Structural characterization of the adenovirus 18 inverted terminal repetition

VIROLOGY

121,

230-239

(1982)

Structural

Characterization

of the Adenovirus

Inverted

Repetition’

CLAUDE F. GARON, IGOR RONINSON,*

Terminal

18

RONALD P. PARR, RADHA PADMANABHAN,* JAMES W. GARRISON, AND JAMES A. ROSE’

Laboratory of Biology of Viruses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 2020.5, and *Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201 Received April 29, 1982; accepted May 24, 1982 Adenovirus (Ad) 18 DNA from two plaque isolates (P-l and P-2) derived from the prototype strain has been analyzed by cleavage with restriction endonucleases and by electron microscopic heteroduplexing techniques. Fragment sizes were determined by contour length measurements, sucrose sedimentation, and agarose gel electrophoresis. A physical ordering of fragments was obtained by comparative digestion of isolated fragments and heteroduplex mapping. End fragments were identified by their susceptibility to exonuclease digestion and by the presence of covalently attached terminal proteins. It was found that P-2 DNA molecules were about 4% longer than P-l molecules, and that the observed length difference was due to a longer inverted terminal repetition (ITR) in P-2 DNA. Both the P-l and P-2 ITR genotypes remained unchanged after five additional virus passages. These findings indicate that infectious Ad18 genomes can carry ITRs of different length. Based on direct nucleotide sequence analysis, the P-l ITR is 165 bases long and possesses extensive homology with the ITR of Ad12. In addition, the P1 ITR contains several short base tracts that are variably present in the ITRs of more distantly related serotypes. One of these tracts (5’. TGACGT) is also found near the ends of DNA from both autonomous and Ad-dependent (AAV) parvoviruses.

al., 1979; Shinagawa and Padmanabhan, 1980; Schwarz et al., 1982). For the major infectious components of encapsidated DNA, the lengths of these repeats have been found to vary from 103 bases (Ad2) to 162 or 164 bases (Ad12). In the case of Ad18, however, it was noted previously that DNA molecules recovered from the main band of CsCl-purified virions possessed occasional ITR variability, with some repetitions approaching 20% of genome length (Garon et ah, 1975). Furthermore, it was observed that increases in ITR length were accompanied by equivalent inward deletions of adjoining sequences, so that total molecular length remained nearly constant. In the present study, we compared the genomes of several plaque isolates derived from the Ad18 prototype strain to determine whether infectious genomes might contain ITRs of different length. Two iso-

INTRODUCTION

DNA molecules extracted from adenovirus (Ad) particles are linear duplexes that contain an inverted terminal repetition (ITR) (Garon et al., 1972; Wolfson and Dressler, 1972). The molecular arrangement of the repetition was initially determined by direct visualization of “panhandle” projections on circularized singlestranded Ad18 DNA (Garon et ah, 1975). More recently, ITRs in the genomes of Ad types 2, 3, 5, 7, and 12 have been analyzed by nucleotide sequencing (Steenbergh et al., 1977; Shinagawa and Padmanabhan, 1979; Arrand and Roberts, 1979; Tolun et ’ The U. S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright carrying this paper, for governmental purposes, is acknowledged. 2To whom requests for reprints should be addressed. 0042-6822/82/120230-10$02.00/O Copyright AI1 rights

0 1982 by Academic Press, Inc. of reproduction in any form reeerved.

230

TERMINAL

REPETITION

lates (P-l and P-Z) were found to have markedly different ITR lengths, and further analysis showed that (i) the approximately eightfold longer P-2 ITR had been generated without any detectable deletion of adjoining internal sequences and (ii) these ITR genotypes remained essentially unchanged after repeated passage. In addition, the base sequence of the P-l ITR was determined and found to possess extensive homology with that of AdI2. MATERIALS

AND

METHODS

Viruses and viral DNA extraction. The prototype strain of Ad18 was originally obtained from Flow Laboratories (Garon et ah, 1975), and a HeLa cell line was provided by J. Williams. An Ad18 stock pool was initially prepared by passage in primary human embryonic kidney (HEK) cells (HEM Research). To obtain genomic isolates, virus was plaqued on HeLa cell monolayers (~2 X lo6 cells; Williams, 1970) at dilutions which yielded no more than two plaques per 60 X 15-mm dish (Falcon). These conditions were used to maximize the probability that plaques would be generated by single particle hits. Plaqued virus was then grown and purified following three to eight subsequent cell passages (five passages in HeLa cells and three passages in KB cells) and viral DNA extracted from C&l-purified virions Sharp et ah, 1976; Janik et al., 1981). DNA preparations were stored in a Tris-EDTA buffer (0.01 M Tris, pH 8.5, and 0.001 M EDTA) at 4°C. For restriction endonuclease analyses, 32Plabeled viral DNA was prepared as before (Rose et aL, 1966). Restm’ction enzyme incubations. Incubation mixtures contained l-5 pg of viral DNA, 0.02 M Tris, pH 7.5, 7 mM MgC12, 2 mM 2-mercaptoethanol, and an excess of BamHI (Miles Biochemicals) or l-5 pg of viral DNA, 0.1 MTris, pH 7.5,5 mMMgCl,, and an excess of EcoRI (Miles Biochemicals). Digestion was stopped by the addition of EDTA and samples then stored at 0°C. Agarose gel electrophoresis. Fifteen-centimeter cylindrical glass tubes containing 0.5-0.8% agarose gels were used for elec-

OF ADENOVIRUS

18

231

trophoresis. Both gel and electrophoresis buffer contained 0.04 M Tris-acetate, pH 8.05, 0.02 M sodium acetate, 2 mM EDTA, and 0.018 M NaCl. Gels were run at 40 V for 14-16 hr at 15°C. Following electrophoresis, gels were either stained for 30 min in a solution containing 0.01 M TrisHCl, pH 8.5, and 0.5 pg/ml of ethidium bromide or, when labeled DNA was used, sliced into l-mm disks and Cerenkov radioactivity determined. Appropriate DNA fragments were electroeluted at 100 V for 3 hr at 15°C and concentrated for subsequent analysis. DNA sequence analysis. The 3’ terminal nucleotide of Ad18 DNA was determined to be a dG residue using deoxynucleotidyl terminal transferase (Roychoudhury et al., 1976; Arrand and Roberts, 1979). This 3 terminal nucleotide was interchanged with a labeled dG residue using the 3, 5 exonucleolytic activity of T4 DNA polymerase in the presence of [cu-32P]dGTP (2000-3000 Ci/mmol, Amersham). The labeled DNA was then cleaved with EcoRI, fractionated on a 1.4% agarose gel, and the C and F fragments eluted from the gel using hydroxyapatite chromatography (Wu et ah, 1976). For sequence analysis, the chemical degradation reactions were carried out exactly as described by Maxam and Gilbert (1977). The reaction products were fractionated on 12 and 20% polyacrylamide slab gels containing 50 mM Tris-borate, pH 8.3, 1 mM EDTA, and 7 M urea at 600-1200 V for 8 to 72 hr. The same Tris-borate buffer without urea was used as reservoir buffer. After electrophoresis, the gels were exposed to X-ray film with an intensifying screen (DuPont Cronex, Hi-speed) at -20°C for 3 to 5 days. Electron microscopy. The denaturation and renaturation of DNA and mounting procedures for microscopy, using either the aqueous or the formamide techniques, were carried out as described by Davis et ah (1971). Grids were examined in a Siemens Elmiskop 101 electron microscope at 40 kV accelerating voltage. Electron micrographs were taken on Kodak electron image plates at magnifications of 40006000. The magnification was calibrated for each set of plates with a grating replica

232

GARON

(E. F. Fullam, No. lOOO), and contour lengths were measured with a Numonics graphic calculator interfaced to a Wang 2200 computer. RESULTS

Selection of Ad18 Plaque Isolates Five plaques were initially isolated from the parent strain under conditions expected to generate plaques derived from single particle hits (see Methods). After three passages in HeLa cells, virus was purified and DNA extracted and screened in the electron microscope to assess ITR length. Two isolates, designated P-l and P-2, were selected for further study because of greatest apparent difference in the lengths of their repeats. In the case of P-l (as well as the remaining isolates) the ITR was not visable, whereas the putative ITR of P-2 molecules was seen as an obvious projection equivalent to about 4% of molecular length. These genotypic features were essentially unchanged after five additional passages in HeLa and KB cells. Restvictim

Endmuclease

Analyses

When P-l DNA was cleaved with either EcoRI or BarnHI, seven fragments were detected by agarose gel electrophoresis. Fragments ranged in size from 7.92 to 0.19 X lo6 daltons and were individually puriTABLE SIZES OF EcoRI DNA fragment A B C D E F Gb Total Intact

3.93 1.82 1.65 1.34 0.72 0.49

P-l

k + f f f f

0.10 0.05 0.07 0.05 0.04 0.04

9.95 9.9

a Sedimentation coefficients bFragment G was analyzed

7.70 3.57 3.23 2.63 1.41 0.96 19.50 19.4

fied from gels whose concentrations were adjusted for maximal separation. Sizes of these fragments are given in Tables 1 and 2. The various estimates of sizes of the larger fragments are in good agreement. However, owing to its small size (about 288 base pairs), we estimated the molecular weight of EcoRI G from its mobility alone. We also deduced the presence of two fragments of similar size (and hence identical mobilities) for BamHI (D1 and Dz, Table 2) based on a comparison of contour length measurements with the 32P distribution in uniformly labeled fragments. The locations of restriction fragments derived from P-l DNA were determined by partial digestion patterns, reciprocal restriction digests of isolated fragments, and heteroduplex mapping (Fig. 1). Identification of end fragments was also confirmed by a method which takes advantage of the fact that the covalently attached terminal protein (Robinson et al., 1973) prevents these fragments from migrating to their appropriate positions in an agarose gel. This is shown in Fig. 1 (gel 2) by the loss of the EcoRI C and F bands and the appearance of ethidium bromide staining material at the top of the gel. Further confirmation was obtained by demonstrating the susceptibility of the C and F fragments to digestion with exonuclease III (Garon et al., 1975; data not shown). Although P-2 DNA was found to be ap1

FRAGMENTS

Fraction length

Length bm)

ET AL.

39.3 18.2 16.5 13.4 7.2 4.9 100 100

OF P-l

DNA

=P distribution

Relative mobility

40.4 18.6 15.8 13.2 7.2 4.6

1.00 1.65 1.80 2.10 3.15 3.70 5.00

100 100

measured in neutral sucrose gradients. in a separate gel system due to its small

size.

(iz) 7.92 3.64 3.09 2.59 1.41 0.90 0.19 19.74 19.4

S” 19.1 16.1 15.4 14.0 11.9 11.3

28.1

TERMINAL

REPETITION

OF ADENOVIRUS

TABLE

DNA fragment A B c D, + D2 E F Total Intact P-l ’ Sedimentation

2

SIZES OF BumHI

FRAGMENTS

(Pm)

MW (X10”)

Fraction length

2.95 * 0.17 2.63 t 0.11 1.74 t 0.06 1.18 t 0.05 0.39 k 0.02 0.28 i 0.05 10.35 9.9

5.78 5.15 3.41 2.31 0.76 0.55 20.27 19.4

Length

coefficients

measured

OF P-l

DNA 32p distribution

MW (X10”)

29.0 25.0 16.7 22.6 3.9 2.8 99.9 100

5.68 4.90 3.27 4.43 0.76 0.55 19.59 19.4

29.5 26.3 17.4 11.8 3.9 2.8 103.50 100

in neutral

sucrose

proximately 4% longer than P-l DNA (10.3 t 0.3 vs 9.9 k 0.2 pm), the EcoRI and BamHI restriction endonuclease cleavage patterns of this DNA differed from those

233

18

D A

L b

E

--

0.2

1-

B

6 L-

~1

0.6

FRACTIONAL

FIG. 1. Restriction analysis of P-l DNA. phenol extraction procedure; the DNA in guanidinum-sucrose gradients. The failure positions in gel 2 is due to the presence representations of the EcoRI and BumHI

D, L

0.4

26.0

of P-l molecules only in the size of the fragments that mapped at one specific terminus (Figs, 2 and 3). The increase in the size of the EcoRI terminal fragment F is

A

F

17.6 17.5 15.8 14.0 10.1 8.9

gradients

G C

S”

C

E -~1

F

~

,_~

0.8

Barn

D2 L

Eco-RI HI

~~~

1.0

LENGTH

The DNA used in gel 1 was purified by a proteinase Km gel 2 was obtained from virus previously disrupted on of EcoRI fragments C and F to migrate to their specific of the covalently attached terminal protein. Schematic restriction maps are shown below.

GARON

234

16-

A

A

ET AL.

BCD

E

F

1412-

I

lo8-

8m 64-

, i

0

25

50

75

100

125

GEL SLICE NUMBER FIG. 2. Separation by gel electrophoresis of the EcoRI restriction endonuclease fragments of P1 (panel A) and P-2 (panel B) DNA. Digested 3ZP-labeled DNA was analyzed on 0.85% agarose gels. Gels were stained with ethidium (inset) and subsequently sliced, solubilized, and counted. Fragment G ran out of the gel under the conditions of electrophoresis.

readily shown from its altered migration rate but no related change in size of the larger terminal fragment C was apparent (Fig. 2). Similarly, gel analysis of BumHI cleaved DNA showed a decreased migration rate for only a single terminal fragment (Dz) which clearly separated it from internal fragment D1 (Fig. 3). Careful calibration of mobilities of DNA fragments and 32P distribution patterns in several experiments consistently indicated that

the increased length of P-2 DNA could be accounted for solely on the basis of an alteration in the size of a single terminal fragment. A concomitant alteration in the length of any internal fragment could not be detected.

Electron

Microscopy

The above findings were supported by further analysis of DNA molecules in the electron microscope. In contrast to the sin-

TERMINAL

AB

REPETITION

C

OF ADENOVIRUS

235

18

DID?

6

I .

0

8t

6

AB

C

T

E

D2 D1

F

32lI

0

25

50

75

100

1:25

GEL SLICE NUMBER FIG. 3. Separation by gel electrophoesis of the BamHI restriction 1 (panel A) and P-2 (panel B) DNA. “P-Labeled DNA was digested Fig. 2.

gle-stranded circular molecules produced with denatured and reannealed P-l DNA, nearly all single-stranded circular P-Z molecules (>95%) had a visible duplex projection equivalent to 3-476 of total genome length (Fig. 4A). Additional evidence that these projections resulted from annealing of the ITR with sequences at the opposite terminus was obtained by demonstrating that duplex projections of similar length also could be generated by appropriate denaturation and reannealing of

endonuelease and analyzed

fragments of Pas described in

the purified BamHI terminal fragments (A and Da) or the purified EcoRI terminal fragments (C and F) (Fig. 4B). Heteroduplex molecules formed between P-l and P-2 DNA showed no evidence of internal nonhomology or rearrangements. Especially frequent were relaxed, doublestranded circular structures characterized by the presence of two projections, one single-stranded and one duplex (Fig. 4C). The duplex projection presumably represents the annealed ends (i.e., the ITR) of

236

GARON

FIG. 4. Electron denaturation and be found on nearly fragments C and stranded circles

ET AL.

microscopic analysis of Ad18 DNA. Single-stranded circular P-2 molecules after reannealing at low concentrations (panel A). A single duplex projection could every (-95%) single-stranded circle (arrows). Hybridization of purified EcoRI F generated duplex segments similar in size to the duplex projections on single(arrows, panel B). When equal quantities of P-l and P-2 DNA were mixed,

TERMINAL

REPETITION

a P-Z strand, whereas the single-stranded projection represents an equal length of unpaired P-l terminal sequences. We could not detect the unpaired, short P-l ITR in these preparations. Consistent with the single-stranded character of one of the visible projections was the finding of only one projection when heteroduplexes were spread under aqueous conditions (Fig. 4D). The structure seen below the circular heteroduplex represents a collapsed singlestranded P-Z circle (bush) whose duplex ITR segment is readily visible and equivalent in length to the projection on the heteroduplex molecule. P-Z circular homoduplexes could be easily distinguished from circular heteroduplexes by the presence of two duplex projections. These molecules were often supercoiled structures with visible regions of strand separation, a consequence of winding constraints that arise when two single-stranded circles anneal (Fig. 4E; Garon et al., 1975). The hybridized P-2 ITRs are clearly seen as adjacent duplex projections.

Nucleotide

Sequence of the P-I ITR

Labeled P-l DNA was cleaved with E’coRI, and the terminal C and F fragments separated by agarose gel electrophoresis were eluted and subjected to the chemical degradation procedure as described under Methods. The terminal nucleotide sequences read off polyacrylamide gels are shown in Fig. 5, together with a comparison of sequences previously derived for the termini of Ad12 DNA (Shinagawa and Padmanabhan, 1980). The P-l ITR is 165 nucleotides long, and this sequence is identical at both termini. Nucleotide 166 introduces the unique region at each end of the P-l molecule, since significant base similarity was not seen in fragments C and F beyond that point. By contrast, Ad12, also one of the highly oncogenic Group A serotypes (Green, 1970),

OF ADENOVIRUS

18

237

has a 162-nucleotide-long ITR that differs from that of P-l at 10 specific nucleotides within the repetition. Thus, the ITRs of Ad12 and the Ad18 P-l isolate possess a high degree of homology. Homologies within or near the ITRs of more distantly related Group B (Ad2, Ad5) and Group C (Ad3, Ad7) serotypes are confined to only a few short base tracts (Fig. 5, underlined and boxed sequences; Steenbergh et al., 1977; Tolun et al.; 1979; Shinagawa and Padmanabhan, 1980). DISCUSSION

It was observed previously that in DNA extracted from virions, increases in Ad18 ITR length occurred at the expense of adjoining internal sequences (Garon et al., 1975). Clearly, such genomes would no longer be expected to be infectious. In the present study, however, we provide evidence that infectious Ad18 genomes can carry ITRs of different length. It is therefore not surprising that the longer ITR of P-2 DNA exists without any apparent loss of adjoining internal sequences. Because DNA molecules were obtained from CsClpurified main bands of virus particles in both this and the earlier study, their maximum length may well have been limited by packaging constraints. It follows, then, that such constraints would also limit the length of ITRs in genomes carried by infectious particles. Whether the P-2 ITR approaches the upper limit of permissible length is not known. Results reported by others indicate that infectious genomes of Ad16 (Hammarskjijld and Winberg, 1980) and Ad12 (Schwarz et al., 1982) also can possess more than one specific ITR length. Although this may be true, accumulated base sequence data suggest that for DNA found in the major virion component of each.serotype, a certain ITR length eventually predominates (Steenbergh et al., 1977;

denatured, and reannealed, circular heteroduplex molecules were formed (panels C and D) together with supercoiled homoduplex molecules (panel E). Duplex projections arising from the annealed inverted repeats of P-2 molecules can be seen in panels C (ds), D, and E (arrows). The DNA shown in all panels except D was mounted using the formamide technique. Panel D DNA was spread under aqueous conditions.

FIG. 5. Base sequence of the P-l terminal repetition. The ITR sequence found for Ad12 DNA is shown for comparison (Shinagawa and Padmanabhan, 1980). Astericks indicate base differences and a (+) denotes an additional A (base 63) in the P-l sequence. Base tracts variably found in the ITRs of other Ad serotypes are underlined, and the hexanucleotide found in the terminal sequences of both Ad and parvovirus DNA is enclosed in a box.

Shinagawa and Padmanabhan, 1979; Arrand and Roberts, 1979; Tolun et aL, 1979; Shinagawa and Padmanabhan, 1980; Schwarz et al., 1982). Predominance of a particular ITR length might relate to its frequency in the population of molecules eligible for encapsidation or to a selective advantage in packaging genomes of certain size or both. It is notable that closely related serotypes appear to carry ITRs of similar or nearly similar length (i.e., Ad2, Ad5: 103 bp; Ad3, Ad7: 136 bp; and Ad12, Ad18 (P-l): 162 or 164 and 165 bp, respectively). So far, the ITR base sequences of closely related Ad serotypes are highly homologous, whereas considerable heterology is observed when more distantly related serotypes are compared. Thus, among human Ad serotypes, at least, large portions of the ITR are not generally conserved. Only three short base tracts are found in common in the ITRs of Group A, B, and C serotypes (Fig. 5: bases g-22,34-41, and the distal hexanucleotide). ITRs of other serotypes not included in these groups may be more divergent. In this regard, the Ad4 ITR contains only the proximal tract (bases 9-22; R. Padmanabhan, unpublished results; J. Engler, personal communication) and those of Ads 9 and 10 contain only a portion of the proximal tract (bases 9-18; J. Engler, personal communication). The constant position and retention of this sequence in the ITRs of all serotypes thus far examined, including

simian Ad?‘, has suggested a possible role in initiation of DNA synthesis (Tolun et ah, 1979; Shinagawa and Padmanabhan, 1980). Another tract, a hexanucleotide (5’. . . TGACGT; Fig. 5, box), also can be found in the terminal DNA sequences of all Ad serotypes. For Ad3, Ad7, and Ad12, it terminates the ITR, whereas for Ad2, Ad5, and Ad18 (P-l), it lies two bases short of ITR termination. In the case of Ad4, the hexanucleotide does not occur within the ITR but lies four bases distal to the repeat and is present only at the right end of the genome (R. Padmanabhan, unpublished results). In Ads 9 and 10, it occurs within the repeat but not near its termination (J. Engler, personal communication). Thus, although this sequence is apparently conserved, it is not invariably located within the ITR, and it does not necessarily occupy a position close to the ITR termination site. It is intriguing that the 3’hairpin terminal sequences of four rodent parvovirus genomes (Astell et ah, 1979) also contain this hexanucleotide (bases 76-81) as does the left terminal DNA sequence (Lusby and Berns, 1982) of the Ad-dependent parvovirus, AAV2 (bases 161-166). REFERENCES ARRAND, J. R., and ROBERTS, R. J. (1979). The nucleotide sequences at the termini of Ad2 DNA. J. Mol. Biol. 128, 577-594. ASTELL, C. R., SMITH, M., CHOW, M. B., and WARD, D. C. (1979). Structure of the 3’ hairpin termini of

TERMINAL

REPETITION

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ROYCHOUDHURY,R., JAY, E., and Wu, R. (1976). The terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase. Nucleic Acids Res. 3, 863-877. SCHWARZ, E., REINKE, C., YAMAMOTO, N., and ZUR HAUSEN, H. (1982). Terminal rearrangements in the genome of adenovirus type 12 mutants adapted to growth in two human tumor cell lines. Virology 116, 284-296. SHARP, P. A., MOORE, C., and HAVERTY, J. L. (1976). The infectivity of adenovirus 5 DNA-protein complex. Virology 75, 442-456. SHINAGAWA, M., and PADMANABHAN, R. (1979). Nucleotide sequence at the inverted terminal repetitions of adenovirus type 2 DNA. B&hem. Biophys. Res. Cbmmun. 87, 671-678. SHINAGAWA, M., and PADMANABHAN, R. (1980). Comparative sequence analysis of the inverted terminal repetitions from different adenoviruses. Proc. Nat. Acad.

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TOLUN, A., ALESTROM, P., and PETTERSSON, U. (1979). Sequence of inverted terminal repetitions from different adenoviruses: Demonstration of conserved sequences and homology between SA7 termini and SV40 DNA. Cell 17, 705-713. WILLIAMS, J. F. (1970). Enhancement of adenovirus plaque formation on HeLa cells by magnesium chloride. J. Gen Virol. 9, 251-255. WOLFSON, J., and DRESSLER, D. (1972). Adenovirus2 DNA contains an inverted terminal repetition. Proc. Nat. Acad. Sci. USA 69, 3054-3057. Wu, R., JAY, E., and ROYCHOUDHURY,R. (1976). NUcleotide sequence analysis of DNA. Methods Cancer Res. 12, 88-176.