JOURNAL OF VIROLOGY, Aug. 1988, p. 2903-2915 Vol. 62, No.

8
0022-538X/88/082903-13$02.00/0
Copyright © 1988, American Society for Microbiology

Nucleotide Sequence and Genomic Organization of Aleutian Mink

Disease Parvovirus (ADV): Sequence Comparisons between a
Nonpathogenic and a Pathogenic Strain of ADV
MARSHALL E. BLOOM,* SOREN ALEXANDERSEN, SYLVIA PERRYMAN, DAVID LECHNER,
AND JAMES B. WOLFINBARGER

18 February 1988/Accepted 2 May 1988

A DNA sequence of 4,592 nucleotides (nt) was derived for the nonpathogenic ADV-G strain of Aleutian mink
disease parvovirus (ADV). The 3' (left) end of the virion strand contained a 117-nt palindrome that could
assume a Y-shaped configuration similar to, but less stable than, that of other parvoviruses. The sequence
obtained for the 5' end was incomplete and did not contain the 5' (right) hairpin structure but ended just after
a 25-nt A+T-rich direct repeat. Features of ADV genomic organization are (i) major left (622 amino acids) and
right (702 amino acids) open reading frames (ORFs) in different translational frames of the plus-sense strand,
(ii) two short mid-ORFs, (iii) eight potential promoter motifs (TATA boxes), including ones at 3 and 36 map
units, and (iv) six potential polyadenylation sites, including three clustered near the termination of the right
ORF. Although the overall homology to other parvoviruses is <50%, there are short conserved amino acid
regions in both major ORFs. However, two regions in the right ORF allegedly conserved among the
parvoviruses were not present in ADV. At the DNA level, ADV-G is 97.5% related to the pathogenic ADV-Utah
1. A total of 22 amino acid changes were found in the right ORF; changes were found in both hydrophilic and
hydrophobic regions and generally did not affect the theoretical hydropathy. However, there is a short
heterogeneous region at 64 to 65 map units in which 8 out of 11 residues have diverged; this hypervariable
segment may be analogous to short amino acid regions in other parvoviruses that determine host range and
pathogenicity. These findings suggested that this region may harbor sonme of the determinants responsible for
the differences in pathogenicity of ADV-G and ADV-Utah 1.

In recent years, the autonomous parvoviruses have be- termini. All the parvoviruses sequenced to date have two
come recognized as major etiological agents of a variety of major open reading frames (ORFs). A left ORF that is
animal and human diseases (6, 16, 31, 44, 69, 72). Most of the governed by a promoter at -4 map units (MU) (12, 23, 27,
parvoviruses cause an acute disease picture the pathogenesis 30, 42, 55, 57, 65) specifies at least one nonstructural protein
of which can be directly related to sites of viral replication, necessary for viral replication and gene regulation, and a
but the Aleutian mink disease parvovirus (ADV) is some- right ORF, the promoter for which lies at -38 to 40 MU,
what unique in that it causes both a severe acute pneumonia encodes the sequences for a set of overlapping structural or
in newborn kits (1, 2, 4, 5) as well as a chronic disorder of the capsid proteins (12, 18, 21, 27, 31, 42, 46, 49, 53, 55, 57, 64,
immune system in adult mink (3, 16, 20, 21, 38, 58, 60). 65, 68).
Although every ADV strain tested induces pneumonia in Sequence data has also provided interesting information
mink kits (1, 5), strains differ markedly in pathogenicity or on the interrelationships among the various parvoviruses.
virulence for adult mink (16, 20, 21, 38, 58-60). The virulent For example, although minute virus of mice (MVM) (9, 52,
ADV-Utah 1 strain (60) causes full-blown Aleutian disease in 66), bovine parvovirus (BPV) (29), B19 parvovirus (68), and
all genotypes of adult mink (16, 21, 22, 60), whereas the the dependovirus adeno-associated virus (AAV) (71) share
ADV-G strain, which was derived in cell culture from little overall homology, several short stretches of amino
ADV-Utah 1, has lost pathogenicity for mink (20). Conse- acids in both structural and nonstructural proteins seem to
quently, it has been recognized that structural differences at be conserved. These findings suggest an important role for
a genomic level may have an important role in determining these regions, although their function is not as yet clear.
the virulence of ADV. Previous work demonstrates differ- In this report, we describe the DNA sequence of the
ences between pathogenic and nonpathogenic strains of nonpathogenic ADV-G strain of ADV and the basic genomic
ADV (17, 18), but the relationship of these differences to organization that is evident. In common with the other
virulence is uncertain. autonomous parvoviruses, the ADV genome contains large
In other parvovirus models, elucidation of the viral DNA left and right ORFs, and there are promoterlike sequence
sequence has provided an important framework for analyz- motifs located at 3 and 36 MU. Furthermore, although the
ing how viral genes or their products might affect the overall levels of DNA and putative amino acid homology to
outcome of virus host interactions (31, 69, 70). The complete several representative members of the parvovirus family are
nucleotide sequence for several autonomous parvoviruses low, there are highly conserved regions in both the left and
has been determined and indicates several common features right ORFs. In addition, several features of the ADV genome
(9, 26, 29, 52, 62, 65, 66, 68, 71). The linear single-stranded seem to be significantly different from the other parvoviruses
genomes are all approximately 5,000 nucleotides (nt) in and these unusual features may be partly responsible for the
length and contain palindromic sequences at both 5' and 3' unique pathogenic properties of ADV. We also report partial
sequence data for the highly pathogenic ADV-Utah 1 strain,
and although the two strains of ADV show very high
*
Corresponding author. homology, we found an 11-amino-acid area in the ORF for
2903
2904 BLOOM ET AL. J. VIROL.

the capsid proteins that shows marked divergence between national Biotechnologies). The data obtained from these
the two strains. This region may be partly responsible for the templates provided the initial sequence information. At-
marked differences in pathogenicity seen between these two tempts to develop minus-sense single-stranded template
ADV strains. molecules by cloning this same DNA segment in M13mpl9
did not yield stable full-length templates.
MATERIALS AND METHODS In other experiments, ADV-G RF DNA was dC tailed by
using terminal deoxytransferase (48), and it was annealed to
Materials. All enzymes were bought from either Bethesda pEMBL8 that had been previously linearized with PstI and
Research Laboratories, Inc. (BRL), Gaithersburg, Md., dG tailed (a generous gift from Bruce Chesebro). The hybrid
New England BioLabs, Inc., Beverly, Mass., or Interna- molecules were transformed into strain JM109, and carbeni-
tional Biotechnologies, Inc., New Haven, Conn., and were cillin-resistant colonies were screened for the presence of
used according to the recommendations of the manufactur- ADV sequences by colony hybridization. The plasmid clone
ers. Vector DNAs were purchased from either BRL containing the largest ADV insert (pADVG IQ-6) produced
(M13mpl8 and M13mpl9) (74) or Promega-Biotec (pGEM3) deleted molecules on batch propagation in JM109 and was
(51), Madison, Wis. pEMBL8 DNA (34) was a gift from subsequently transferred into a recA derivative of E. coli
Francis Nano. 32P- and 35S-labeled deoxynucleotide triphos- MM294 [recA F- endAl hsdR17 (r- m+) supE44, thi-J Ql-;
phates were bought from Du Pont-New England Nuclear obtained from Francis Nano] (40). The generation of deleted
Corp., Boston, Mass. Unlabeled deoxy- and dideoxynucleo- plasmid molecules in this strain was greatly reduced, and
tide triphosphates were obtained from Pharmacia-P-L Bio- pADVG IQ-6 DNA prepared from these bacteria served as
chemicals, Piscataway, N.J. Synthetic nucleotide primers template for most of the sequences reported in this article.
were either purchased (BRL or Promega-Biotec) or were Physical mapping indicated that with the exception of the
prepared on an oligonucleotide synthesizer (SAM-1; Bio- right-hand end, this DNA was the same size as ADV-G RF
search, San Rafael, Calif.). These latter oligomers (generally DNA isolated by Hirt extraction (19). Subsequently, how-
20 bases in length) were separated from failure sequences ever, sequence comparisons with other clones indicated that
either by preparative electrophoresis in denaturing poly- 18 base pairs (bp) (nt 2469 to 2487) were missing from this
acrylamide gels or by high-performance liquid chromatogra- clone. Therefore, three other clones derived from the tailing
phy. experiments were prepared for analysis of sequences bor-
Viruses, cells, and viral DNA. The ADV-G strain (20) of dering this deletion, and all three contained the 18 bp which
ADV was propagated in Crandell feline kidney cells (CRFK) were included in the presented sequence. DNA from the
as previously described (4, 20) except that no antibiotics same three clones was used to confirm the 3' (left) terminal
were used in the growth medium. Duplex monomer replica- sequence.
tive-form (RF) DNA was isolated by a modified Hirt proce- Additional attempts to develop clones containing the
dure (19); however, the denaturation step for the enrichment extreme 5' (right) terminus were made by adding EcoRI
of covalently linked hairpin forms was omitted. Instead, the linkers (48) to ADV-G virion DNA that had been made
partially purified RF DNA was resolved on a low-melting- double stranded with the Klenow fragment of E. coli DNA
temperature agarose gel and isolated on NACS columns polymerase (17, 19, 48). After several cycles of EcoRI
(BRL). Alternatively, the RF DNA was purified by two digestion and microdialysis, the DNA was ligated into
cycles of agarose gel electrophoresis and electroelution, EcoRI-digested pGEM3 DNA and transformed into E. coli
using an Elutrap apparatus (Schleicher & Schuell, Inc., DB1256 (recA recB21 recC22 sbcBJ5 hsdR F- proA2 his4
Keene, N. H.). Single-stranded virion DNA was prepared as thi-l argE3 lacYl galK2 ara-14 xyl-S mtl-i str-31 tsx-33;
previously noted (17, 19, 20). obtained from Grant McFadden) (33). This strategy yielded
Molecular cloning techniques. Unless specifically noted, clones extending from the ADV EcoRI site at 53 MU (17, 19,
the machinations employed for molecular cloning of DNA, 49) rightward in the direction of 100 MU. No clones repre-
agarose gel electrophoresis, restriction endonuclease diges- senting the portion from 0 to 53 MU were obtained. Selected
tions, and large-scale preparation of plasmid DNA were the clones were analyzed by restriction mapping and plasmid
same as previously used (2, 17, 22, 48, 49). Colony hybrid- DNA sequencing.
ization was performed by a modification of an alkaline The ADV-Utah 1 DNA used for sequence determination
blotting procedure (63); colony replicas were transferred to was the previously described recombinant plasmid contain-
82-mm (0.45 ,um pore size) nylon filters (Hybond-N; Amer- ing the BamHI-HindIII fragment (15 to 88 MU) of ADV-
sham Corp., Arlington Heights, Ill.) and were laid colony Utah 1 molecularly cloned into pUC18 (17).
side up on Whatman 3MM filter paper saturated with 0.5 M DNA sequencing techniques. All sequencing was per-
NaOH. After 5 min, the filters were blotted briefly on dry formed by the dideoxy-chain terminating method (67) with
3MM paper and subsequently were replaced on the 0.5 M either 32p_ or 35S- (14, 74) labeled deoxynucleotides as label.
NaOH-saturated paper for an additional 5 min. After a brief Templates for sequencing reactions were either single-
rinse in 2x SSPE (48)-0.1% sodium dodecyl sulfate, the stranded bacteriophage DNA isolated from M13 clones (74)
filters were reacted with the appropriate ADV DNA or RNA or, in most cases, purified double-stranded plasmid DNA
probes (17, 22). (28). All M13 bacteriophage-derived sequences were con-
The purified BamHI-HindIII (15 to 88 MU) fragment of firmed by sequencing plasmid DNA, and almost all regions
pADV-G (17) was subcloned into M13mpl8 in Escherichia were sequenced at least twice in both directions. Initially,
coli JM109 [recAl endAl gyrA96 thi hsdRJ7 supE44 relAl the appropriate commercially available primers were used,
Ql- A(lac-proAB) F'traD36 proAB AlacIqZMJ5) (74); the but most of the reactions were performed with ADV-specific
sense of the M13 templates from these recombinant bacte- oligonucleotide primers, the sequences of which were based
riophages was plus in relation to the ADV sequences. A upon ADV DNA sequences that were already obtained.
series of M13 clones processively deleted (32) from the Utilization of these ADV-specific primers made it possible to
HindIII site were derived from this recombinant bacterio- obtain extended sequence information on a single template
phage by using a commercially available kit (Cyclone; Inter- preparation. In some instances, sequencing reactions were
VOL. 62, 1988 SEQUENCE ANALYSIS OF ADV 2905

run using a deaza-nucleotide kit (American Bionetics, Inc., right of the Hindlll site at nt 4170. The same result was
Emeryville, Calif.) to minimize secondary structure. For obtained with clones produced in DB1256 (33), a strain with
sequencing the extreme left (3') end, ADV-G RF DNA was a recB recC sbc genotype (24). Because we were unable to
included in a standard double-stranded dideoxy sequencing arrange our right-hand terminal sequence in a hairpin, we
reaction, using an ADV-specific 20-nt primer complemen- concluded that most if not all of the palindrome at that end
tary to nt 122 to 142 of the plus strand. was absent in the clones we have obtained to date. As a
DNA sequence analysis. DNA sequences were analyzed on result, we were unable to determine whether the 5' and 3'
a personal computer (IBM XT; International Business Ma- termini are different, as in the case of MVM (9, 31, 66), or the
chines, Inc., Boca Raton, Fla.), using the Microgenie se- same, as in the case of B19 parvovirus (68) and AAV (71).
quence analysis program package (Beckman Instruments, We observed an imperfect A+T-rich direct repeat of 25 nt
Inc., Palo Alto, Calif.). Homology comparisons of coding that ended at nt 4591:
regions (ORFs) and terminal regions were performed by 4469 ATAAACCTACATTCTATACTATCTA 4493
using the Alignment function of the Analysis mode of this *** ** **** ******** ***
program. The melting temperatures (Tm of 3' hairpins were 4569 ATATACTAACAT-CTATACTA-CTA 4591
calculated by the method of Wetmur and Davidson (48, 73).
Published sequences of BPV (29), feline panleukopenia virus Similar repeats have been found near the right-hand termi-
(FPV) (26), and the human parvovirus B19 (68) were gra- nus of several other autonomous parvoviruses (31).
ciously provided on computer disks by Muriel Lederman, Summing the 4,592 bases of DNA with the presumed 200
Jon Carlson, and Carol Astell, respectively. Other sequence bases missing at the right end gives a size of -4,792 nt, very
data (MVM [9] and AAV [71] were contained in the data close to previous estimates (19). This makes ADV-G several
base of the Microgenie program. hundred bases shorter than any of the other nondefective
parvoviruses [MVM(i), 5,085 nt] (9), but -100 nt longer than
RESULTS the dependovirus AAV (4,681 nt) (11, 71). On the basis of
these considerations, we used 4,800 nt as the denominator
Determination of ADV-G sequence. The primary aim of when expressing genome coordinates in map units.
these experiments was to determine the genomic nucleotide Identification of potential promoters and transcriptional
sequence of the ADV-G strain (20) of ADV. Using as control elements. Analysis of the sequences of other auton-
templates the DNAs detailed in Materials and Methods, we omous parvoviruses suggests the existence of two functional
obtained 4,592 bases of DNA sequence. We have adopted promoters (10, 45), referred to as TATA boxes, at 4 and at 38
the convention that defines the 3' end of the minus-sense to 40 MU (12, 23, 27, 30, 31, 42, 46, 55, 57, 65). We identified
virion strand as the zero map position or left-hand end (8, eight potential TATA boxes (TATAA and TATTAA) in
70); thus, nt 1 would be the first base at the 5' end of the ADV-G (Fig. 1 and 3). Speculating that ADV-G had two
plus-sense strand. The sequence is given in Fig. 1 for the functional protnoters at -4 MU and at -38 to 40 MU,
plus strand. analogous to those of other parvoviruses, the nt 154 TATAA
The genomic termini of all parvoviruses contain self- (3 MU) and the nt 1729 TATTAA(36 MU) are the most likely
complementary sequences capable of forming hairpin struc- candidates.
tures. The sequences at the left-hand end (3' end of the Six repeats of the sequence motif AATAAA, which gen-
minus-sense virion strand) can be arranged in a Y-shaped erally occurs 10 to 30 nt upstream from the start of the 3'
configuration (31), and those at the right-hand end can be poly(A) tracts in mRNA (15, 50), were identified (Fig. 1 and
arranged in a simple U-shaped structure (31). The left-hand 3). The coordinates are nt 665, 818, 2546, 4136, 4394, and
or 3' terminus of ADV-G contains a palindrome that can be 4468. Several of these motifs are clustered at the right end of
arranged as shown in Fig. 2. The first 9 bases were deleted the genome, and one occurs near the middle of the genome,
from the longest molecular clones, and that sequence was as described for other parvoviruses (11, 29, 31, 55, 71).
obtained by direct dideoxy sequencing of purified RF DNA. Sequence elements in addition to TATA boxes and AA
The most stable configuration of the 3' palindrome was TAAA motifs are essential in determining functional promot-
compared with that described for MVM (Fig. 2) (9, 31, 66). ers and polyadenylation sites (10, 15, 43, 45, 50). We have
Several features were noteworthy: (i) a 7-bp A+T-rich presented a detailed analysis of this material in a manuscript
extreme 3' terminus, (ii) a duplex stem (29 bp in ADV-G and dealing with the ADV transcription program (S. Alexander-
44 bp in MVM), (iii) a short mismatched gap or bubble in the sen, M. E. Bloom, and S. Perryman, submitted for publica-
duplex stem (nt 20 to 21 with 95 to 97 in ADV-G; nt 25 to 26 tion) and thus, have deferred detailed discussion of those
with 88 to 90 in MVM), and (iv) one G+C-rich arm of 5 to 6 points in this report. For similar reasons, we have not
bp (nt 76 to 86 in ADV-G; nt 60 to 71 in MVM). The other discussed potential splice donors and acceptors (29, 54).
arm in the observed ADV-G sequence was considerably Definition of ORFs in the ADV-G genome. The structural
longer, less highly base paired and G+C poorer than that for organization of parvovirus genomes reveals that all have two
MVM. This same left-hand end region was sequenced in a major ORFs. (By informal convention [31, 70], the capsid
total of four separate clones, and none gave a different proteins have been designated from the largest to the small-
sequence than that shown. Taken together, these data imply est as VP1, VP2, VP3, and the nonstructural proteins are
that the structure of the 3' terminus of ADV-G is very similar designated NS-1 and NS-2. For simplicity, we will conform
but not identical to that of MVM and other autonomous to these designations.) The left ORF contains the sequences
parvoviruses. Furthermore, the calculated Tm (48, 73) of the for the largest of the nonstructural proteins, NS-1 (26, 30,
palindrome observed for ADV-G (37.3°C) is significantly less 31), and the right ORF codes for the bulk of the sequences
than that for MVM (48.7°C). Thus, the 3' hairpin of ADV-G contained in the structural or capsid proteins (31). In addi-
is less stable than that of MVM. tion, several smaller ORFs seem to be a common feature and
All ADV-G clones containing extreme right-hand end likely encode portions of additional NS proteins (30, 31). All
sequences were approximately 200 bp shorter than ADV-G of these coding regions occur on the strand complementary
RF DNA (data not shown). This truncation mapped to the to the virion DNA, i.e., the plus-sense strand (31).
 left hand (3 ) terminal palindrome 2401 AGGAAATGGA TTCTACTGAA GCTGAACAAA TGGACACTGA GCAAGCAACT AACCAAACTG
ATTAATTCTC AACCAATATT CGTTAGCAAC CAACACCAGC TCGCTTCGCT CGCGCACCTT
Itkrt 2461 CTGAAGCTGG TGGTGGGGGG GGTGGGGGTG GTGGGGGTGG TGGTGGTGGT GGTGGGGTTG
61 CGGCGCTGGT GTTGGGCGCT TCGCGCTTGC TAACTTCATA TTGGTTGAGA ATTAATCCGT
poly A
LORF TATA 154 2521 GTAACAGCAC TGGCGGCTTT AATAACACAA CAGAATTCAA AGTAATAAAC AATGAAGTGT
121 GTCTTTCCTG TGGAATGAGG AAGTAGTGTG GTATATAAGC AGAGGTTGCT TGGAGCAAAG
2591 ATATTACTTG TCACGCTACT AGAATGGTAC ACATTAACCA AGCTGACACA GACGAATACT
181 CACAGACCGG TTACAGCAAA GTAACATGGC TCAGGCTCAA ATTGATGAGC AGAGGAGACT

2641 TGATATTTAA TGCTGGTAGA ACTACTGATA CCAAAACACA TCAGCAAAAA CTAAACTTAG

241 GCAGGACCTG TATGTGCACT TGAAGAAGGA GATTAACGAC GGTGAAGGAG TTGCCTGGTT
2701 AATTTTTTGT ATATGATGAT TTTCACCAAC AAGTAATGAC ACCTTGGTAT ATAGTAGATA
301 GTTCCAACAA AAGACCTACA CCGACAAGGA CAACAAACCA ACCAAAGCAA CACCGCCACT

2761 GCAACGCTTG GGGTGTATGG ATGAGTCCTA AAGACTTTCA ACAAATGAAA ACACTGTGTA

361 GAGGACAACC TCTTC1GACC TAAGGTTAGC TTTTGACTCT ATTGAAGAGA ATTTAACAGC

421 TTCTAATGAA CACTTAACTA ACAATGAGAT AAACTTTTGT AAACTAACCT TGGGGAAGAC 2821 GTGAAATTAG TTTGGTTACT TTGGAACAAG AAATAGACAA TGTAACCATA AAAACTGTAA

481 GTTGCTGTTA ATTGATAAGC ATGTAAAAAG CCACAGATGG GATAGTAACA AAGTTAACTT 2881 CAGAAACCAA CCAAGGTAAC GCATCTACCA AGCAATTCAA CAATGACTTA ACTGCGTCGT

541 AATTTGGCAA ATAGAAAAAG GAAAAACTCA GCAATTTCAT ATTCACTGTT GCTTAGGTTA 2941 TACAGGTTGC TTTAGATACT AACAACATAC TGCCATATAC TCCAGCTGCG CCGTTGGGGG

601 CTTTGATAAG AATGAAGATC CTAAGGATGT TCAAAAATCC TTAGGTTGGT TTATGAAAAG 3001 AAACACTGGG CTTTGTTCCT TGGAGAGCAA CCAAACCAAC CCAATATAGG TATTATCATC

poly A TATA
661 ACTAAATAAA GACCTAGCAG TTATCTATAG TAACCATCAT TGTGACATAC AAGATATTAA 3061 CATGTTACAT TTACAACAGA TATCCTAACA TTCAAAAAGT TGCAACAGAA ACACTAACCT
715
721 GGATCCTGAA GATAGAGCTA AGAACCTAAA AGTGTGGATT GAAGATGGAC CTACTAAGCC 3121 GGGATGCAGT ACAAGATGAT TACCTTAGTG TGGATGAACA GTACTTTAAC TTTATTACTA
poly A TATA 3197
781 TTACAAATAT TTTAACAAAC AAACCAAACA AGACTACaAT AAACCAGTTC ACTTGAGAGA 3181 TAGAGAACAA CATACCTATT AACATTCTCA GAACGGGAGA TAACTTTCAT ACAGGCTTGT

1441 CTATACATTC ATATACCTGT TTAACAAAGA TAAGATAAAT ACAGATAGTA TGGATGGTTA 3241 ATGAGTTTAA CAGTAAACCA TGTAAACTAA CCTTAAGCTA TCAAAGTACA GGCTACCTCC

901 CTTTGCTGCT GGTAACGGTG GCATTGTTGA CAACCTAACT AACAAAGAAC GAAAAACTTT 3301 TCTCTGCAAA CCAAAGACAG ATACAACACA CAAAGTAACC TCAAAAGAAA CGTTGCTTGG
TATA 994
961 AAGAAAAATG TACTTAGATG AGCAGAGTTC AGATATAATG GATGCTAATA TAGACTGGGA 3361 ACGGAGCTGA CCTAATTTAC ATACAAGGAC AAGATAATAC CAGACTAGGT CACTTTTGGG

1021 AGATGGCCAA GACGCGCCAA AAGTAACTGA CCAAACTGAC TCAGCAACCA CAAAAACAGG 3421 GTGAGGAAAG AGGTAAGAAA AACGCAGAGA TGAACAGAAT TAGACCTTAC AACATAGGTT

1081 AACTAGTTTG ATTTGGAAAT CATGTGCTAC TAAAGTAACC TCAAAAAAAG AAGTTGCTAA 3481 ACCAATATCC TGAATGGATA ATACCAGCAG GGTTACAGGG TAGTTACTTT GCTGGAGGAC

1141 TCCAGTTCAG CAACCTTCTA AAAAACTGTA CTCAGCTCAA AGTACTTTAG ATGCATTGTT 3541 CAAGACAGTG GAGTGACACA ACCAAAGGTG CAGGTACACA CAGTCAACAC TTACAACAGA
TATA 1234
1201 TAACGTTGGT TGCTTTACTC CAGAAGATAT GATTATAAAG CAAAGTGACA AATACCTTGA 3601 ACTTTAGTAC TAGGTACATC TATGACAGAA ACCACGGTGG AGACAACGAG GTAGACCTAT

1261 ACTATCTTTA GAACCAAACG GGCCTCAAAA AATTAACACT TTACTTCACA TGAACCAAGT

3661 TAGATGGAAT ACCCATTCAT GAAAGAAGTA ACTACTACTC AGACAATGAG ATAGAGCAAC
TATA 1357
1321 AAAGACATCA ACCATGATTA CTGCTTTTGA TTGTATTATA AAATTTAATG AAGAGGAAGA
3721 ATACAGCAAA GCAACCAAAG TTACGTACAC CACCCATTCA CCACTCAAAA ATAGACTCGT
TATA 1402
1381 TGACAAACCT TTGCTAGCAA CTATAAAAGA CATGGGACTT AATGAACAAT ACCTTAAGAA
3781 GGGAAGAAGA AGGTTGGCCT GCTGCTTCAG GCACACACTT TGAAGATGAG GTTATATACC
1441 GGTACTATGT ACCATCCTAA CCAAGCAAGG TGGAAAGAGA GGTTGTATTT GGTTCTATGG
3841 TAGACTACTT TAACTTTAGT GGTGAACAGG AGCTAAACTT TCCACATGAA GTATTAGATG
1501 ACCGGGGGGC ACTGGAAAAA CCTTGCTAGC ATCTTTAATA TGTAAAGCAA CAGTAAACTA
3901 ATGCTGCTCA GATGAAAAAG CTACTTAACT CATACCAACC AACAGTTGCT CAAGACAACG
1561 TGGTATGGTT ACTACAAGCA ATCCAAACTT TCCATGGACT GACTGTGGCA ATAGAAACAT
3961 TTGGTCCTGT ATACCCGTGG GGACAGATAT GGGACAAGAA ACCTCATATG GATCACAAAC
1621 CATTTGGGCT GAAGAGTGTG GTAACTTTGG TAACTGGGTT GAAGACTTTA AAGCCATTAC
4021 CTAGCATGAA CAACAACGCT CCATTTGTAT GTAAAAACAA CCCTCCAGGT CAACTCTTTG
TATA 1729
1681 TGGAGGTGGT GATGTAAAAG TAGACACCAA GAACAASCAA CCTCAATCTA TTAAAGGCTG pol y
4081 TTAAACTAAC AGAAAACCTC ACTGATACAT TTAACTATGA TGAAAATCCA GACAGAATAA
1741 TGTGATTGTA ACAAGCAACA CCAACATAAC CAAAGTAACT GTTGGATGTG TGGAAACAAA A
4141 AAACCTATGG TTACTTTACT TGGAGAGGCA AGCTTGTACT AAAAGGCAAA CTAAGCCAAG
1801 CGCTCACBCA GAGCCACTTA AACAGAGGAT GATTAAGATA CGTTGCATGA AAACCATCAA
4201 TAACATGCTG GAATCCTGTT AAGAGAGAAC TCATAGGAGA ACCTGGTGTA TTTACTAAAG
1861 CCCTAAAACT AAAATAACAC CAGGCATGTT AAAAAGATGG CTAAATACCT OGGATAGACA
4261 ACAAGTATCA CAAACAGATA CCAAACAACA AAGGTAACTT TGAAATAGGG TTACAATATG
end L
1921 ACCAATTCAA CTAAGCCATG AGATGCCTGA ACTGTACTTA GGTAAGTGCC GTTGGTAAGT end RORF
ORF I.t rt MORF I 4321 GAAGAAGTAC TATCAAATAT ATCTACTAAA GTAACCTGTG TACTATGTTA CTATGTTACT
1981 AAQpCATTTT AAATGCCAAC TTTAAACCAA CATCAATTTA TGAGGTTACT TTACTTTACA poly A
start MORF2 4381 ATGATAATAT CTCAATAAAA GTTACATGAA TAGTGAACAA CCTAAATACT GTGTACTTCC
2041 GAGACTACTG GACCAAACTC GAGTGCCACA ACTGCCACGA AGAATACTGG CAACTCACAA poly A
4441 TTATTTTACC AGAAAGTGGC GGATTAAAA_T AAACCTACAT TCTATACTAT CTATATACTA
2101 CCTACTACTG CAAAGAGTGC AGAAAGTGTG AACACGGAAA ACTGCGACAC ACCAAAAAGG
Od_MORF 1 4501 CTAACTAACC TATAGGTTAC TTTGCTTTGA TATACTGATG TAGGAATACA GGATACTAAC
2161 AGTGCGAGCA GTGTGCCTGC AAAGCAGCAC AAGAGACCTC GGCAT>AGTA AAAGTAAATA
end MORF2
start RORF 4561 ATTTATATAT ATACTAACAT CTATACTACT AA
2221 ACCTACTTAA AGTAACCTAA Qe;CATAACA CTTTACTTTC CTTGTACTTA TGTTACTTTA

2291 CTTTAGTTCC TCAGCACTAT CCTGGGAAAA AGAGAAGTGC TCCAAGACAC GTGTTTATTC

2341 ASCAAGCAAA AAAGAAGAAG CAAACTAACC CTGCGGTCTA CCACGGAGAG GACACCATAG

FIG. 1. Derived DNA sequence of the plus strand of ADV-G. Several features are indicated: the extent of the terminal palindrome at the
left-hand end (i.e., 3' end of virion strand); the positions of potential promoterlike TATA sequences; the locations of AATAAA
polyadenylation signals; and the boundaries of the right, left, and the two mid- (MORF1 and MORF2) ORFs.
2906
VOL. 62, 1988 SEQUENCE ANALYSIS OF ADV 2907

c
A The left ORF of the other parvoviruses contains a con-
oco served domain at -30 MU of approximately 60 amino acids
*o referred to as the GKRN region (29, 31, 68). When the
* Cc
* theoretical translations of the left ORFs of ADV-G and
AA
o
MVM are aligned, the presence of this GKRN element in the
D-GATHNDYAIW
C,. C.

ADV sequence (31 MU) is evident (Fig. 5A). Several short

C- T C stretches within this domain show extensive amino acid
C / sequence conservation among ADV-G, MVM (Fig. 5A), and
A/~~~~~~~~A
T AC t tTTis the other parvoviruses (data not shown), although the over-
C AACATT TATACCAACCTTAATTAOOCAC* all relatedness to MVM of the entire GKRN region is only
ac Nj\b
* soI AO " sE
52%.
00-^
A
The right parvoviral ORFs have been alleged to contain a
total of six conserved amino acid regions (29, 31, 68). In
acronymic form, these have been designated NPYL, TPW,
PIW, PGY, GGG, and YNN (29). Not all of these domains
could be found in the ADV-G sequence we obtained. Re-
Aa IMIVLEITHAMD(2)TUIS
gions that likely corresponded to the GGG, TPW, YNN, and
the PIW elements were characterized and identified in an
alignment of the ADV-G and MVM right ORFs (Fig. SB).
C
SSS SST
c TCA CACSTCACT?ACOT ACATOOTT@STCAST?CTAAAA.T The amino acid homologies of these regions to the corre-
AOT *?TCA SA AITSCA TOTACCAACCAOTCAAOa??1?TTCTATT C
sponding ones of MVM are: GGG, 73%; TPW, 78%; YN4 ,
C
76%; and PIW, 67%. Amino acid sequences corresponding
to the NPYL or the PGY motifs could not be clearly
FIG. 2. Structures of the terminal palindrome sequences at the identified in the right ORF of ADV-G. In fact, they could not
left or 3' end of the virion or minus strand of ADV-G and MVM. The be found in any of the three theoretical translations of the
sequences have been arranged to show maximal base pairing, that entire ADV-G sequence, suggesting that they are not fea-
for MVM is based on that reported for the prototypic strain of tures of ADV-G.
MVM, MVM(p) (9, 31). The A+T-rich extreme 3' termini and their Comparisons of ADV-G sequence to partial sequence of
complements are indicated by the solid lines. ADV-Utah 1. The ADV-G strain is a cell culture derivative of
the highly virulent ADV-Utah 1 strain that lost pathogenicity
for adult mink after serial passage in CRFK cells (20). We
When the sequence of the ADV-G plus strand was trans- previously cloned the 15- to 88-MU segment (BamHI-
lated in the three possible frames beginning with nt 1, 2, or 3, HindIII fragment) of ADV-Utah 1 directly from DNA iso-
respectively, a pattern similar to that described above was lated from the tissues of infected mink (17). Using the
obtained. A diagram depicting the location of the ORFs ADV-specific primers developed for sequencing ADV-G, we
along with the respective ATGs and stop signals is depicted also sequenced this segment of ADV-Utah 1 and compared it
in Fig. 3. Those ATGs embedded in the most common with the corresponding ADV-G segment (nt 721 to 4176).
context for initiation (ANNATGG) (45) are signified. The The ADV-Utah 1 BamHI-HindIII segment contains 3,454 nt
location of restriction enzymes with one or two recognition (1 less than in the corresponding segment of ADV-G), and
sites is also displayed in Fig. 3, and a partial list of enzymes the overall relatedness to ADV-G at the DNA level is very
without recognition sites in ADV-G is listed in the legend to high (97.5%). Assuming the overall genomic lengths of both
that figure. viruses are -4,800 nt, this comparison encompasses approx-
The left (left ORF), right (right ORF), and the 2 mid- imately 76% of the genomes. A total of 73 nucleotide
(MORFi and MORF2) ORFs were translated into amino differences were observed between the two strains (Fig. 6).
acids; the protein sequences along with estimated molecular One short segment in the right ORF (64 to 65 MU) shows
masses and nucleotide coordinates are shown in Fig. 4. In marked heterogeneity between ADV-G and ADV-Utah 1;
addition, the boundaries of the ORFs within the actual this region, bounded by nt 3094 and 3130, contains 16 base
sequence are indicated in Fig. 1. changes, a single-base deletion at nt 3094, and a single-base
The left ORF is 1,859 nt in length and has a theoretical addition at nt 3112 (ADV-G coordinates).
molecular mass of 70,927 daltons. This value is close to that Because we had determined the ORFs of ADV-G, it was
reported for the 71,000-dalton mass of the ADV-G NS-1, p71 of interest to examine the ORFs of the two ADV strains. The
(5, 21). The right ORF is 2,105 nt long and could specify a ADV-Utah 1 sequence ends at the HindIII site at amino acid
protein of 79,970 daltons, a value intermediate to the two 645 of the right ORF; as a result, the extreme 3' portion of
structural polypeptides of ADV-G (5, 20, 21). The 2 mid- this ORF was not available for analysis. Nevertheless, we
ORFs could encode polypeptides less than 10,000 daltons in compared this sequence with the first 645 residues of the
size. ADV-G right ORF; an analysis that encompasses 92% of the
Relationship of ADV-G to other parvoviruses. In order to 702 residues in the ADV-G right ORF. The theoretical sizes
investigate the relationship of ADV-G to several other for these truncated right ORF translation products are 73,388
members of the parvovirus family, we compared the homol- and 73,516 daltons for ADV-G and ADV-Utah 1, respec-
ogies of the various ORFs at both the DNA and amino acid tively. The putative proteins are 95.8% related and have 15
level. In addition, we also searched the first 200 nt to see isolated single-amino-acid changes, several of which are
whether significant homologies existed for the left-hand located in the conserved regions discussed above (Fig. 7). In
palindrome. None of these comparisons (AAV, B19 parvo- addition, the region at 64-65 MU alluded to in the previous
virus, BPV, MVM, and FPV) yielded a relatedness of paragraph exhibits several singular features in the putative
greater than 50%o (data not shown), thus suggesting that protein translations (Fig. 6 and 7). In a short cluster of 11
ADV is not closely related to any of these parvoviruses. amino acids positioned just downstream from the YNN
2908 BLOOM ET AL. J. VIROL.

0 C. 0 0
NUCLEOTIDE 0 0 0
C. 0 0
.

MAP UNIT
S.
0
I 1
20
IlI40 ll ll
60
lI l
80
lI 10C
3.

STOP
FRAME I
ATG

FRAME 2 STOP
ATG

STOP MORF2
FRAME3
AMG III
~j1I
2
11111 II1~ I
2 2 2 2 2
G0TORF
t Jt ~~~t
t tt t t t t tt

~~~~~~
4 ~~~~ 4
#~~~~~~~~~~~~~~~~~~mC44~~~~InCMc
4
l

S1~~~~~~4 I I I A

0
U 0 0' le
Sc
RESTRICTION *m ZZ .z x - 0
MAP

_ a, ,a 'N. 0
,- C4
a x _
z
a

n0 Z

FIG. 3. Schematic representation of the genomic organization of ADV-G. A line diagram was prepared that displayed the major features
of the ADV-G genome, including the ORFs and potential transcription-translation control signals in ADV-G. The location of termination
codons (STOP) in the three potential reading frames is indicated by vertical marks above a horizontal line for each frame; some of these lines
represent multiple STOPS too close to depict graphically. The boundaries of the ORFs are denoted by open boxes. Vertical lines below the
horizontal lines indicate potential initiation (ATG) codons, and those ATGs in an optimal context for initiation (ANNATGG) (44) are marked
( I ). The approximate position of promoterlike TATA boxes (TATAA and TATTAA) and polyadenylation signals (AATAAA) are denoted
by arrows. The dotted portion of the horizontal lines represents the presumed unsequenced segment of the genome. The restriction map for
those restriction endonucleases having one or two recognition sites in ADV-G is also shown. The following is a partial list of enzymes with
no sites in ADV-G: AatIII, AhaII, AsuIl, BanI, Bcll, BglI, BglII, Clal, KpnI, Mlul, NarI, NotI, PvuI, Sacl, SacIl, Sall, SmaI, SphI, Stul,
XbaI, XmaIII.

element, eight residues have diverged between the two ADV ual amino acid changes in this segment of the left ORF (Fig.
strains, suggesting that this might be a hypervariable region 7), three of which were rated as conservative (data not
(Fig. 7, inset A). shown). There was one change (nt 1646, F to L) which
The effect of these amino acid changes on the theoretical occurred within the GKRN region; however, this did not
hydropathy (41) of the right ORFs was also examined (Fig. affect the hydropathy or the predicted protein structure (data
8). As expected from the high level of relatedness, the not shown). MORF1 and MORF2 had one and three single-
general pattern of both strains is very similar, consisting of amino-acids changes, respectively.
alternating hydrophobic and hydrophilic regions. Further-
more, the 15 amino acid changes not located in the hyper- DISCUSSION
variable segment are found both in hydrophilic (9 of 15) and
hydrophobic (6 of 15) areas. However, the amino acid Two topics have been presented in this report. The first
changes within the hypervariable region produce a small, but reports the nearly complete DNA sequence of the ADV-G
obvious perturbation of this plot. strain of ADV and relationships to other parvoviruses. The
We also compared the available left ORF of ADV-Utah 1 second set of results is a comparison of the ADV-G sequence
with that of the ADV-G left ORF beginning at the BamHI with a partial sequence of the virulent ADV-Utah 1 strain.
site at nt 721. The single-nucleotide deletion of an A corre- The first sections in this report point out structural simi-
sponding to ADV-G nt 806 shifts the downstream portion of larities as well as differences among ADV-G and the other
the left ORF to ADV-Utah 1 from this point into another parvoviruses (9, 29-31, 65, 66, 68, 71). The overall levels of
translational reading frame. This deletion was observed on homology are low, confirming previous suggestions that
repeated sequencing of this region in both directions, but we ADV is not closely related to other parvoviruses (49, 58, 59).
could not determine whether the deletion was real or repre- On the other hand, the gross organization of the ADV
sented a cloning artifact, because only a single clone of genome resembles in many but not all ways that recently
ADV-Utah 1 was available for analysis (17). Assuming that a summarized for MVM and other members of the group (31,
single base was deleted during cloning and that the left ORF 69, 70).
of ADV-Utah 1 is single and continuous like that of ADV-G The terminal structures are known to play a pivotal role in
and all the other parvoviruses (31), there were four individ- parvoviral DNA replication, and the variation that can be
VOL. 62, 1988 SEQUENCE ANALYSIS OF ADV 2909

TRANSLATION OF ADV-G LEFT ORF TRANSLATION OF ADV-G RIGHT ORF

(NT 116-1975, FRAME 2, MOLECULAR WEIGHT-70,927 DALTONS) (NT 2241-4346, FRAME 3, MOLECULAR WEIGHT=79,970 DALTONS)
60 60
SVSFLWNEEVVWY ISRGCLE OSTDRLQQSNMAQAO I DEOR RLQDLYVOLKKE INDGEGVA HHNTLLSLYLCYFTLVPOHY PGKKRSAPRHVF IOOAKKKK OTNPAVYHGEDT IEEMDSTE
120 120
WLFQQKTYTDKDNKPTKATP PLRTTSSDLRLAFDS I EENL TASNEHLTNNE INFCKLTLG AEOMDTEOATNQTAEAGGGG GGGGGGGGGGVGNSTGGF NNTTEFKV INNEVY I TCHAT
190 180
KTLLL I DKHVKSHRWDSNKV NLIWOIEKGKTQOFHIHCCL GYFDKNEDPKDVQKSLGWFM RMVHINOADTDEYL IFNAGR TTDTKTHQQKLNLEFFVYDD FHOOVMTPWY I VDSNAWGVW
240 240
KRLNKDLAV I YSNHHCDI QD IKDPEDRAKNLKVWIEDGPT KPYKYFNKQTKODYNKPVHL MSPKDFOOMKTLCSEISLVT LEQE I DNVT IKTVTETNOGN ASTKQFNNDLTASLOVALDT
300 300
RDYTF I YLFNKDK INTDSMD GYFAAGNGGIVDNLTNKERK TLRKMYLDEQSSDIMDANI D NNILPYTPAAPLGETLGFVP WRATKPTOYRYYHPCY I YNR YPNIOKVATETLTWDAVQDD
360 360
WEDGODAPKVTDOTDSATTK TGTSL IWKSCATKVTSKKEV ANPVQOPSKKLYSAOSTLDA YLSVDEQYFNFI T IENNIPI NILRTGDNFHTGLYEFNSKP CKLTLSYQSTRCLGLPPLCK
420 420
LFNVGCFTPEDMI IKOSDKY LELSLEPNGPOKINTLLHMN OVKTSTMITAFDCI IKFNEE PKTDTTHKVTSKENGADL IY IOGODNTRLGHFWGEERGKK NAEMNR IRPYNIGYQYPEWI
480 480
EDDKPLLAT IKDMGLNEQYL KKVLCT ILTKOGGKRGCIWF YGPGGTGKTLLASLICKATV IPAGLOGSYFAGGPROWSDT TKGAGTHSQHLQONFSTRY I YDRNHGGDNEVDLLDGIPIH
540 540
NYGMVTTSNPNFPWTDCGNR NI IWAEECGNFGNWVEDFKA I TGGGDVKVDTKNKQPOSIK ERSNYYSDNE IEQHTAKOPK LRTPPIHHSK I DSWEEEGWP AASGTHFEDEV I YLDYFNFS
600 600
GCVIVTSNTNI TKVTVGCVE TNAHAEPLKORMIK IRCMKT INPKTK I TPGMLKRWLNTWD GEQELNFPHEVLDDAAQMKK LLNSYOPTVAODNVGPVYPW GQIWDKKPHMDHKPSMNNNA
660
ROP IOLSHEMPELYLGKCRW PFVCKNNPPGOLFVKLTENL TDTFNYDENPDRIKTYGYFT WRGKLVLKGKLSQVTCWNPV
COMPOSITIONs 620 AMINO ACIDS KREL IGEPGVFTKDKYHKOI PNNKGNFE IGLQYGRST I KY IY
ACIDIC (ASP + GLU) 77(12.4%) COMPOSITION. 702 AMINO ACIDS
BASIC (ARS + LYS) 84(13.5%) ACIDIC (ASP + GLU) e81(1i.5%)
AROMATIC (PHE + TRP + TYR) 53 e. 5%) BASIC (ARG + LYS) 66(9.4%)
HYDROPHOBIC (AROMATIC + ILE + LEU + MET + VAL) 1ee(30.3%) AROMATIC (PHE + TRP + TYR) e81 1 .5%)
HYDROPHOBIC (AROMATIC + ILE + LEU + MET + VAL) 207(29.4%)

TRANSLATION OFADV-G MID-ORF2

(NT 1993-2209, PHASE 1, MOLECULAR WEIGHT-7,918 DALTONS) (NT 1983-2204, PHASE 3, MOLECULAR WEIGHT-8,922 DALTONS)
60 60
MPTLNDHOFMRLLYFTETTG PNSSATTATKNTGNSOPTTA KSAESVNTENCDTPKRSASS H ILNANFKPTSIYEVTLLYR DYWTKLECHNCHEEYWOLTT YYCKECRKCEHGKLRHTKKE

VPAKQHKRPRHE CEOCACKAAOETSA
COMPOSITION. 72 AMINO ACIDS COMPOSITION. 74 AMINO ACIDS
ACIDIC (ASP + GLU) 5(6.9%) ACIDIC (ASP + GLU) 10(13.5%)
BASIC (ARG + LYS) 9(12.5%) BASIC (ARG + LYS) 11(14.9%)
AROMATIC (PHE + TRP + TYR) 3(4.1%) AROMATIC (PHE + TRP + TYR) 9(12.2%)
HYDROPHOBIC (AROMATIC + ILE + LEU + MET + VAL) 10( 13.9%) HYDROPHOBIC (AROMATIC + ILE + LEU + MET + VAL) 18(24.3%)

FIG. 4. Theoretical translations of the coding regions of ADV-G. The DNA sequence of the major ORFs was translated into amino acids,
using the single-letter code. The nucleotide coordinates, translation frames, and calculated molecular masses in daltons are shown. In
addition, the numbers and compositions of the residues are listed.

tolerated is of great interest (11, 31, 47). Unfortunately, growth in the lungs of poikilothermic newborn mink (1, 2, 4),
these sequences have proven extremely difficult to molecu- and it is conceivable that one function of the reduced
larly clone and to maintain in procaryotic vectors (24, 52). temperature is to help stabilize the left-hand hairpin and
The structure of the 3' ADV-G terminus is very similar to facilitate replication.
that described for MVM (Fig. 3), but the calculated Tm of the The sequence of ADV-G at the right-hand end does not
ADV-G 3' terminus is 37.3°C, significantly below that of contain a hairpin but terminates just downstream of a 25-bp
MVM (48.7°C), and the ADV-G hairpin is thus less stable. A+T-rich direct repeat. Similar direct repeats occur imme-
Because of the severe difficulties encountered by others (24, diately before the right-hand palindrome of MVM, H-1 virus,
29, 52, 62) in analyzing this structure and because our clones FPV, and canine parvovirus (CPV) (31), and we concluded
containing the 3' end were not produced in E. coli strains that the -200-bp deletion at this end of the ADV-G sequence
tolerant of parvoviral palindromic structures (24), it is con- likely contained the right-hand hairpin. Similar results were
ceivable that the sequence we have presented may contain obtained when ADV DNA was molecularly cloned into E.
some errors, but the several clones we sequenced gave the coli DB1256, which has a recB recC sbc genotype and is
same result. The host range of ADV is greatly restricted in tolerant of vaccinia virus palindromes (33). This might
vivo and in vitro (4, 20, 58), and we recently showed that suggest that the ADV right-hand terminus will prove more
intracellular levels of RF DNA vary markedly between difficult to clone than those of the other parvoviruses,
permissive and nonpermissive systems (2-4, 22). Perhaps a nevertheless, additional cloning experiments with other re-
relatively unstable 3' terminus impairs the ability of ADV to combination-deficient E. coli strains (JC8111) that are de-
replicate promiscuously and thus limits populations of sus- monstrably tolerant of parvoviral palindromes (24) are in
ceptible host target cells. Interestingly, the two systems for progress. Perhaps clones derived from these bacteria will
highly permissive ADV replication are at reduced tempera- provide definitive information on the terminal fine structure
tures, i.e., propagation in CRFK cells at 32°C (20, 59) and of ADV.
2910 BLOOM ET AL. J. VIROL.

A. B.
(PGY REGION) (NPYL
ADVG -SVSFLWNEEVVWYISRGCL EGSTDRLOOSNMAOAQIDEO RRLODLYVO-LKKEINDGEG ADVB HHNTLLSLYLCYFTLVP--- --O-------------N--- Y-----POK----KRSAPRH
MYm RALRARLRTSHVTYVSHGWS VLKMISGSGSLNOGA--KRK WAWFKVYKGLLKSVTYLFFH NYM NHLVLGWVPPGYKYLGPGNS LDoGEPTNPSDAAAKEHDEA YDOGY IKSGKNPYLYFSAADO
REGION)
ADVG VAWLFOoK---TYTDKDN-- --KPTKATPPLRTTSSDLRL AFDSIEENLTASNEHLTNNE ADVG VFPIOO---AK----K----- ---K-----K-OT--NP--- AV-YHGEDT-----I -----
MVM SVSRDAGKESNOLTMAGNAY SDEVLGATNWLKEKSNOEVF SFVFKNEN-VOLN--GKDIG MVM
*- -
*-R*P*T* * * * * *
RFI DOTKDAKDWGGKVGHYF FRTKRAFAPKLATDSEPGTS GVSRAGKRTRPPAYI FI NOA

ADVG INFCKLTLGKTLLL IDKHVK SHRWD-S-NKVNLIWGIEKG KTQGOFHIHCCLGYFDKNEDP ADVG ---EEMDSTEAEO-----M-D -TEO-----ATNOTA--E-A GGGGGGGGGGGGGGGGVGNS

MVM WNSYKKELOEDELKSLORGA ETTWDOSEDMEWETTVDEMT KKOVF- IFDSLVKKCLFEVL MVM RAKKKLTSSAAOQSSOTMSD GTSOPDSGNAVHSAARVERA ADGPGGSGGGGSGGGGVGVS

ADVG KDVOKSLGWFMKRLNKDLAV IYSNH-HCDIQDIKD-PEDR AK-NLKVWIIEDGPTKPYKYF ADVB TG-GFNNTTEFKVINNEVYI TCHATRMVHINOADTDEYLI FNAGRTTDTKTHOOKLNLEF

MVM NTKNIFPGDVNWFVOHEWGK DOGWHCHVLI-GGKDFSOAO GKWWRROLNVYWSRWLVTAC MVM TGSYDNOTHYRFLGDGWVEI TALATRLVHLNMPKSENYCR IRVHNTTDT---SVKGN---
TPW REBGION
ADVG NKO-TKODYNK-PVHLRD-Y TFIYLFNKDKINTDSMDGYF AAGNGGIVDNLTNKERKT-L ADVB FVYDDFHOOVMTPWYIVDSN AWGVWMSPKDFOOMKTLCSE ISLVTLEOEI DNVTIKTVTE
MVM NVOLTPAERIKLREIAEDNE WVTLLTYKHKOTKKDYTKCV LFGNMIAYYFLTKKKISTSP MVM MAKDDAHEOIWTPWSLVDAN_ AWGVWLOPSDWOYICNTMSQ LNLVSLDOE IFNVVLKTVTE
YNN REGION
ADVG RKMYLDEOSSD-IMDANIDW EDGODAPKVTDOTDSATTKT GTSLIWKSCATKVTSKKEVA ADVG TNOGNASTKOFNNDLTASLQ VALDTNNILPYTPAAPLBET LGFVPWRATKPTOYRYYHPC
MVI PRDGGYFLSSDSGWKTNFLK E-GERHLVSKLYTDDMRPET --------VETTVTTAOETK MVM ODLGGGAIKIYNNDLTACMM VAVDSNNILLPYTPAANSMET LGFYPWKPT IASPYRYY-FC

ADVG NPVDoPSKKLYSAOSTLDAL FNVGCFTPEDMI IKOSDKYL ELSLEPNG-POKINTLLHMN ADVO YIYNRYPNIOKVATETLTWD AVODDYLSVDEOYFNFITIE NNIPINILRTGDNFHTGLYE

MVM RGRIO-TKKEVSIKTTLKEL VHKRVTSPEDWMMMQPDSYI EMMAOPGGENLLKNTLEICT MVM --VDR--- DLSV-TYENOEG TVEHNVMGTPKGIPOFFTIE NTOOITLLRTGDEFATGTYY

ADVG OVKTSTMITAFDCI IKFNE- -EEDDKPLLAT---- I-KDM GLNEQYLKKVLCTILTKOGG ADVG FNSKPCKLTLSYOSTRCLGL PPL--CKPKTDTTHKVTSKE NGADLIYIOGODNTRLGHFW
MVM LTLART-KTAFDLILEKAET SKLTNFSLPDTRTCRIFAFH GWNYVKVCHAICCVLNROGG MVM FDTNSVKLTHTWOTNRQLGO PPLLSTFPEAD-TDAGTLTA OG----SRHG--TTOMGVNW

ADVG KRGCIWFYGPGGTGKTLLAS LICKATVNYGRVTTSNPNP WTDCGNRN! IWAEECGNFGN ADVG GEERGKKNAEMNRIRPYNIG YOYPEWI IPAGLOGSYFAGG PROWSDTTKGAGTHSOHLOO
0*S * ** *-- * * *- * --- *- - - -- -- ----
MVM KRNTVLFHGPASTGKSI IAG AIAGAVGNVGCYNAANVNFP FNDCTNKNLIWVEEAGNFGG MVM VSE-----A--IRTRPAOVG FCoPNNDFEASRAG-PFA-A PKVPADITOG---- VDKEA

ADVG WVEDFKAITGGGDVKVDTKN K-OPOSIKGCVIVTSNTNIT KVTVGCVETNAHAEPLKORM ADVG NFSTRYIYDRNHGGDNEVDL LDGIPIHERSNYYSDNEIEO HTAKOPKLRTPPIHHSK IDS

*R* * * E* *- -- *-- * *-E- * * *-
MVM
*V *---
QVNgFKAICSGOTI RI DOKG KGSKO IEPTPV IMTTNEN IT VVR IGCEERPEHTOP IRDRM mvm
* *-
-NG- E- -*
NGSVRYSYGKOHG---ENWA SHG-PAPER--YTWD-E---
*
-TSFGSGRDT --- KDGFIOS
-

ADVG IKIRCMKT---- INPKTKIT PGMLKRWLNTWDROPIOLSH EMPELYLGK-CRW .ndADV ADVO WEEEGWPAASGTHFEDEVIY LDYFNFSGEOELNFPHEVLD DAAQMKKLLNSYOPTVAODN
MVM LNIHLTHTLPBDFBLVDKNE WPMIICAWLVKNGYOSTMAS- --YCAKWGKVPDWSENWAEP MVM APLVVPPPLNG ------- I- LTNANPIG--TKNDIH---- --- FSNVFNSYGPLTAFSH
PIW REGION
MVM KVPTPINLLBSARSPFTTPK STPLSGNYALTPLASDLEDL ALEPWSTPNTPVABTAETON ADVG VGPVYPWGGIWDKKPHMDHK PSMNNNAPFVCKNNPPGOLF VKLTENLTDTFNYDEN---P
MVM PSPVYPOGOIWDKELDLEK PRLHITAPFVCKNNAPGOML VRLGPNLTD--OYDPNGATL
MVI TGEAGSKACODGOLSPTWSE IEEDLRACFGAEPLKKDFSE PLNLD ndgMVM
ADVG DRIKTYGYFTWRGKLVLKGK LSOVTCWNPVKREL IGEPGV FTKDKYWIKQIPNNKGNFE IG
MVM SRIVTYGTFFWKGKLTMRAK LRANTTWNPV-YOVSAEDNG NSYMSVTKWLPTATGNMOSV

ADVG LOYGRSTIKYIY undADV

mIVM PLITRPVARNTY wndMVMI

FIG. 5. Comparison of amino acid sequences of the left and right ORFs of ADV-G and MVM(p). The left (A) and right (B) ORFs of ADV-G
and MVM(p) were translated into amino acids, using the single-letter code. The optimal overlap was determined by using the Alignment
maneuver of the Comparison mode of the Microgenie sequence program. Identical residues are indicated (*). The locations of conserved
regions (GKRN in the left ORFs; PGY, NPYL, GGG, TPW, YNN, and PIW in the right ORFs) (29, 31) are also defined.

Purified ADV-G virions contain two major capsid proteins consensus [G:(X)4:G:K:T/S:(X)5-6:I/LNV] for the purine tri-
with molecular masses of 85 (VP1) and 75 kilodaltons (VP2) phosphate-binding site has been proposed by Cotmore and
(20, 21), and in addition, a nonstructural protein of 71 Tattersall (31), and this consensus is preserved in ADV (Fig.
kilodaltons (NS-1) is found in ADV-infected cells (21). These 4 and 5A, left ORF amino acid 464 and following).
proteins must be coded for by ORFs within the primary Additional NS proteins (30, 31) have not been formally
sequence, although noncontiguous coding regions are likely identified in ADV-infected cells, but the presence of smaller
juxtaposed by RNA processing (27, 31, 53, 55). ADV-G, like mid-ORFs suggests that existence of such gene products,
the other parvoviruses, contains major left and right coding and, in fact, mRNA and cDNA analyses reinforce this
regions as well as several smaller ORFs located in the middle suggestion (Alexandersen et al., submitted).
of the genome. Our results to date suggest that the genomic We previously showed that coding sequences for the ADV
localization of these gene products in ADV grossly resem- capsid proteins are found to the right of the EcoRI site (49)
bles that of the other parvoviruses. at nt 2553 and furthermore that all VP2 sequences are
NS-1 is the largest gene product of the left ORF (26, 30, contained within VP1 (21). Therefore, it seemed reasonable
31). Its emerging role is that of a multifunctional protein to assume that the right ORF does in fact code for the bulk
involved both with DNA replication as well as regulation of of the capsid proteins. The theoretical translation of this
viral and cellular promoters (12, 30, 31, 64). The conserved ORF (Fig. 5) yielded a protein with a mass (79,970 daltons)
GKRN element (Fig. 5A) found in the available NS-1 se- intermediate to those observed for VP1 (85,000 daltons) and
quences (29, 31, 68) resembles portions of several ATP- and VP2 (75,000 daltons). VP1 and VP2 are products of a spliced
GTP-binding proteins (7, 31) and has led others to speculate mRNA, the boundaries of which cannot be defined by simply
that NS-1 may be one of the presumed viral proteins directly examining the genomic sequence, and consequently, we
involved with DNA replication (7, 31). A minimal amino acid have deferred discussing the arrangement of the ORFs into
VOL. 62, 1988 SEQUENCE ANALYSIS OF ADV 2911

A5V- P T101 UTAH I ALTERATION POSITION my_-_a_ ITIO" --UTAH 1 ALTERAJIDN--- CO ioN
LEFT OPEN REACINS FRANE 2454 A 6 to D 2
2679 N to A 1,2
A 756 I to V 1
A BO rading froam shift 204 A 3
T 860 C delete polyA site 3 A 0 to K
6 118e A S to N a 2749 Y to F 6
T 1339 6 I to M 3 2777 S 3
T 14. C F to L 1
£ j705___ T 2 2~ 3
237 S 3
MID OPEN READINS FRAMES 2904 S6 3
3094
A 2141 MORFI S to B a 3100 a
MOFE E to 3 3103
6 2179 C RF2 C to 2 3104
A 2181 6 rF2 K to E 1 3105
A 2211 B 3 3109
3111
3112
_AQ_ II 6 non-codin. reion 3114 SEE FIGURE 7, INSET A
3117
RIGHT OPEN READING FRA 3118
3119
A 2247 S N to D 3123
C 2240 A S to Y 3124.
6 2333 A 3127
C 23 6 3126
6 2390 A 3129
C 2394 A 3130 6
A 2415 G T to A 3144 3
G 2421 C A to P 3158 3
A 2429 G 3176 C 3
T Z471 6 3459 I to v 1
G 2477 A 3590 H to 0 3
G 2497 A G to S 3705 C N to H 1
T 2492 349 A 3
G 2495 3872 A 3
T 2507 3873 T
A 2409 3876 N to E 1,3
T 2415 3976 G1
GJ
C 2633 3911 3
3977 A 3
4005 N to D I

FIG. 6. Sequence differences between ADV-G and ADV-Utah 1. The DNA sequence of ADV-G from the 5' end of the BamHI site (nt 721)
to the 3' end of the HindIll site (nt 4176) was compared with the sequence of a BamHI-HindIII molecular clone of ADV-Utah 1 (17). DNA
sequence differences are listed, with the nucleotide coordinate of ADV-G as a reference point; where appropriate, the ADV-G nucleotide
immediately 5' to any insertion or deletion is denoted. The location of these changes with respect to ORFs or noncoding regions is given. In
addition, the position of the nucleotide change within codons and any amino acid changes are also described. Adjacent nucleotide differences
that altered single codons (nt 2679-2680 and nt 3876-3878) are grouped with a bracket. The region from nt 3094 to nt 3130 contains extensive
changes, with a complex effect on the coding sequence; this set of differences is also grouped with a bracket, and a comparison of this region
in the two ADV strains is depicted in detail in inset A of Fig. 7.
ADVG HHNTLLSLYLCYFTLVPOHY POKKRSAPRHVFIOOAKKKK OTNPAVYHGEDTIEEMDSTE
UTAH HHDTLLYLYLCYFTLVPOHY PGKKRSAPRHVFIOOAKKKK OTNPAVYHGEDTIEEMDSAE
the probably viral polypeptides to a report dealing with ADV
one REGION
ADVG AEOMDTEOATNOTAEAGGGG GGGGGGGGGGGGVGNSTGGF NNTTEFKVINNEVYITCHAT transcription (Alexandersen et al., submitted).
UTAH PEOMDTEOATNOTAEAGGGG GOSGGGGGGGGGVGNSTGGF NNTTEFKVINNEVYITCHAT
The amino terminus of VP1 for MVM occurs in a different
TPW REGION
ORF and, as noted (31, 42), is joined to the bulk of the capsid
ADVG RPIVHINQADTDEYLIFNAGR TTDTKTHOOKLNLEFFVYDD FHOQVMTPWYIVDSNAWGVW coding region by mRNA splicing. This VP1-specific segment
UTAH RMVHINOADTDEYLIFNADR TTDTKTAOKKLNLEFFVYDD FHQOVMTPWFIVDSNAWGVW contains for MVM, AAV, BPV, FPV, and B19 parvovirus
YNN REG ION two conserved sequences (31) denoted NPYL (-47 amino
ADVG MSPKDFOQMKTLCSE ISLVT LEOEIDNVT IKTVTETNOGN ASTKOFNNDLTASLOVALDT acids in size) and PGY (-30 amino acids), the functions of
UTAH MSPKDFOOMKTLCSEISLVT LEOEIDNVTIKTVTETNOGN ASTKOFNNDLTASLOVALDT which are obscure (31). Analogs to these two amino acid
1I--inset A--I elements could not be identified in ADV (Fig. SB). Perhaps
ADVG NNILPYTPAAPLGETLGFVP WRATKPTOYRYYHPCYIYNR YPNIOKVATETLTWDAVODD
the absence of the PGY and NPYL regions is partly respon-
UTAH NNILPYTPAAPL1ETLGFVP WRATKPTOYRYYHPCYIYNR YPNIOKLGOEOLEWT6TODD sible for some of the unusual biological properties of ADV,
ADVO YLSVDEOYFNFITIENNIPI NILRTGDNFHTGLYEFNSKP CKLTLSYOSTRCLGLPPLCK
such as the inability to spread well in culture (20, 59). The
UTAH YLSVDEOYFNFITIENNIPI NILRTGDNFHTGLYEFNSKP CKLTLSYOSTRCLGLPPLCK
effect of mutations in this region would therefore be inter-
esting to observe in MVM or other parvoviruses.
ADVG PKTDTTHKVTSKENOADLIY IOGODNTRLGHFWGEERGKK NAEMNRIRPYNIGYOYPEWI The final set of data that we present here is a comparison
UTAH PKTDTTHKVTSKENGADLIY IOGODNTRLGHFI4GEERGKK NAEMNRVRPYNIGYOYPEWI
of the ADV-G sequence to a partial sequence of the virulent
ADV-Utah 1 (Fig. 7 and 8). The two strains are highly
ADVG IPAGLOGSYFAGGPROlWSDT TKGAGTHSOHLOONFSTRYI YDRNHGGDNEVDLLDGIPIH related (>95% at both the DNA and amino acid levels). A
UTAH IPAGLOGSYFAGGPROWSDT TKGAGTHSOWLOONFSTRYI YDRNHGGDNEVDLLDGIPIH deletion of a single base occurs in the sequence of ADV-
Utah 1 in the position corresponding to nt 806 of ADV-G that
ADVO ERSNYYSDNE IEOHTAKOPK LRTPPIHHSK IDSWEEEGWP AASGTHFEDEVIYLDYFNFS shifts the left ORF of ADV-Utah 1 from translational frame
UTAH ERSNYYSDHEIEOHTAKOPK LRTPPIHHSKIDSWEEEGWP AASGTHFEDEVIYLDYFNFS
PIW REGION
ADVG 6EOELNFPHEVLDDAAOMKK LLNSYOPTVAODNVGPVYPW GOIWDKKPHMDHKPSMNNNA
UTAH GEOELEFPHEVLDDAAOMKK LLNSYOPTVAODNVGPVYPW GOIWDKKPDMDHKPSMNNNA FIG. 7. Comparison of the right ORFs of ADV-G and ADV-Utah
1. The right ORF of ADV-G from the start at nt 2241 to the amino
ADVO PFVCKNNPPGOLFVKLTENL TDTFNYDENPDRIKTYGYFT WRGKL acid corresponding to the 3' end of the Hindlll site (nt 4176) was
UTAH PFVCKNNPPGOLFVKLTENL TDTFNYDENPDRIKTYGYFT WRGKL
aligned with the theoretical translation of the analogous segment of
ADV-Utah 1, using the single-letter amino acid code. Nonidentical
inset A
or unmatched residues are emphasized (*). (Inset A) Detail of
nucleotide and amino acid changes at the localized area of marked
I leGlnLysVaIAI*ThrGluTh rL&uThrTrpAspAlaValGln
ADVG 3090 ATTCAAAAAGTTGCAACAGAAAC-ACTAACCTGGGATSCAGTACAA 3134 difference described in the text. To better illustrate the effects of
UTAH ATTC-AAAAGCTGGSGCAGGAOCAATTAOAATGGACTGSTACACAA
nucleotide changes on amino acid sequence, the three-letter amino
IeSInLySLeuGlyGlnBluGlnL-uGluTrpThrGlyThrSln acid code was used.
2912 BLOOM ET AL. J. VIROL.

RESIDUES 1 -201

3J0
I 1 I
2.0 2.0

1.0

-1.0 .1.0

2.0 -2.0

-3 .0

RESIDUES 201 - 401

3.0 111111..
2.0 20,

1.0I

2.0 ''-2.0
-3.0 -3.0

RESIDUES 401 - 601

2.0 2.0

-2.0 -2.0

-30.0 30

RESIDUES 601 e 702

.30
30

1.0

-2.0

FIG. 8. Comparative hydropathy plot of the right ORFs of ADV-G and ADV-Utah 1. Hydropathy plots of the entire 702-amino-acid right
ORF of ADV-G (nt 2241 to 4346) and the 645-amino-acid right ORF sequence available for ADV-Utah 1 were constructed with the Microgenie
sequence program, using the procedure of Hopp and Woods (41). The plots were compared, and the regions unique to ADV-Utah 1 are
signified with a solid line. The positions of the individual amino acid changes between ADV-G and ADV-Utah 1 are indicated by vertical
marks above the plot. Those portions above the horizontal axis were hydrophilic, and those below were hydrophobic. The apparent
divergence at the end of the ADV-Utah 1 plot was due to the fact that the available right ORF of ADV-Utah 1 ends at residue 645.
VOL. 62, 1988 SEQUENCE ANALYSIS OF ADV 2913

2 to frame 1. Although we did not have additional molecular observed, but on the other hand, the unsequenced regions
clones of ADV-Utah 1 to compare, we speculated that this may also play a role in pathogenicity. Further molecular
change was a cloning artifact because the left ORFs of analysis will hopefully provide additional insight into these
ADV-G and all other parvoviruses are continuous. Addi- problems.
tional support for this notion may be obtained from the
observation that p71, the NS-1 product of the ADV left ORF ACKNOWLEDGMENTS
(M. E. Bloom, S. Alexandersen, and J. B. Wolfinbarger, We acknowledge the assistance of Anders Cohn, Oskar Ruger-
Second Parvovirus Workshop, 1988) is the same size in both Kaaden, Sven Bergstrom, Camille Locht, Eric Huggans, Kenneth
ADV-G and ADV-Utah 1 (-71,000 daltons) (5, 61), close to Robbins, and Danny Weidbrauk in the initial stages of this work.
the theoretical coding capacity of the total left ORF of Other members of the staff of the Rocky Mountain Laboratories
ADV-G. provided helpful discussion throughout the work. Robert Evans and
The major differences between ADV-G and ADV-Utah 1 Gary Hettrick aided in the production of the illustrations, and Irene
relate to the ability of the former to grow well in CRFK but Cook Rodriguez assisted with preparation of the manuscript.
poorly in mink (20) and the capacity of the latter to grow and This work was supported in part by the Danish Fur Breeders
to cause fulminant disease in mink (20, 21, 38, 58, 60) but not Association Research Foundation. Soren Alexandersen is an NIH
to replicate productively in CRFK cells (20, 39). There are Visiting Associate on leave from the Institute of Veterinary Pathol-
only 23 amino acid changes between the right ORFs of the ogy, Royal Veterinary and Agricultural University of Copenhagen,
Denmark.
two ADV strains; however, a short amino acid stretch at
64-65 MU shows a marked divergence between the two LITERATURE CITED
strains (8 of 11 amino acids) (Fig. 6, 7, and 8). The observa- 1. Alexandersen, S. 1986. Acute interstitial pneumonia in mink
tion of this short divergent segment is particularly intriguing kits: experimental reproduction of the disease. Vet. Pathol. 23:
because hypervariable regions have been reported in the 579-588.
capsid genes for two strains of MVM [the immunosuppres- 2. Alexandersen, S., and M. E. Bloom. 1987. Studies on the
sive MVM(i) and the prototypic MVM(p)] (9, 66) and for sequential development of acute interstitial pneumonia caused
CPV-FPV (56). In both instances, the regions map to a by Aleutian disease virus in mink kits. J. Virol. 61:81-86.
similar location in the right ORF. This hypervariability 3. Alexandersen, S., M. E. Bloom, and J. Wolfinbarger. 1988.
correlates with the viral host range (CPV-FPV) (56) and the Evidence of restricted viral replication in adult mink infected
with Aleutian disease of mink parvovirus. J. Virol. 62:1495-
ability of the virus to initiate a productive rather than 1507.
restrictive infection [MVM(i)-MVM(p)] (44, 72; P. Tatter- 4. Alexandersen, S., M. E. Bloom, J. Wolfinbarger, and R. E. Race.
sall, R. Moir, and E. Gardner, Second Parvovirus Work- 1987. In situ molecular hybridization for detection of Aleutian
shop, 1987). Perhaps this region in ADV functions in an mink disease parvovirus DNA by using strand-specific probes:
analogous fashion and is a major determinant of host range identification of target cells for viral replication in cell cultures
and pathogenicity. and in mink kits with virus-induced interstitial pneumonia. J.
It is possible to discriminate ADV strains antigenically by Virol. 61:2407-2419.
using monoclonal antibodies, although most monoclonal 5. Alexandersen, S., A. Uttenthal-Jensen, and B. Aasted. 1986.
antibodies react with all ADV strains (61). Therefore, it is Demonstration of non-degraded Aleutian disease virus (ADV)
interesting to speculate that the sequence variation observed proteins in lung tissue from experimentally infected mink kits.
Arch. Virol. 87:127-133.
in the right ORFs of ADV-G and ADV-Utah 1 may relate to 6. Anderson, M. J. 1987. Human parvovirus infections. J. Virol.
these minor differences in antigenicity. The amino acid Methods 17:175-181.
changes in the right ORFs were found both in hydrophobic 7. Anton, I. A., and D. P. Lane. 1986. Non-structural protein 1 of
and hydrophilic regions (Fig. 8). Although single-amino-acid parvoviruses: homology to purine nucleotide using proteins and
changes can greatly influence pathogenicity and antigenicity early proteins of papovaviruses. Nucleic Acids Res. 14:7813.
(25, 35, 36), it is difficult to predict from the primary 8. Armentrout, R., R. Bates, K. Berns, B. Carter, M. Chow, D.
nucleotide sequence the location of major epitopes (13, 37) Dressier, K. Fife, W. Hauswirth, G. Hayward, G. Lavelle, S.
or the effect of a single change (25, 33, 35, 37). Nevertheless, Rhode, S. Strauss, P. Tattersall, and D. Ward. 1978. A standard
nomenclature for restriction endonuclease fragments, p. 523-
it would be interesting to synthesize peptides that spanned 526. In D. Ward and P. Tattersall (ed.), Replication of parvovi-
these variable regions and to determine whether they could ruses. Cold Spring Harbor Laboratory, Cold Spring Harbor,
induce virus strain-specific responses or modify the outcome N.Y.
of ADV infection. 9. Astell, C. R., E. M. Gardiner, and P. Tattersall. 1986. DNA
Finally, when infectious ADV clones are constructed, it sequence of the lymphotropic variant of minute virus of mice,
will be possible to construct ADV-G/ADV-Utah 1 chimeras MVM(i), and comparison with the DNA sequence of the fibro-
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and an unusual left-hand terminus tentatively suggest struc- antigenic structure. Science 229:932-940.
tural features that might account for the unusual nature of 14. Biggin, M. D., T. J. Gibson, and G. F. Hong. 1983. Buffer
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