polymerases, but the sequence of its exonuclease motifs (exo I, exo II, and ExoIII) most closely resembles that of E. co/i DNA poly- merase I (66, 67). The A185R gene is expressed in the virus-infected cells early in infection; the mRNA was first detected 15 min after infection, which is consistent with the timing of initiation of PBCV-1 DNA synthesis (l-2 h p.i. (17)). The genomic DNA of Chlorella viruses is a large linear dsDNA molecule with hairpin ends (68,

69). DNA replication of this uniquely structured molec- ule requires some special enzymatic activities (70-72). Therefore, the reaction mode of this DNA polymerase is of great interest. In this context, it is also noteworthy that two ORFs, A478L and A583L of PBCV-1, show some sequence homology with topoisomerase I and II, respectively, of various organisms (Table l), which may also be involved in DNA replication.

mRNA capping enzyme The ORF A103R of PBCV-1 encodes an mRNA capping enzyme that cata- lyzes the transfer of GMP to the 5’-diphosphate end of RNA (73). With respect to its size (3%kDa monomer), amino acid sequence and biochemical properties (74), the A103R product is more closely related to the yeast RNA-guanylyltransferases than it is to the multifunc- tional capping enzymes coded for by other large DNA viruses, poxviruses and African swine fever virus. It is conceivable that this similarity of A103R to cellular guanylyltransferases is dictated by a unique virus-host dynamic, whereby capping of Chlorella viral mRNAs entails the interaction of a virus-encoded component with triphosphatase and methyltransferases encoded by the host.

In poxviruses and African swine fever virus, mRNA capping enzyme is encapsidated with polyA-polymerase (75) as well as RNA polymerase (76), which are intro- duced into host cells at the time of infection. Whether this enzyme is incorporated into the CVK2 virion is not yet known.

Hyaluronan synthase An ORF (A98R) of PBCV-I encodes active hyaluronan synthase (HAS) (77, 78). Hyaluronan (hyaluronic acid) is a simple linear polysac- charide chain composed of alternating /31 ,Cglucuronic acid (GlcA) and p 1,3-N-acetylglucosamine (GlcNAc) moieties, and is a ubiquitous constituent of the extracel- lular matrix, particularly of soft connective tissues in vertebrates (79). This polysaccharide influences the growth

VOL. 88, 1999

and migration of cells in the processes of embryonic development (80), oocyte maturation (81), angiogenesis, wound healing (82), and tumor progression (83). HAS adds sugar residues from UDP-GlcA and UDP-GlcNAc. PBCV-1 also encodes two other enzymes required for hyaluronan synthesis (84), namely, glutamine: fructose- 6-phosphate amidotransferase (GFAT, ORF AlOOR) and UDP-glucose dehydrogenase (UDP-GlcDH, ORF A609L), which produce the sugar precursors glucosamine-6- phosphate and UDP-GlcA, respectively. The hyaluronan polysaccharide begins to accumulate as hair-like fibers on the outside of the host Chlorellu cell wall by 1%30min p.i. (78). The biological functions for the PBCV-l-encoded hyaluronan are not yet known.

Aspartate transcarbamylase (ATCase) An ORF, A169R, of PBCV-1 encodes functional ATCase that cata- lyzes the committed step in the de nova biosynthetic pathway of pyrimidines. The amino acid sequence in- cludes the ATCase catalytic motif and is highly homolo- gous to a variety of plant enzymes. The viral enzyme is expressed early and transiently in infection (85), which is consistent with its function as an enzyme responsible for the synthesis of pyrimidine precursors for DNA syn- thesis. This is the first report of a functionally active ATCase encoded by a viral genome.

The above-mentioned Chlorella viral enzymes re- present only a few examples. As seen in Table 1, there are many possible enzymes encoded by the Chlorella viral genome, some of which may show novel and in- teresting properties that could be of interest to research- ers in both basic and applied fields of science.

FUTURE PROSPECTS As many as 702 ORFs found in the PBCV-1 genome

contain genes for a variety of functions, some of which seem irrelevant to viral replication. However, these genes should be considered carefully if they consistently occur in the genome regardless of their viral origin. In fact, a high degree of colinearity of gene arrangement was re- vealed between PBCV-1 and CVK2 (86). All such genes can be tested for their expression by Northern blot analy- sis with total RNA extracted from virus-infected cells at various times p.i. As hybridization probes, an appropri- ate part of the gene can be amplified by PCR with oligonucleotide primers synthesized based on the nucleo- tide sequence available for PBCV-1.

If Chlorella viral proteins of interest are packaged into the virion, they can be recovered easily from the viral particles and collected by centrifugation after cell lysis. To date, the identification of individual structural pro- teins in the Chlorella viral particle remains unsatisfac- tory. However, most of the proteins can be efficiently sepa- rated on SDS-PAGE gels, so that it may not be difficult to determine the N-terminal amino acid sequence for individual proteins. Based on the N-terminal amino acid sequence, the corresponding gene can be identified in the PBCV-1 genome. Some viral structural proteins may be replaced with useful foreign proteins using recombinant DNA technology. In this context, the ORF245 product that is fused to the C-terminus of the CVK2 chitosanase protein and functions as a virion-targeting signal as described above (44) is of great interest. This protein may be used as an optional Chlorella virus virion-target- ing signal for various foreign proteins. From the applica- tion point of view, a particle like the Chlorella virus

CHLORELLA VIRAL ENZYMES 359

virion, in which multienzyme complexes are systematical- ly organized, could serve as good material for the con- struction of an efficient bioreactor system.

ACKNOWLEDGMENT

The work in the laboratory of T. Yamada was supported by the New Energy and Industrial Technology Development Organization (NEDO)/Research Institute of Innovation Technology for the Earth (RITE). T. Y. was also supported in part by a grant from the Technical Research Center, Chugoku Electric Co., Inc.

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