Mol Gen Genet (1993) 241:554-563

© Springer-Verlag 1993

Characterization of the terminal inverted and their neighboring tandem repeats in the Chlorella CVK1 virus genome

repeats

Takashi Yamada, Takanobu Higashiyama

Faculty of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima 724, Japan

Received: 16 March 1993/Accepted: 28 May 1993

Abstract. A unique group of large icosahedral viruses that infect a unicellular green alga (Chlorella sp. NC64A) were isolated from freshwater sources in Japan. These viruses contain a linear double-stranded DNA (dsDNA) genome with hairpin ends. A physical map was con- structed for the genomic DNA of CVK1 (Chlorella virus isolated in Kyoto, no. 1) by pulsed-field gel electrophore- sis of restriction fragments. The nucleotide sequences around both termini of the CVK1 DNA revealed the presence of inverted terminal repeats (ITR) of approxi- mately 1.0 kb. Adjacent to the ITR, unique sequence ele- ments of 10 to 20 bp were directly repeated 20 to 30 times in tandem array. Several copies of these repeat elements were deleted in virus mutants that were occasionally gen- erated from Chlorella cells that were in a putative CVK1 carrier state. These repeats might represent a hot spot of rearrangement in the CVK1 genome.

Key words: Chlorella virus - Linear dsDNA genome - Hairpin terminus - Terminal inverted and tandem re- peats

Introduction

We have recently isolated and characterized large (130-200 nm diameter), icosahedral, plaque-forming vi- ruses from freshwater sources in Japan that replicate in the unicellular eukaryotic green alga Chlorella strain NC64A (Yamada et al. 1991, 1993). Noteworthy features of these viruses include a bacteriophage-like infection process, a large linear double-stranded DNA (dsDNA) genome, methylated nucleotides in the genome and many protein components in the viral capsid (Yamada et al. 1993). These features are similar to those reported for PBCV-1, which is a virus isolated from an endosymbiotic

Communicated by M. Sekiguchi

Correspondence to: T. Yamada EMBL/GenBank/DDBJ accession nos. D14469 and D14470

Chlorella-like alga in Paramecium bursaria (Van Etten et al. 1982b, 1991). The DNA genome of PBCV-1 was first characterized as a large circular molecule based on its restriction site map (Girton and Van Etten 1987). It is now described as a linear, nonpermutated 333 kb dsDNA molecule with covalently closed hairpin ends (Rohozinski et al. 1989). The termini of this genome are identical inverted repeats of at least 2185 bp and contain two potential open reading frames (Strasser et al. 1991). The genomic DNA of our Japanese viruses is also a linear double-stranded molecule but it varies in size from 330 to 380 kb depending on the viral species (Yamada et al. 1993). One of the Japanese viruses, CVK1 (Chlorella virus isolated in Kyoto, no. 1; a small plaque-forming type) has a 350 kb linear dsDNA genome (Yamada et al. 1991). When Chlorella strain NC64A cells were infected with CVK 1, a few resistant algal colonies appeared on a confluently lysed plate. The resistant Chlorella cells occa- sionally produced irregularly shaped virus plaques (Yamada et al. 1991).

In this report, we characterize the terminal hairpin structure and inverted repeat sequences of the CVK1 genome. Surprisingly, some 10-20 bp sequence elements are repeated many times in tandem array flanking both inverted terminal repeats (ITRs). Several sets of the re- peats are deleted in some viral mutants.

The Chlorella virus system described in this paper allows for the rapid and large-scale production of virus. Viral infection can be synchronized so that viral DNA replication and gene expression can be effectively studied in vivo as well as in vitro.

Materials and methods

Culture conditions and isolation of viral DNAs. The growth of the host (Chlorella sp. NC64A) on MBBM medium and the production and purification of CVK1 and the other viruses were as described previously (Yamada et al. 1991 ; Van Etten et al. 1982a). Viral DNA for structural analyses was isolated from purified CVK1

particles by heating at 60 ° C for 15 min in 0.01 M TRIS- HC1 buffer, pH 7.4 containing 0.1 M NaC1, 1 mM ED- TA, and 0.1% Sarkosyl (Fluka AG). This was followed by the addition of Hoechst 33258 dye (1 gg/ml) and the sample was centrifuged at 60 000 g for 15 h on preformed 30% to 60% CsC1 gradients, equilibrated with 0.01 M TRIS-HC1, pH 7.4. The single fluorescent DNA band was isolated and the dye was removed by three extrac- tions with isopropanol (Sarkosyl-treated DNA). In some cases, the DNA preparation was treated with proteinase K(1 mg/ml, Nippon Gene Co.) in 0.01 M TRIS-HC1 buffer, pH 7.4 containing 0.1 M NaC1, 1 mM EDTA at 50°C for 1 h (proteinase-treated DNA). For com- parison, DNA was directly extracted from the purified virus particles by phenol extraction without treatment with proteinase K and Sarkosyl (phenol-treated DNA). Viral DNA for restriction analyses and cloning was prepared as described previously (Yamada et al. 1991).

DNA manipulation and Southern blot hybridization. Di- gestion of DNA with restriction enzymes, agarose gel electrophoresis, and Southern blot hybridization were carried out according to Sambrook et al. (1989). Probes for hybridization of Southern blots were obtained by restriction enzyme digestion of the CVK1 DNA and its subclones. Labeling of probes and immunodetection were carried out with a kit from Boehringer Mannheim. Restriction enzymes were purchased from Toyobo except for Sse8387I (Takara Shuzo Co.).

Pulsed-field gel electrophores&. The purified virus parti- cles were embedded in 0.7% low melting point agarose (InCert agarose, FMC) prepared in 0.125 M EDTA, pH 7.5. After solidification, the gel was incubated over- night at 50 ° C with a lysis solution containing proteinase K (1 mg/ml), 0.01 M TRIS-HC1, pH 8.0 and 1% Sar- kosyl. DNA gel blocks were cut to fit into the wells of an agarose gel [1% agarose in 0.5 x TBE (45 mM TRIS, 45 mM boric acid, 1 mM EDTA, pH 8.3)]. Contour- clamped homogeneous electric field (CHEF) gel electro- phoresis (Higashiyama and Yamada 1991) was carried out at 13 ° C with a switching interval of 30 s at 6.6 V/cm for 24 h in 0.5 x TBE.

For digestion with restriction enzymes, the sample DNA in an agarose block was pretreated with phenyl- methylsulfonyl fluoride (PMSF) and washed with TE buffer according to Smith et al. (1988). After digestion with restriction enzymes, the DNA fragments were separated by CHEF electrophoresis as above and analyzed to construct the physical maps.

Digestion of viral DNA with exonuclease III, E-exonu- clease, and Bal31 nuclease. Viral DNA (Sarkosyl-treated DNA) was treated with exonuclease III (Nippon Gene), ~-exonuclease (BRL), or Bal31 nuclease (Takara Shuzo) according to Sambrook et al. (1989). The reaction mix- ture for exonuclease III contained 50 mM TRIS-HC1, pH 8,0, 5 mM MgCI2, 10 mM 2-mercaptoethanol, and exonuclease III (2.5 U). For ~ exonuclease digestion, the reaction mixture contained 67 mM glycine-KOH, pH 9.4, 2.5 mM MgC12, 50 gg/ml bovine serum albumin,

555

and ~ exonuclease (2.5 U). Bal31 digestion was carried out in a reaction mixture containing 20 mM TRIS-HC1, pH 8.0, 0.6 M NaC1, 12 mM CaCI2, 12 mM MgCI2, 1 mM EDTA, and Bal31 nuclease (2.5 U). The reaction was halted by heating at 70 ° C for 5 rain. After extraction with phenol, DNA was digested with EcoRI and subject- ed to agarose gel electrophoresis. As an internal control, pUC18 DNA (0.5 pg) linearized with HincII was in- cluded in the reaction mixture.

Alkaline agarose gel electrophoresis. The 4.7 and 2.1 kb EcoRI fragments of CVK1 DNA (Sarkosyl-treated DNA) were cut from the agarose gel, eluted by electro- phoresis, and concentrated by ethanol precipitation. The fragments (0.5 ~tg) were subjected to alkaline agarose gel electrophoresis according to Sambrook et al. (1989).

Sequencing of DNA fragments. Restriction fragments of both terminal regions of the CVK1 DNA were cloned into M13 mpl8 and mp19, respectively. Single-stranded DNA was sequenced by the chain termination procedure with a kit (Auto Read sequencing kit, Pharmacia) using an Automated Laser Fluorescent (A.L.F.) DNA se- quencer (Pharmacia). Sequences were compiled and analyzed using GENETYX software (SDC Software De- velopment Co.) on an NEC PC9801 DA computer.

Results

Mapping of the CVK1 DNA genome

DNAs isolated from all Chlorella viruses isolated in Ja- pan give single bands, 330-380 kb in size, on CHEF gel electrophoresis (Yamada et al. 1991, 1993). The DNA of CVK 1 showed a size of 350 kb in the middle of this range

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Fig. 1. Contour-clamped homogeneous electric field (CHEF) gel electrophoresis of the Chlorella virus CVK1 DNA digested with restriction enzymes. Lane 1, ladder of )~ DNA; Lanes 2-5, CVK1 DNA digested with (2) NotI; (3) SfiI; (4) NotI and SfiI; (5) no enzyme. Stars 1 and 2 indicate products of incomplete digestion with NotI of 350 kb (310 + 40 kb) and 120 kbp (80 + 40 kb), respec- tively

556

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Fig. 2. Restriction map of CVK1 DNA. In the upper part, size is given in kilobase pairs (kbp). SseI, Sse8387I. EeoRI frag- ments from both termini are enlarged in the lower part. Arrow indicates the invert- ed terminal repeat. Smallfilled squares in- dicate the portion deleted in CVKIR2 and CVK1R3 DNA

A B C D

1 2 3 4 I 2 3 4 5 1 2 3 4 5 1 2 3 4

Fig. 3A-D. Electrophoretic analysis of the terminal structure of CVKI DNA. A EcoRI digestion of DNAs prepared from the virus particles by treatment with 0.1% Sarkosyl (lane 2), with proteinase K (1 mg/ml) at 37 ° C for 1 h (lane 3) and by extraction with phenol (lane 4). Lane 1 contains )~ DNA digested with HindIII. B CVK1 DNA (20 gg, Sarkosyl-treated DNA) was treated with exonuclease III by the standard method (Sambrook et al. 1989) for 0 min (lane 2), 2 min (lane 3), 4 min (lane 4) and 6 rain (lane 5). The reaction was stopped by heating at 70 ° C for 5 rain. After extraction with phenol, the DNA was digested with EcoRI and subjected to agarose gel electrophoresis. As an internal control, pUC18 DNA (0.5 ~g)

linearized with HincII was included in the reaction mixture. The arrow indicates the pUC18 DNA. C CVK1 DNA (20 ~tg, Sarkosyl- treated DNA) was treated with )~-exonuclease (Sambrook et al. 1989) for 0 min (lane 2), 2 rain (lane 3), 4 min (lane 4) and 6 rain (lane 5) before digestion with EcoRI as above. The arrow indicates the pUC18 DNA (0.5 gg) internal control. D CVK1 DNA (20 gg, Sarkosyl-treated) was treated with Bal31 exonuclease for 0 min (lane 2), 10 min (lane 3) and 20 rain (lane 4) before EcoRI digesiton as above. The internal control is the same as in B. Arrowheads indicate the bands that disappeared afte Bal31 treatment. The terminal bands are indicated by white dots

(Fig. l, lane 5). W h e n CVK1 D N A was digested with NotI , two fragments , o f 310 and 40 kb, appeared on the gel (Fig. 1, lane 2). Similarly, with SfiI, three bands (220, 120 and 9.5 kb) and with NotI + SfiI, four bands (220, 80, 40 and 9.5 kb) were separated by C H E F gel electro- phoresis (Fig. 1, lanes 3 and 4). F r o m these results, a physical m a p could be cons t ruc ted for the C V K 1 D N A , and this is shown in Fig. 2 (upper part). The sites for NaeI, which gave five f ragments o f 105, 80, 65, 55 and 45 kb, and Sse8387I, which gave three f ragments o f 170, 100 and 80 kb were also located on the m a p by analyzing

C H E F gel electrophoret ic pat terns o f f ragments p roduced by partial digestion and /o r digestion in com- binat ion with NotI and SfiI (Fig. 2, upper part).

Terminal hairpin structure o f the viral D N A

Various specific terminal structures such as cohesive ends, direct or inverted repeats, hairpins and terminal proteins are generally required for the replication o f linear d s D N A molecules (Kornberg 1980). Terminal pro-

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Fig. 4. Alkaline agarose gel electrophoresis of the terminal EeoRI fragments of CVK1 DNA. Alkaline denaturation and treatment of DNA with T4 DNA ligase and are schematically explained in the lower part. Lane 1, ~ DNA digested with HindlII; 2 and 4, 2.1 kb EcoRI fragment derived from the right terminus of CVK1 DNA; 3 and 5, 4.7 kb EcoRI fragment from the left terminus; 6, 2.1 kb EcoRI fragment after treatment with T4 DNA ligase and EcoRI; 5, 4.7 kb EcoRI fragment after T4 DNA ligase and EcoRI treat- ments. After electrophoretic separation, DNA bands were blotted onto a nylon filter and hybridized with digoxygenin-dUTP-labeled 2.1 kb EcoRI fragment (lanes 4-7). Triangles indicate the single- stranded DNA bands of 2.1 kb (lane 4), 4.7 kb and 9.4 kb (lane 5), 4.2 kb (lane 6) and 9.4 kb (lane 7)

teins are absent from the CVK1 genome. This was ev- ident from the experiments shown in Fig. 3A, where D N A was isolated from the virus particles by three dif- ferent methods prior to comparison of EcoRI digests: (i) Sarkosyl treatment, (ii) proteinase treatment and (iii) phenol extraction. If proteins are covalently attached to both termini, two bands derived from those regions by restriction enzyme digestion should migrate differently depending on whether or not D N A was subjected to prior proteinase treatment. As a positive control, plas- mid pSLA2 from Streptomyees rocei (Hirochika and Sakaguchi 1982) was examined under our conditions. This plasmid is a linear dsDNA molecule of 17 kb in size. Its 5' end is covalently bound to a terminal protein, as are the D N A genomes of adenovirus (Rekosh et al. 1977)

Fig. 5A, B. Comparison of' mutant viral genomes. A DNAs isolated from mutant viruses that occasionally produced plaques on lawns of the lysis-resistant algal strains K1R2 (CVK1R2) (lanes 3 and 6) and K1R3 (CVK1R3) (laned 4 and 7) were digested with BamHI (lanes 3 and 4) and EcoRI (lanes 6 and 7) and compared with BamHI-digested (lane 2) and EcoRI-digested (lane 5) CVK1 DNA. Lane 1 contains % DNA digested with HindlII. B The same gel as in A was blotted onto a nylon filter and hybridized with the 2.1 kb EcoRI fragment of CVK1 DNA. (Fig. 2). Arrowheads indicate the altered bands

and bacteriophage ~29 (Salas et al. 1978). Without prior proteinase treatment, pSLA2 DNA did not enter the aqueous phase after phenol extraction. Moreover, an ap- proximately 400 bp BglII fragment derived from both termini of the pSLA2 DNA could not enter the agarose gel under ordinary conditions of electrophoresis (see also, Hirochika and Sakaguchi 1982). For the CVK1 DNA, no change was observed in the banding patterns; the 4.7 and 2.1 kb fragments, which are derived from the termini as shown below (Fig. 3A), were not affected by prior protease treatment. The terminal regions of intact viral DNA are, however, structurally protected from di- gestion with exonuclease III, which is 3' end specific and )~-exonuclease, which is 5' end specific. As shown in Fig. 3B and C, both enzymes rapidly digested the control, linearized pUC18 DNA, whereas the CVK1 D N A (not- ably the 4.7 and 2.1 kb EcoRI fragments) were not signifi- cantly affected. Nevertheless, Bal31, another exonuclease that degrades single strands and hairpin structures, could attack the CVK1 D N A (Fig. 3D). Two bands of 4.7 kb and 2.1 kb were preferentially lost from EcoRI digests of CVK1 D N A that had first been treated with Bal31 for 20 rain (arrowheads in lane 4). These results imply that the two bands are derived from the terminal regions and have some structural modification such as a hairpin.

The 4.7 and 2.1 kb fragments were cut from the aga- rose gel and subjected to alkaline agarose gel electro-

558

A 10 20 30 40 50 60 70 80 90 i00 TGCATCATAA CCATAATAAG CTGAGTGATG CAGTGGCATG GTTCCAGTAT CATCA.ATGAC GTCAAGGTTT GCACCGCTTC GACGAGCAGC TTCACACATG

110 120 130 140 150 160 170 180 190 200 CATCGTGGCC ATTAAAAACC GCACGATGAA GAfiGTGTGCA TCCCGAATTA TCTTTGATGT CAATATTTGC ACCCGCATCA ATGAGCATCT TTACGCATGC

210 220 230 240 250 260 270 280 290 300 ATCATAACCA TAATGAATCA CGTAATGCAA CGCGGTCCAC CTTGAACCAC CAACGATGTT CAGGTCGGCA CCTGCTTCAA TGAGCGTTTT CAAACAAACG

310 320 330 340 350 360 370 380 390 400 CCATGGCCAT TTTTTGCTGC AGCGATTATA CTTTTGCAAT TGTGAACCAT TGTATTGTAT TGTGTTTGAA TTGTGTTTGT ATTGTGTTTG TATTGTGTTT

410 420 430 440 450 460 470 480 490 500 GT~TTGTGTT. TGT~TTGTGT TTGT~TTGTG. TTTGTATTGT~ GTTTGTATTG• TGTTTG~TT GTGTTTGT~T TGTGTTTGT~ .TTGTGTTTGT .~TTGTGTTTG

510 520 530 540 550 560 570 580 590 600 T_~TTGTGTTTGT~TTGTGTT TGT~TIGTGT TTGT~TTGTG TTTGTOTTGT GTTTGT~TTG TGTTTGT~TT GTGTTTGT~T TGTGTTTGT~ TTGTGTTTGT

610 620 630 640 650 680 670 680 [ i 9 0 700 /

STGGTGTTTG T~TGGCGTTT GT~.TGGOGTT TOT~TGGCGT TTO~TGGTT TTATAG~ATT TTGT~ATTTG T~ATTTGT~A TTTGT~,TTT GT;ATTTGT~

710 720 730 740 750 760 770 780 790 800 CCTGGTGAAA TGACGACTGG AAATGACAAA AAAGAGTATA AAAGGCGGCC ACACGCCTCA GAGGTATCAA ATATTCCAAA GCAAACTTTC AAACTTTCAA

810 820 830 840 850 860 870 880 890 900 AGTTTTTAAA AGCTTTCAAA GCATGAGTCA TTACGAGTAC GACATGCGTC GCATTGAGTA CACCCGCGAG ATGCGCCGCC GCGAGTACAA CCGCGAGATG

910 920 930 940 950 960 970 980 990 1000 GCTCGCCGCA ACGCCATGGC TCAGGAGCGC GAGATGGCAT TCCGCGCCGA GATGCAGCGC CGCCAACACG CGGAGCAGGA GCACCGCAGC GCATGGCCAT

I010 1020 1030 1040 1050 1060 1070 1080 1090 1100 GCATCATCAC AACCAGCAGA TGCATCAGCG TTACGGACCT CCCCGCGACG CTGTTGATGA TGGCTGCGGG TGCTGCATCA TGTGAATGTG AATGTGAATG

1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 rGTGTAATTC TTTCTTGAAC CTTTCGTTGA CGCACAAGGC GCTAACGAAA ATATTTTTAA AAATGTTCAA AAATGTTTGT CATTTGACCC CGGTGGAATT

1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 }ACGACTGGA AATGACAAAT GACATTCTCC CGAAATGCAT AAAAGGCGTG TGTGTGTGGG TGAGTGTATC AAATTATTAT TAACAATGAT GTCTGCTATC

1310 1320 ~AGAACCTCT TCGA Fig. 6A, B. Nucleotide sequences of both terminal regions of CVK1 DNA. A A 1314 base sequence from the EcoT22I-TaqI region near the left end (Fig. 2). B A 1482 base sequence from the EcoRI-TaqI region near the right end (Fig. 2). A part of the inverted terminal repeat is boxed. Arrows indicate tandem repetitive elements. Dotted

line in B shows an extended repetition of a sequence around the ITR junction. A 75 base sequence (bracket 1) and a 45 bp sequence (bracket 2) in B is deleted from the corresponding region of the CVK1R2 and CVK1R3 DNAs, respectively

phoresis by the standard method (Sambrook et al. 1989). The 2.1 kb EcoRI fragment separated into two single- stranded bands of around 2.1 kb (Fig. 4, lanes 2 and 4). The 4.7 kb fragment also separated into two bands: one band of 4.7 kb (lane 3) and an additional band of twice this length (lane 5). The results differed when the same fragments were treated overnight with T4 DNA ligase at 16 ° C and then digested with EcoRI; after ligation, the 2.1 kb fragment yielded a 4.2 kb band and the 4.7 kb fragment an approximately 9.4 kb band (Fig. 4, lanes 6 and 7). These results can be interpreted to mean that both termini exist in the form of hairpins with single-strand nicks or gaps. In addition, strong homology was revealed between both terminal fragments because the 2.1 kb fragment hybridized with the 4.7 kb fragment.

Appearance of resistant clones

In the usual infection cycle, the viruses lysed the host cells rapidly and completely, both in liquid cultures and on plates. This was true with CVK2, which produced large plaques (6-10 mm diameter), whereas CVK1, which forms smaller plaques (2-3 mm diameter), took twice as long to lyse the host cells in liquid cultures. The genomes of CVK1 and CVK2 were stable during normal lytic cycles; their restriction fragmentation patterns did not change after many lytic cycles (Yamada et al. 1991). However, a few resistant colonies appeared on the lysed lawn of CVKl-infected cultures, at a low frequency. Three of these resistant algal clones, K1R1, K1R2, and K1R3 were also resistant to other viruses from different sources. K1R1 grew as vigorously as the wild type

B I0 20 30 40 50 60 70 80 90 100

GAATTCAGTA ATTTGTTTTA TAGTGAAATT CCTGCTAGTT CCAGTGATTG TCTTGATTAT CCCGACGAAC TTATGTACGT AGAAACTCAA CCCGAAATGT

11o 12o 180 140 150 160 170 180 190 200 TTTTGTGTTC TCCAGAAGAT CCTACTGACC CTAACACTCCTAC~GACCCT AACACTCCTA C~GACCCTAA CACTCCTAC~ GACCCTAACA CTCCTAC~G~

210 220 230 240 250 260 270 280 290 300 CCCTAACACT CCTACTGACC CTAACACTCC TACTGACCCT AACACTCCTA CTGACCCTAA CACTCCTACT GACCCT~CA CTCCTACTGA CCCTAACACT . . . . ~ ~ . ~ ~ -

310 320 380 340 350 360 370 380 390 400 CCTACTGACC CTAACACTCC TACTGACCCT AACACTCCTA CTGACCCTAA CACTCCTACT CCTAGCACTC CTACTCC~G CACTCCTACT CCTAGCACTC

410 420 Z 430 440 450 | 460 470 480 490 500 CT6CTCC!AG CACTCCTACT CC~GCACTC AC~GCACTC , , , , CACTCCTACT CTACTACTAG CACTCCTACT AC'FAGCACAT CT~TGACAG CTACTACZAG

510 520 530 540 550 560 570 580 590 600 TTCATCATCT AACTTGCCTA TTATTATTGG CGCTAGTGTG GGTGGTGTGG TTCTAATTTG TATCTGCATT GGTGTATTTG TATGCATT~ GAAGAGGAAC

610 620 630 640 650 660 670 680 690 700 AAAACTAATT ATGACCCATC TCTTGATACA TTTGGCCAGA CAGTCGTTGT TGACfiAGCAG CAATTTATCA CGGCACffl'TC TGTACCAACC GGTGCCATTC

710 720 OCGCTCAAGC TATTTCTGGT

810 820 GAGGTGTTGA CGCAAAAGGC

910 920 AGAGTATAAA AGGCGGCCAC

730 740 750 760 770 780 790 800 AATGACCACA CTATTGTGAA TGTTATGTAA GCTGTCTGTG CCTAGTTACC AGTCTTGTGA GTGTTGTAAG TGTCCGAGGC

840 850 / : 860 870 880 890 900 830 GCTAACACTQ AATTT__TJ~TAA_ _AAA_LGT!nJL~ItLG!_CATrr_GJpQq JQG.LCAAALa_Aq~C!_qa_~_A!QAq._~

1

930 940 950 960 970 980 990 1000 ACQCCTCAaA aOTATC~AT Arrcc~Gc ~cT~rCAA ACr~CAAA~ r~ r~A~Q c r ~ c ~ G c ATQAGTCA~

i010 1020 i030 1040 1050 1060 1070 I080 1090 Ii00 ACGAGTACGA CATGCGTCGC ATTGAGTACA CCCGCGAGAT GCGCCGCCGC GAGTACK~CC GCGAGATGGC TCGCCGCAAC GCCATGGCTC AGGAGCGCGA

I I I0 1120 GATGGCATTC CGCGCCGAGA

1210 1220 ACGGACCTCC CCGCGACGCT

1310 1320 CACAAGGCGC TAACGAAAAT

1410 1420 AAATGCATAA AAGGCGTGTG

1130 1140 1150 1160 1170 1180 1190 1200 TGCAGCGCCG CCAACACGCG GAGCAGGAGC ACCGCAGCGC ATGGCCATGC ATCATCACAA CCAGCAGATG CATCAGCO'FI

1230 1240 1250 1260 1270 1280 1290 1300 GTTGATGATG GCTGCGGGTG CTGCATCATG TGAATGTGM TGTGAATGTG TGTAATTCTT TCTTfiAACCT TTCGTTGACG

138o 184o 135o 186o 187o 188o 189o 14oo A_TT_T_T! __~ " _A_T_G _rrct~a_ _ }LG _TrT_G_TCA_ ~T2A_CCC _CG - .G!G_G@_F#_A 9_AC.T_G.G.W__TG_A_C__Ua_TGA CATTCTCCCG

1430 1440 1450 1460 1470 1480 1490 TGTGTGGGTG AGTGTATCAA ATTATI'ATTA ACAATGATGT CTGCTATCAA GAACCTCTTC GA

559

NC64A, while the K1R2 and K1R3 cultures showed some signs of infection: the liquid cultures were less turbid, the cells were more difficult to spin down and more sensitive to cellulase digestion, and the growth rate decreased several fold. Nuclear DNA isolated from these cells hybridized with the CVK1 DNA probe (data not shown). When the cells were plated and incubated for a prolonged period (2 weeks), irregularly shaped, indistinct plaques of various sizes appeared with both K1R2 and K1R3. Single plaques were picked and purified from K1R2 and K1R3 lawns and designated CVK1R2 (ChlorelIa virus from K1R2) and CVK1R3 (Chlorella virus from K1R3), respectively. Virus particles were isolated from liquid NC64A cultures infected with CVK1R2 and CVK1R3 and their genomes were analyzed by restriction enzyme digestion and Southern hybridization. Figure 5A shows electrophoretic separa- tion of BamH! and EcoRI fragments of CVK1R2 and

CVK1R3 DNAs compared with the original CVK1 DNA. Only one band was changed in both mutants: a 4.4 kb band in the BamHI digests and a 2.1 kb band in the EcoRI digests. As shown in Fig. 5B, Southern blot hybridization using the 2.1 kb EcoRI fragment of CVK1 DNA as probe (Fig. 2) revealed that these bands hy- bridized strongly and corresponded to one terminal re- gion of the genome. However, the bands derived from the other terminus (a large BamHl and the 4.7 kb EcoRI band), which hybridized with the probe, were not changed in the viral mutants.

DNA sequence of the terminal regions

The EcoRI fragments derived from both termini of CVK1, CVK1R2, and CVK1R3 DNAs were isolated from agarose gels. By digestion with several restriction

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Fig. 7A, B. Matrix comparison analy- ses of the terminal regions of CVK1 DNA. By using the GENETYX pro- gram, direct repeats were looked for in the left terminal (A) and the right terminal (B) regions. Dot condition: more than 15 matches between 20 nucleotide sequences

561

enzymes, physical maps for these fragments were con- structed and are shown in Fig. 2. The 4.7 kb EcoRI fragments from CVK1, CVK1R2, and CVK1R3 DNAs produced exactly the same restriction patterns with all enzymes tested, while a 560 bp EcolO5I-HincII subfrag- ment of the 2.1 kb EcoRI fragment from CVK1 DNA was shortened to 490 bp in the CVK1R2 and CVK1R3 DNAs, indicating that a deletion of approximately 70 bp had occurred within this portion of the mutant viral DNAs. All restriction fragments were cloned into M13mp18 and mpl9, except for the distal most 400 bp TaqI fragment containing the hairpin loop. Attempts to clone this fragment, including Bal31 digestion, fill-in with Klenow enzyme and random digestion with restriction enzymes, were unsuccessful. This terminal fragment may have unusual base-pairing or secondary structure or may be toxic to Escherichia coli.

Figure 6 shows the nucleotide sequences of both ter- mini of the CVK1, CVK1R2, and CVK1R3 DNAs. As expected, the 1314 base sequences of the left termini from the 4.7 kb EcoRI fragments were exactly the same in the CVK1, CVK1R2, and CVK1R3 DNAs. A 628 base sequence in the 3' region of these sequences was homolo- gous to the corresponding region of the CVK1 DNA right terminus (2.1 kb EcoRI fragment), indicating that it was a part of the ITR. The inverted repeat extended to the 400 bp TaqI fragment, as shown by the fact that fragments from both termini hybridize to each other and have the same restriction patterns (data not shown). Therefore, the size of the CVK1 ITR was estimated to be at most 1.0 kb, which is less than half that of the ITR in the PBCV-1 genome (2185 bp, Strasser et al. 1991).

Flanking the left ITR, there are 30 repeats of related sequence elements (Fig. 6A, positions 360-686; Fig. 7A, Table 1). The ITR ends with two copies of the sequence, 5'-ATTTGTC-3', at its 5' end. The core sequence, 5'- TTGTGTTTGTA-3' , is GT rich and rather similar to the Chlorella telomeric sequence, 5'-TTTAGG- GTTTAGGG-3' (Higashiyama and Yamada, in preparation). Interestingly, an extended tandem array made up of several types of repeat elements is also

Table 1. Direct repetitive sequences in the CVK1 termini

Sequence No. of times Location present

Left terminus TTGTGTTTGAA 1 359-370 TTGTGTTTGTA 21 371-601 TGGTGTTTGTA 1 602-612 TGGCGTTTGTA 3 613-645 TGGTTTTATAGT 1 646-657 ATTTTCTC 1 658-665 ATTTGTC 5 666-700

Right terminus GACCCTAACACTCCTACT 12 127-342 GACCCTAACACTCCTACTCCT 1 343-363 AGCACTCCTACTCCT 4 364-423 AGCACTCCTACTACT 4 424-483 AGCACATCTAATGAC 1 484498

present next to the right ITR (Fig. 6B, positions 127-498; Fig. 7B). The repetitive elements are listed in Table 1. It was found that five copies of a 15 base element were deleted from the corresponding region of the CVK1R2 DNA (Fig. 6B, positions 379-453). This change was consistent with the shorter size of the EcolO5I-HincII fragment from the CVK1R2 DNA. A similar deletion was found in the other mutant virus, CVK1R3: three copies of the same element were deleted from the same region of the CVK1R3 DNA (Fig. 6B, positions 379-423).

Discussion

The genome of CVK1 was characterized as a large (350 kb), linear dsDNA with hairpin termini. Such termi- nal hairpin structures have been found in poxvirus DNA (Geshelin and Berns 1974; Baroudy et al. 1982), the short single-stranded DNA genomes of parvoviruses (Straus et al. 1976; Tattersall and Ward 1976), Paramecium mitochondrial DNA (Prichard and Cummings 1981), yeast mitochondrial DNAs (Dinoual et al. 1993), ex- trachromosomal Tetrahymena rDNA (Blackburn and Gall 1978), yeast chromosomal DNAs (Forte and Fang- man 1979), plasmids of the spirochete Borrelia burgdor- feri (Barbour and Garon 1987), and plasmids of the plant pathogenic fungus Rhizoctonia solani (Miyashita et al. 1990). Hairpins and terminal homologous regions are thought to play key roles in the replication of linear DNAs and some models for replication have been proposed (Baroudy et al. 1982; McFadden 1988; Merch- linski 1990).

Our results from alkaline agarose gel electrophoresis showed that the terminal hairpins of CVK 1 DNA con- tain nicks or gaps (Fig. 4). This contrasts with the case of PBCV-1 where Rohozinski et al. (1989) identified covalently closed hairpin termini in the genomic DNA, based on inability to label the ends and sensitivity of the termini to Bal31 exonuclease. They also described rapid renaturation after denaturing the terminal fragments and electrophoresis of the terminal fragments on an agarose gel, but the data were not shown. Although the nicks or gaps we detected may have been artifacts, we consider it possible that a single-strand nick exists at a precise point within the inverted repeat adjacent to the CVK1 DNA loop. This is because two single-stranded fragments of different sizes were derived from one terminal region (Fig. 4, lane 2), although some fraction of the other terminus contained a closed hairpin loop (Fig. 4, lane 5). Similar hairpin termini with nicks have recently been reported for the linear mitochondrial DNAs of the yeasts Pichia pijperi and P. jadinii (Dinou~l et al. 1993). The ITRs of CVK1 were estimated to be about 1.0 kb in size and are thus less than half the size reported for that of PBCV-1 (Strasser et al. 1991). The biological function of the CVK1 ITR is not known. However, as we have reported elsewhere, the ITR sequence seems to be highly conserved among the viruses isolated in Japan: 10 ran- dom samples of 29 viral species isolated from different places in Japan always showed two restriction fragments

562

that hybridized strongly with a probe containing the terminal repeat (Yamada et al. 1993). This degree of conservation may suggest that the ITR plays an impor- tant role in the viral infection cycle, perhaps during virus D N A replication and/or packaging of D N A into the capsid. However, the ITR sequence of CVK1 D N A has no significant similarities with the terminal sequences of the PBCV-1 (Strasser et al. 1991) or vaccinia virus genome (Wittek and Moss 1980).

A survey of the EMBL/GenBank /DDBJ databases showed no CVKl-l ike sequences, except for an ap- proximately 300 bp sequence in the Thermoproteus tenax virus 1 genome (EMBL accession no. X14855), which was 60 % homologous with the repetitive sequences of the CVK1 right terminus (Fig. 6, positions 226488) . The biological significance of this region is not known. Com- puter analyses failed to find any extended open reading frames in either terminal region, except for those asso- ciated with the tandemly repeated sequences. Northern hybridization experiments with total R N A prepared from host cells at various stages of CVK1 infection showed no expression signals from either terminal region.

Another interesting feature of the CVK1 genome found in this work is the presence of many direct tandem repeats at both terminal regions flanking the ITR. Such repeats are thought of as a common characteristic of the terminal repetitive region of vaccinia virus (Wittek and Moss 1980) and ASV (Sogo et al. 1984). In the case of CVK1, however, the sequence, size, and frequency of repeats are different between both termini, although the total size of the repetitive region is almost the same, about 350 bp. In the left terminal region, the ITR ends with two copies of the repetitive sequence 5 ' -ATTTGTC- ATTTGTC-3 ' (Fig. 6, positions 687-700), whereas a sequence of 65 bp around the right ITR junction, includ- ing a 21 bp divergent sequence and a 44 bp ITR sequence (Fig. 6, positions 834-898), is repeated within the ITR (positions 1322-1387). These complicated structures are reminiscent of the repeat mediated rearrangements pos- tulated to explain the expansion/contraction of the large inverted repeats of Chlorella chloroplast D N A (Yamada 1991). Although it is not known whether expansion/ contraction has been operative in structuring the CVK1 ITR, several copies of a repetitive element were, in fact, deleted from the right terminal region of the viral mutant CVK1R2 and CVK1R3 DNAs. This region of tandem repeats is contained within an approximately 600 bp EcoRI-HincII fragment in the CVK1 genome (Fig. 2). We found by Southern hybridization that the corre- sponding EcoRI-HincII fragment is somewhat larger (ca. 700 bp) in another virus isolate from Kyoto (CVK2, a large plaque forming type) (data not shown). Therefore, this terminal part of the Chlorella virus genome appears to be a hot spot for genomic rearrangements. A similar deletion/reiteration of a set of tandem repeats was report- ed for the ITR ofvaccinia viral D N A (Moss et al. 1981), but larger scale. I n that case, no major differences in plaque sizes, particle to plaque forming-unit ratios or burst sizes were detected between standard and variant viruses. This is also the case for CVK1, CVK1R2, and CVK 1 R3.

These interesting structures and rearrangements of the Chlorella virus genome may be fundamentally related to special mechanisms of hairpin D N A replication.

Acknowledoments. The authors thank H. Kinashi for providing them with the plasmid pSLA2, T. Miyakawa, E. Tsuchiya and D. Hirata for valuable discussions and R. Yamada for preparing the manuscript. This investigation was supported in part by the Technical Research Center, Chugoku Electric Power Co.

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