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Journal of Virology, February 1999, p. 939-947, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Human Adeno-Associated Virus Type 5 Is Only Distantly Related to
Other Known Primate Helper-Dependent Parvoviruses
Ursula
Bantel-Schaal,*
Hajo
Delius,
Rainer
Schmidt, and
Harald
zur
Hausen
Deutsches Krebsforschungszentrum Heidelberg,
Forschungsschwerpunkt Angewandte Tumorvirologie F0400, D-69120
Heidelberg, Germany
Received 14 September 1998/Accepted 29 October 1998
 |
ABSTRACT |
We have characterized 95% (4,404 nucleotides) of the genome of
adeno-associated virus type 5 (AAV5), including part of the terminal
repeats and the terminal resolution site. Our results show that AAV5 is
different from all other described AAV serotypes at the nucleotide
level and at the amino acid level. The sequence homology to AAV2,
AAV3B, AAV4, and AAV6 at the nucleotide level is only between 54 and
56%. The positive strand contains two large open reading frames
(ORFs). The left ORF encodes the nonstructural (Rep) proteins, and the
right ORF encodes the structural (Cap) proteins. At the amino acid
level the identities with the capsid proteins of other AAVs range
between 51 and 59%, with a high degree of heterogeneity in regions
which are considered to be on the exterior surface of the viral capsid.
The overall identity for the nonstructural Rep proteins at the amino
acid level is 54.4%. It is lowest at the C-terminal 128 amino acids
(10%). There are only two instead of the common three putative Zn
fingers in the Rep proteins. The Cap protein data suggest differences
in capsid surfaces and raise the possibility of a host range distinct
from those of other parvoviruses. This may have important implications for AAV vectors used in gene therapy.
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INTRODUCTION |
Adeno-associated viruses (AAVs) are
small, nonenveloped viruses that encapsidate single-stranded DNA of
both polarities in equal amounts. They belong to the
Parvoviridae family and are distinct from other members of
this family by their dependence on helpers for replication (for
reviews, see references 7-10, 31, and
37). Six primate AAV serotypes have been reported in
the literature (2, 5, 42). They are designated types 1 to 6 (AAV1 to AAV6). With the exception of AAV5, which has been isolated
from a penile flat condylomatous lesion (5, 19), all known
AAVs were first found as contaminants in laboratory adenovirus stocks
(1, 29, 34, 42). Up to now, the DNAs of AAV2, AAV3,
AAV4, and AAV6 have been sequenced (17, 36, 41, 42,
51). The sequence identities among the different serotypes are
high. The identities within the genomes of AAV2, AAV3, and AAV6 are
82%, and with AAV4 they still range from 75 to 78% (17, 36,
42). For AAV3, two sequences (designated AAV3A and 3B) have been
published which have differences in 16 nucleotides (36, 42).
In the group of autonomous parvoviruses, the closest relative appears
to be the goose parvovirus. At the genomic level and at the level of
the capsid proteins, homologies with sequenced AAVs of ca. 54% have
been reported (17, 36, 62), and for the nonstructural
proteins of AAV3 the identities are ca. 44% (36).
Two large open reading frames (ORFs) have been identified within the
AAV genome (7, 8, 17, 36, 37, 41, 42, 51). Experimental data
on translation and transcription have mainly been obtained for AAV2,
but predictions based on nucleotide sequence analogy could be made for
the other AAV serotypes. The left ORF of AAV2 encodes the nonstructural
Rep proteins that are transcribed from two separate promoters (p5 and
p19, according to their relative map positions). The transcripts from
both promoters are translated from spliced and unspliced mRNAs,
resulting in four proteins designated Rep78, Rep68, Rep52, and Rep40.
The Rep proteins are regulators of AAV transcription; they are involved in multiple steps of AAV replication, and they play a role in the
production of single-stranded progeny genomes and virus assembly (7-10, 13-16, 27, 30, 33, 37, 38, 43, 55, 56, 60). In
addition, Rep proteins are required for site-specific integration of
AAV DNA into the host cell genome (44, 45, 58). Furthermore, they are able to modulate transcription from heterologous promoters (6, 9, 21-23, 25, 26, 28, 32, 37, 61). The degree of
sequence conservation for the Rep proteins is high among AAV2, AAV3A,
AAV3B, AAV4, and AAV6. The Rep78 proteins from these viruses are
reported to be 89 to 93% identical to each other (17, 36, 42). This is thought to mirror their important basic functions in
the AAV life cycle (7-10, 17, 37).
The AAV cap gene is located in the right half of the AAV
genome and codes for the three capsid proteins VP1, VP2, and VP3, with
VP3 being the smallest but most abundant; VP1 has the highest molecular
weight but is present in a much smaller quantity (as shown for AAV2,
AAV3, and AAV5 [7-10, 19]). The respective mRNA is
translated from the p40 promoter. As has been shown for AAV2, the Cap
proteins differ from each other due to alternative splicing and by the
use of an unusual start codon (ACG) for VP2 (7-11, 37). In
contrast to the Rep proteins, the reported degree of sequence
conservation among the capsid proteins is smaller. This is likely to
provide a basis for differences in host range and host cell
specificities (17, 36, 37, 42).
AAVs are interesting for several reasons. First of all, they have
oncosuppressive properties (3, 22-26, 52, 57; for a
review, see reference 40), and they are useful as
general transduction vectors for gene therapeutic approaches in human cells (for reviews, see references 31, 37, and
48). Much work has been done with vectors derived
from AAV2 (31, 37). However, vectors derived from other AAV
serotypes could provide several additional advantages, including the
dependence on different cell receptors, resulting in transduction into
different cell types, and the resistance to neutralizing antibodies
directed against AAV2 (17, 35, 36, 42, 53).
Here we report the partial sequence of AAV5 covering about 95% of the
AAV5 genome (4,404 nucleotides [nt]), with the exception of the
terminal hairpin structures but including the terminal resolution site
and its inverted counterpart (50). The data show that the
genomic organization of AAV5 is similar to that of other AAVs but that
the interserotype homology is reduced to values of between 54 and 56%
when AAV5 is included into alignments. The differences concern both
ORFs. The overall percentage of identical amino acids in the structural
proteins is less than 45% and, in contrast to the case with other
AAVs, the overall percentage of identical amino acids encoded by the
left-sided ORF is also strongly reduced from 83.4 to 54.4%. Thus, AAV5
is clearly distinct from the other known AAV serotypes.
| |
MATERIALS AND METHODS |
Cell culture and virus stocks.
HeLa cells were cultured as
monolayers in Dulbecco's modified Eagle medium (DMEM; Sigma,
Deisenhofen, Germany), supplemented with 5% heat-treated fetal calf
serum and glutamine and penicillin-streptomycin at standard
concentrations. These cell cultures were used to propagate AAV5 with
the helper-adenovirus type 2 (5). Preparations of AAV5
stocks were essentially done as described previously (5), except for the ammonium sulfate precipitation, which was replaced by a
centrifugation step at 13,000 rpm for 60 min in the Sorvall SS34 rotor
(Sorvall Instruments).
Preparation of viral DNA and cloning of restriction
fragments.
Viral DNA was isolated from purified virus particles by
using alkaline conditions (0.1 N NaOH for 45 min at room temperature). The solution was neutralized, and the DNA was purified with the Geneclean II kit for removing proteins (Bio 101, Inc., La Jolla, Calif.). Restriction fragments (BamHI, EcoRI,
SacI, and XhoI) were cloned into bacterial
plasmids by standard protocols. pBluescript II(KS+) and pUC18 were used
as bacterial vectors for cloning in the bacterial strains HB101 and
XL1-Blue.
DNA sequencing.
The major part of the sequence determination
was done radioactively on plasmid clones of AAV5 by using the dideoxy
termination method (46) with general vector primers and
primers derived from the 3' part of the newly determined sequences. The
terminal sequences and sequences showing deviations between different
subclones were determined directly on the viral DNA by cycle sequencing by using the fluorescent dye terminator method (ABI PRISM Big Dye ready
reaction terminator cycle sequencing kit) on a model 377 automatic
sequencer (Perkin-Elmer/Applied Biosystems) according to the
manufacturer's protocol. The partial terminal sequences were confirmed
by LION Bioscience, Heidelberg, Germany.
Sequences were aligned with NCBI's MACAW program (Multiple Alignment
Construction and Analysis Workbench). Either blocks of similarity or
identities of the aligned sequences were shaded.
Nucleotide sequence accession numbers.
The partial AAV5
nucleotide sequence determined in this study is available through EMBL
databank under accession no. Y18065.
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RESULTS |
Nucleotide sequence and genomic organization.
The partial AAV5
genome is presented in Fig.
1 and is also available
through the EMBL databank.
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FIG. 1.
Partial sequence of AAV5. The 4,404 bases obtained by
sequencing of AAV5 are shown. The putative promoters (p5, p19, and
p40), polyadenylation signal (polyA), start and stop codons for
nonstructural (Rep) and capsid (VP) proteins, splice sites (arrows),
and the trs are underlined and marked by boldface letters. The position
of the inverted terminal sequence counterpart of the terminal
resolution site is also indicated by "trs."
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The major part of the sequence was determined by radioactive sequencing
of cloned restriction fragments derived from viral
DNA isolated from
purified AAV5 particles. Sequence differences
in overlapping parts of
the subclones were resolved by fluorescent
cycle sequencing directly on
the single-stranded viral DNA. Viral
DNA containing the termini could
neither be stably propagated
in bacteria nor could the terminal hairpin
structures be sequenced
by cycle sequencing, since the problem of
polymerase stalling
in the palindromic structure could not be
resolved.
By analogy to other published AAV sequences the sequence presented here
(Fig.
1) includes the terminal resolution site (trs
[
50]) and part of the inverted terminal structures
which end
at the 5' and 3' ends at symmetrical positions (Fig.
2). As far
as the sequence was
determined, the 5' and 3' inverted repeat
stretches are identical
except for a G at position 38, where a
T at position 4365 replaced the
expected C (Fig.
2A). The location
of the TATA boxes, splice sites, and
start and stop codons indicate
a genomic organization similar to that
of other sequenced AAVs
(
11,
17,
36,
41,
42,
51). Promoters
are found around
the expected map position for p5, p19, and p40, and
there is only
one single polyadenylation site at map position 4284 (AATAAA)
of the partial AAV5 sequence. Translation start and
stop codons
for all putative AAV5 nonstructural proteins (Rep) and
capsid
proteins (VPs), including the unusual ACG start codon for VP2
and one common stop codon shared by all three known capsid proteins,
are at the expected locations.
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FIG. 2.
Partial sequence of inverted terminal repeats of AAV5.
(A) Part of the inverted terminal repeats comprising the first 47 nt
that could be deduced at the 5' end (nt 1 to 47) and the last 49 nt of
the 3' end (nt 4356 to 4404). The lowercase letters "g" and "t"
mark the nucleotide pair at positions 38 and 4365 that does not match
the repeat. The putative trs (50) and the respective
inverted sequence are shown in boldface letters. Note that the
underlined sequences may fold back in AAV5 DNA and form a 7-bp
double-stranded loop. (B) Alignment of AAV sequences surrounding the
trs and the respective inverted positions in the 5' and 3' ends of the
DNA molecules. The trs consensus sequence and the respective inverted
sequences are shown in boldface letters. Dashes indicate gaps
introduced in the alignment. Nucleotides included in the alignment were
as follows: AAV2, 104 to 133 and 4547 to 4576; AAV3B, 103 to 132 and
4590 to 4619; AAV4, 104 to 133 and 4635 to 4664; AAV6, 104 to 133 and
4551 to 4580; AAV5 partial sequence, 1 to 30 and 4373 to 4402.
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Although up to now we were not able to determine the complete terminal
sequences of AAV5, the approximate length of the genome
could be
deduced. On the basis of the position of the trs (Fig.
2), about 100 additional nucleotides are expected at each end
of the DNA molecule.
Thus, some 150 to 200 nt presumably would
complete the 4,404 nt of the
partial sequence. The resulting total
of 4,550 to 4,600 nt is in
agreement with the size 4.5 to 4.6
kb derived from agarose gels and the
mapping of restriction fragments
(
5).
To obtain data on binding sites for transcription factors, we made use
of the TRANSFAC database (
59). As with other AAVs
(
8,
17,
36,
42,
51), several putative binding sequences,
such as
CCAAT, GGGCGG, and GGTGGT boxes were found for
AAV5. Others,
such as the cyclic AMP-responsive element (CRE;
TGACGTCA [
20]),
were found in AAV5 but not
in AAV2 DNA or were located at unusual
map positions, e.g., the
consensus sequence GTGACGT for the transcription
factor EivF
(
18,
39). In all AAVs sequenced up to now this
sequence was
found upstream of the p5 region (
8,
17,
36,
42,
51), but in
the case of AAV5 it is at map position 1740
and thus is upstream of the
p40 promoter. Other recognition sites,
such as the sequence targets for
YY1 (CGACATTTT or CTCCATTTT)
near the p5 promoter
of AAV2 or the p7 promoter of AAV4 (
17,
49), were not found
in AAV5
DNA.
Similarities among AAV genomes.
To find regions of local
similarity among the different sequences and to define blocks of
aligned sequence segments, we made use of the MACAW alignment program
(National Center for Biotechnology Information). The different multiple
alignments are shown in schematic form in Fig.
3.
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FIG. 3.
Block alignment of AAV genomic sequences. The complete
sequences of AAV2, AAV3B, AAV4, and AAV6 and the partial sequence of
AAV5 were aligned with the MACAW program. Segment pair overlap with a
minimum segment pair score of 60 was used as a search method to define
nucleotide blocks of local similarity. The aligned blocks were linked
into the alignment (solid bars). To determine the start and end
positions for the alignment of AAV5, the trs (50) were
selected by sequence analogy. The scale of alignment is 5,000 nt.
Nucleotide positions on the individual genomes are in relation to this
scale. The panels show the alignment of AAV2, AAV3B, and AAV6 (A); the
alignment of AAV2, AAV3B, AAV4, and AAV6 (B); and the alignment of
AAV2, AAV3B, AAV4, AAV6, and AAV5 (C). The positions of the conserved
genetic elements are marked, including the p5, p19, and p40 promoters
(position of TATA boxes); the intron splice sites (GT/AG [arrows]);
the polyadenylation signal (polyA); the translation start (up
arrowheads) and stop (down arrowheads) sites for the Rep (Rep 78, 68, 52, and 40) and capsid (VP1 to VP3) proteins; and the terminal
resolution site and its inverted counterpart (both marked trs). Note
that all of the elements in panel A were within blocks defined by the
program and that in panel B only the start of VP3 was excluded, while
in panel C several were no longer in regions that met the criteria
required for block selection [p5, p40, start Rep 78 and 68, stop Rep
78 and 52, start VP1 and VP3, the splice signals, and the poly(A)
site]. Such positions were marked manually. Note that the p40 sites in
panel C are located in stretches of high dissimilarity and were not
aligned.
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When the sequences of AAV2, AAV3B, and AAV6 are compared (score cutoff,
60), almost the entire genomes could be well aligned
into blocks, with the exception of small regions between the terminal
repeats and the transcribed part of the genomes, some heterogeneity
in
the splice region, and one interval corresponding to AAV2 nt
3548 to
3610 (Fig.
3A). These results are in agreement with the
82%
homology reported by Muramatsu et al. between AAV2 and AAV3A
(
36) and by Rutledge et al. among AAV2, AAV3B, and AAV6
(
42).
AAV4 is more distantly related to the genomes of AAV2, AAV3B, and AAV6
(
17,
42). Thus, the inclusion of AAV4 in the alignment
results in the extension and addition of regions of low similarity,
especially in the right half of the genomes (Fig.
3B).
Since we did not succeed in reading the entire sequence of the AAV5
genome, we used the terminal resolution site and the respective
inverted sequence (
50) (Fig.
2B) as reference positions for
aligning the partial AAV5 sequence to the published DNA sequences
of
AAV2, AAV3B, AAV4, and AAV6 (
11,
17,
36,
41,
42,
51).
Alignment of all five sequences resulted in a further reduction
of
regions with similarities above the cutoff (Fig.
3C). The scattered
blocks of sequence similarity around the splice region in the
left half
of the genome (Fig.
3A and B) are replaced by a large
interval spanning
more than 400 nt (nt 1778 to 2249 on the genome
of AAV2 corresponding
to nt 1684 to 2130 on the incomplete AAV5
genome). This region is also
covered by a
BamHI restriction fragment
(
5) used
to design AAV5-specific primers (
54).
We also used pairwise alignments to find out whether AAV5 may be more
closely related to one or more of the other members
of the AAV family.
But all homologies of the four pairs were between
54 and 56%, and the
obtained patterns (data not shown) were comparable
to the picture shown
for the overall alignment in Fig.
3C. Thus,
AAV5 appears to be more
distant from the rather closely related
group of AAV2, AAV3, AAV4, and
AAV6. This finding is in agreement
with the hybridization data reported
in 1984 (
5).
Nonstructural (Rep) protein coding region.
By analogy with
other AAVs, the AAV5 left-sided ORF encodes the putative unspliced
nonstructural Rep proteins. The complete ORF (nt 239 to 2068) encodes a
protein of 610 amino acids, and the additional ATG start codon (nt 899)
would give rise to a smaller unspliced protein of 390 amino acids (nt
899 to 2068). Thus, both putative unspliced AAV5 Rep proteins are
slightly smaller than those reported for the other AAV serotypes
(8, 17, 36, 37, 42).
When the aligned left-sided ORFs of AAV2, AAV3A, AAV3B, AAV4, and AAV6
were compared at the amino acid level, the overall
identity was 83.4%
(data not shown; see also Fig.
4A).
However,
the percentage of identical amino acid residues was reduced to
54.4% when AAV5 was included in the alignment (see also Fig.
4B
and
5). When Fig.
4A is compared with Fig.
4B, it becomes evident
that the amount of identical amino acids was
reduced throughout
the sequence, but it was most striking for the
sequences from
amino acid 483 of AAV5 (corresponding to residue 487 of
AAV2)
to the C-terminal amino acid 610 (621 of AAV2). Whereas
without
AAV5 the identity in this part of the aligned sequences could
still be attributed to 91 of 135 (67.4%) of the amino acids, the
value
was only 13 identical residues of 128 (10.1%) when it was
included
(see Fig.
5).
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FIG. 4.
Comparison of the left-sided ORFs of AAV. The left-sided
ORFs of AAV were aligned with the MACAW program. Segment pair overlap
with a minimum segment pair score of 60 was used as a search method to
define amino acid (aa) blocks of local similarity. The aligned blocks
were linked and identical amino acid residues were shaded. Shown are
alignments of AAV2, AAV3A, AAV3B, AAV4, and AAV6 (A) and AAV2, AAV3A,
AAV3B, AAV4, AAV6, and AAV5 (B).
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FIG. 5.
Putative amino acid sequences of the Rep ORFs. The Rep
ORFs of AAV2, AAV3A, AAV3B, AAV4, AAV6, and AAV5 were compared by using
the MACAW alignment program. Identical amino acids are shown with white
letters on a black background. Dashes indicate gaps in the sequence
added by the alignment program. The putative ATP-binding site (ATP) and
Zn fingers (three pairs of black dots above the sequences for all AAVs
except AAV5, and two pairs of black triangles below the sequences for
AAV5) are marked. ×, start of the two unspliced Rep proteins.
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The differences between the AAV5 noncapsid proteins and the respective
proteins of the other AAV serotypes may affect the
secondary structure
and functional domains. While the putative
ATP-binding site
(
47) is well conserved in all AAVs, one of
the three
putative Zn fingers at the carboxyl terminus (for a
review, see
reference
10) is missing in the AAV5 Rep proteins
(Fig.
5). The distances between the three Zn-binding motifs (CXXH/CXXC)
in AAV2 are 9, 4, and 9 amino acids. The respective two motifs
in AAV5
have distances of 9 and 4 amino
acids.
Capsid protein coding region.
The right-sided ORF (nt 2087 to
4258) encodes a capsid protein (VP1) of 724 amino acids, with a
predicted molecular mass of 80 kDa. The VP2 capsid (nt 2495 to 4258),
which by analogy with other AAVs is presumably initiated at an ACG
triplet, and the VP3 capsid (nt 2663 to 4258) would comprise 588 and
532 amino acids, respectively, with corresponding molecular masses of
65 and 59 kDa. Thus, all of the molecular masses predicted for the AAV5 capsid proteins are smaller than those reported for other AAVs (8, 17, 36, 37, 42).
In a group alignment of the amino acid sequences of the capsid proteins
of AAV2, AAV3, and AAV6, very high identities of more
than 80% were
found (Table
1). The similarity was
considerably
decreased to 59.7% when AAV4 was included in the
alignment (Table
1). The pairwise
alignments yielded identities between 61.5%
for AAV2 versus AAV4 and
64.8% for AAV3B versus AAV4 (Table
1).
Blocks of very high similarity
were obtained from amino acids
1 to 173 (AAV2) and amino acids 599 to
735 upon both pairwise
and overall alignments of the respective
sequences.
The results of pairwise alignments of capsid ORFs of AAV2, AAV3A,
AAV3B, AAV4, and AAV6 to AAV5 showed identities between
51.4% for AAV4
versus AAV5 and 58.8% for AAV6 versus AAV5 (Table
1), and the
alignment of all six capsid ORFs resulted in a reduction
of overall
amino acid identity to 44.9% (Fig.
6;
Table
1). This
is in correlation to the increased heterogeneity seen
upon alignment
of the respective nucleotide sequences (Fig.
3C).
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FIG. 6.
Alignment of the right-sided cap ORFs of AAV. The
right-sided ORFs of AAV were aligned with the MACAW program. Segment
pair overlap with a minimum segment pair score of 60 was used as the
search method to define amino acid blocks of local similarity.
Identical amino acid residues are shown with white letters on a black
background. VP1, VP2, and VP3 indicate the beginnings of the respective
capsid proteins.
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As can be seen from the results listed in Table
1, including either
AAV4 or AAV5 in the group alignments strongly reduced
the percentage of
identities between the capsid ORFs of the AAV
serotypes 2, 3A,
3B, and 6 (from 80.1 to 59.7 and 53.2%, respectively).
However, the
pairwise alignment of the capsid ORFs of AAV4 and
AAV5 yields already a
lower degree of similarity (51.4; Table
1). Thus, the capsid ORFs of
both AAV4 and AAV5 differ from the
other serotypes as well as from
each other. Therefore, the percentage
of identical amino acids
decreases further (44.9%) when both AAV4
and AAV5 are included in the
group alignment (Table
1).
| |
DISCUSSION |
We have determined the sequence of 4,404 nt (about 95% of the
estimated size of 4.5 to 4.6 kb) of the AAV5 genome, but up to now we
were not able to resolve the complete sequence of the terminal repeats.
Using the trs (50), the respective inverted sequence, and
the TATAA box in the p5 promoter as reference points, we have made
alignments of the AAV5 sequence to the published sequences of AAV2,
AAV3B, AAV4, and AAV6 (17, 36, 41, 42, 51). The overall
organization within the AAV5 genome is similar to that of other AAVs
with three putative promoters, one single polyadenylation site, and two
large ORFs.
As reported by others, the identities on the nucleotide level between
AAV2, AAV3, and AAV6 exceeded 82% and were still as high as 75% when
AAV4 was included (17, 36, 42). In contrast to the good
sequence conservation between the other AAV serotypes, AAV5 is clearly
more distantly related, and the overall identity is reduced to about
56% when its nucleotide sequence is included in the alignment. Thus,
the distance between AAV5 and the other AAV serotypes is in the same
range as that reported for the other AAVs compared to their closest
relatives among autonomous parvoviruses, namely, the goose and duck
parvoviruses (17, 36, 62).
The differences in the nucleotide sequence near the p5 and the p40
promoters appear to be connected to differences in the adenovirus
transcription factor recognition sites. Thus, the EivF element involved
in E1A-responsive transactivation of the adenovirus E4 promoter
(18, 39) is shifted from the common region upstream of the
p5 promoter to a position upstream of the p40 promoter in AAV5, and the
YY1 sites (49) are not found in the AAV5 genome. If and how
these consensus sequences and other sequences (e.g., the CRE element
[20]) are involved in adenovirus transactivation remains to be determined.
Within the coding regions, stretches of dissimilarity were most
prominent at AAV5 nt 1684 to 2130 (AAV2 nt 1778 to 2249), 2504 to 2692 (AAV2 nt 2623 to 2839), and 3393 to 3848 (AAV2 nt 3529 to 3993). The
last two affect all three capsid proteins. Thus, the capsid proteins of
AAV5 differ significantly from the respective proteins of the other
serotypes, and the overall percentage of identical amino acids is less
than 45% (Table 1). Since the respective differences comprise regions
which are supposed to be exposed at the virus surface (12),
tissue tropism, cellular receptor(s), and resistance towards AAV
neutralizing antibodies might be different from those of other AAVs.
Thus, the host range of AAV5 may be distinct. This should be of
interest for the construction of AAV gene transduction vectors and for
their application in gene therapy (31, 37, 48).
The region of nucleotide sequence dissimilarity at AAV5 map positions
1684 to 2130 (AAV2 nt 1778 to 2249) reflects differences in the Rep
proteins. This certainly represents the most striking finding, since
the sequence of Rep proteins was thought to be highly conserved because
of the essential functions that these proteins have in the AAV life
cycle. Whether the altered sequence of the AAV5 Rep proteins and the
lack of a third Zn finger motif leads to structurally and functionally
different proteins remains to be elucidated. It is noteworthy that the
alignment of each of the AAV sequences to the respective sequences of
goose parvovirus (4, 62) showed a high sequence conservation
around the Rep ATP-binding site (47). This result
(4) is in agreement with the high degree of sequence
similarity seen at the respective positions when the Rep ORFs of all
AAVs are aligned (Fig. 5). Thus, while the C-terminal sequences and
the Zn fingers may be more flexible, the high degree of conservation of
the ATP-binding site suggests its unique structure and points to its
central role in the viral life cycle.
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FOOTNOTES |
*
Corresponding author. Mailing address: Deutsches
Krebsforschungszentrum Heidelberg, Forschungsschwerpunkt Angewandte
Tumorvirologie F0400, Im Neuenheimer Feld 242, D-69120
Heidelberg, Germany. Phone: 49-6221-424823. Fax:
49-6221-424822. E-mail:
u.bantel-schaal{at}dkfz-heidelberg.de.
| |
REFERENCES |
| 1.
|
Atchison, R. W.,
B. C. Casto, and W. M. Hammon.
1965.
Adenovirus-associated defective virus particles.
Science
194:754-756.
|
| 2.
|
Bachmann, P. A.,
M. D. Hoggan,
J. L. Melnick,
H. G. Pereira, and C. Vago.
1975.
Parvoviridae.
Intervirology
5:83-92[Medline].
|
| 3.
|
Bantel-Schaal, U.
1995.
Growth properties of a human melanoma cell line are altered by adeno-associated parvovirus type 2.
Int. J. Cancer
60:269-274[Medline].
|
| 4.
| Bantel-Schaal, U., and H. Delius. 1998. Unpublished results.
|
| 5.
|
Bantel-Schaal, U., and H. zur Hausen.
1984.
Characterization of the DNA of a defective human parvovirus isolated from a genital site.
Virology
134:52-63[Medline].
|
| 6.
|
Beaton, A.,
P. Palumbo, and K. I. Berns.
1989.
Expression from the adeno-associated virus p5 and p19 promoters is negatively regulated in trans by the rep protein.
J. Virol.
63:4450-4454[Abstract/Free Full Text].
|
| 7.
|
Berns, K. I.
1990.
Parvovirus replication.
Microbiol. Rev.
54:316-329[Abstract/Free Full Text].
|
| 8.
|
Berns, K. I.
1995.
Parvoviridae: the viruses and their replication, p. 1017-1041.
In
B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fundamental virology, 3rd ed. Lippincott-Raven Publishers, Philadelphia, Pa.
|
| 9.
|
Berns, K. I., and M. A. Labow.
1987.
Parvovirus gene regulation.
J. Gen. Virol.
68:601-614[Abstract/Free Full Text].
|
| 10.
|
Carter, B. J.,
J. P. Trempe, and E. Mendelson.
1990.
Adeno-associated virus gene expression and regulation, p. 227-254.
In
P. Tijsen (ed.), Handbook of parvoviruses, vol. 1. CRC Press, Boca Raton, Fla.
|
| 11.
|
Cassinotti, P.,
M. Weitz, and J. D. Tratschin.
1988.
Organization of the adeno-associated virus (AAV) capsid gene: mapping of a minor spliced mRNA coding for virus capsid protein 1.
Virology
167:176-184[Medline].
|
| 12.
|
Chapmann, M. S., and M. G. Rossmann.
1993.
Structure, sequence, and function correlations among parvoviruses.
Virology
194:491-508[Medline].
|
| 13.
|
Chejanovsky, N., and B. J. Carter.
1989.
Mutagenesis of an AUG codon in the adeno-associated virus rep gene: effects on viral DNA replication.
Virology
171:120-128[Medline].
|
| 14.
|
Chejanovsky, N., and B. J. Carter.
1990.
Mutation of a consensus purine nucleotide binding site in the adeno-associated virus rep gene generates a dominant-negative phenotype for DNA replication.
J. Virol.
51:329-339.
|
| 15.
|
Chiorini, J. A.,
S. M. Wiener,
R. M. Kotin,
R. A. Owens,
S. R. M. Kyöstiö, and B. Safer.
1994.
Sequence requirements for stable binding and function of Rep68 on the adeno-associated virus type 2 inverted terminal repeats.
J. Virol.
68:7448-7457[Abstract/Free Full Text].
|
| 16.
|
Chiorini, J. A.,
L. Yang,
B. Safer, and R. M. Kotin.
1995.
Determination of adeno-associated virus Rep68 and Rep78 binding sites by random sequence oligonucleotide selection.
J. Virol.
69:7334-7338[Abstract].
|
| 17.
|
Chiorini, J. A.,
L. Yang,
Y. Liu,
B. Safer, and R. M. Kotin.
1997.
Cloning of adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4 particles.
J. Virol.
71:6823-6833[Abstract].
|
| 18.
|
Cortes, P.,
L. Buckbinder,
M. A. Leza,
N. Rak,
P. Hearing,
A. Merino, and D. Reinberg.
1988.
EivF, a factor required for transcription of the adenovirus EIV promoter, binds to an element involved in E1a-dependent activation and cAMP induction.
Genes Dev.
2:975-990[Abstract/Free Full Text].
|
| 19.
|
Georg-Fries, B.,
S. Biederlack,
J. Wolf, and H. zur Hausen.
1984.
Analysis of proteins, helper dependence, and seroepidemiology of a new human parvovirus.
Virology
134:64-71[Medline].
|
| 20.
|
Hardy, S., and T. Shenk.
1988.
Adenoviral control regions activated by E1A and the cAMP response element bind to the same factor.
Proc. Natl. Acad. Sci. USA
85:4171-4175[Abstract/Free Full Text].
|
| 21.
|
Heilbronn, R.,
A. Bürkle,
S. Stephan, and H. zur Hausen.
1990.
The adeno-associated virus rep gene suppresses herpes simplex virus-induced DNA amplification.
J. Virol.
64:3012-3018[Abstract/Free Full Text].
|
| 22.
|
Hermonat, P. L.
1991.
Inhibition of H-ras expression by the adeno-associated virus Rep78 transformation suppressor gene product.
Cancer Res.
46:3373-3377.
|
| 23.
|
Hermonat, P. L.
1994.
Down regulation of the human c-fos and c-myc protooncogene promoters by adeno-associated virus Rep78.
Cancer Lett.
81:129-136[Medline].
|
| 24.
|
Hermonat, P. L.
1994.
Adeno-associated virus inhibits human papillomavirus type 16: a viral interaction implicated in cervical cancer.
Cancer Res.
54:2278-2281[Abstract/Free Full Text].
|
| 25.
|
Hermonat, P. L.,
R. T. Plott,
A. D. Santin,
G. P. Parham, and J. T. Flick.
1997.
Adeno-associated virus Rep78 inhibits oncogenic transformation of primary human keratinocytes by a human papillomavirus type 16-ras chimeric.
Gynecol. Oncol.
66:487-494[Medline].
|
| 26.
|
Hermonat, P. L.,
A. D. Santin, and R. B. Batchu.
1996.
The adeno-associated virus Rep78 major regulatory/transformation suppressor binds Sp1 in vitro and evidence of a biological effect.
Cancer Res.
56:5299-5304[Abstract/Free Full Text].
|
| 27.
|
Hölscher, C.,
J. A. Kleinschmidt, and A. Bürkle.
1995.
High-level expression of adeno-associated virus (AAV) Rep78 and Rep68 protein is sufficient for infectious-particle formation by a rep-negative AAV mutant.
J. Virol.
69:6880-6885[Abstract].
|
| 28.
|
Hörer, M.,
S. Weger,
K. Butz,
F. Hoppe-Seyler,
C. Geisen, and J. A. Kleinschmidt.
1995.
Mutational analysis of adeno-associated virus Rep protein-mediated inhibition of heterologous and homologous promoters.
J. Virol.
69:5485-5496[Abstract].
|
| 29.
|
Hoggan, M. D.,
N. R. Blacklow, and W. P. Rowe.
1966.
Studies of small DNA viruses found in various adenovirus preparations: physical, biological, and immunological characteristics.
Proc. Natl. Acad. Sci. USA
55:1467-1474[Free Full Text].
|
| 30.
|
Hunter, L. A., and R. J. Samulski.
1992.
Colocalization of adeno-associated virus Rep and capsid proteins in the nuclei of infected cells.
J. Virol.
66:317-324[Abstract/Free Full Text].
|
| 31.
|
Kotin, R. M.
1994.
Prospects for the use of adeno-associated virus as a vector for human gene therapy.
Hum. Gene Ther.
5:793-801[Medline].
|
| 32.
|
Kyöstiö, S. R.,
R. A. Owens,
M. D. Weitzmann,
B. Antoni,
N. Chejanovsky, and B. J. Carter.
1994.
Analysis of adeno-associated virus (AAV) wild-type and mutant Rep proteins for their abilities to negatively regulate AAV p5 and p19 mRNA levels.
J. Virol.
68:2947-2957[Abstract/Free Full Text].
|
| 33.
|
McCarty, D. M.,
D. J. Pereira,
I. Zolotukhin,
X. Zhou,
J. H. Ryan, and N. Muzyczka.
1994.
Identification of linear DNA sequences that specifically bind the adeno-associated virus Rep protein.
J. Virol.
68:4988-4997[Abstract/Free Full Text].
|
| 34.
|
Melnick, J. L.,
H. D. Mayor,
K. O. Smith, and F. Rapp.
1965.
Association of 20 millimicron particles with adenoviruses.
J. Bacteriol.
90:271-274[Free Full Text].
|
| 35.
|
Mizukami, H.,
N. S. Young, and K. E. Brown.
1996.
Adeno-associated virus type 2 binds to a 150-kilodalton cell membrane glycoprotein.
Virology
217:124-130[Medline].
|
| 36.
|
Muramatsu, S.-I.,
H. Mizukami,
N. S. Young, and K. E. Brown.
1996.
Nucleotide sequencing and generation of an infectious clone of adeno-associated virus 3.
Virology
221:208-217[Medline].
|
| 37.
|
Muzyczka, N.
1992.
Use of adeno-associated virus as a general transduction vector for mammalian cells.
Curr. Top. Microbiol. Immunol.
158:97-129[Medline].
|
| 38.
|
Prasad, K.-M. R., and J. P. Trempe.
1995.
The adeno-associated virus Rep78 protein is covalently linked to viral DNA in a preformed virion.
Virology
214:360-370[Medline].
|
| 39.
|
Raychaudhuri, P.,
R. Rooney, and J. R. Nevins.
1987.
Identification of a E1A-inducible cellular factor that interacts with regulatory sequences within the adenovirus E4 promoter.
EMBO J.
6:4073-4081[Medline].
|
| 40.
|
Rommelaere, J., and J. J. Cornelis.
1991.
Antineoplastic activities of parvoviruses.
J. Virol. Methods
33:233-251[Medline].
|
| 41.
|
Ruffing, M.,
H. Heid, and J. A. Kleinschmidt.
1994.
Mutations in the carboxy terminus of adeno-associated virus 2 capsid proteins affect viral infectivity: lack of an RGD integrin-binding motif.
J. Gen. Virol.
75:3385-3392[Abstract/Free Full Text].
|
| 42.
|
Rutledge, E. A.,
C. L. Halbert, and D. W. Russell.
1998.
Infectious clones and vectors derived from adeno-associated virus (AAV) serotypes other than AAV type 2.
J. Virol.
72:309-319[Abstract/Free Full Text].
|
| 43.
|
Ryan, J. H.,
S. Zolotukhin, and N. Muzyczka.
1996.
Sequence requirements for binding of Rep68 to the adeno-associated virus terminal repeats.
J. Virol.
70:1542-1553[Abstract].
|
| 44.
|
Samulski, R. J.
1993.
Adeno-associated virus: integration at a specific chromosomal locus.
Curr. Opin. Genet. Dev.
3:74-80[Medline].
|
| 45.
|
Samulski, R. J.,
X. Zhu,
X. Xiao,
J. D. Brook,
D. E. Housman,
N. Epstein, and L. A. Hunter.
1991.
Targeted integration of adeno-associated virus (AAV) into human chromosome 19.
EMBO J.
10:3941-3950[Medline].
|
| 46.
|
Sanger, F.,
S. Nicklen, and A. R. Coulson.
1977.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:5463-5467[Abstract/Free Full Text].
|
| 47.
|
Saraste, M.,
P. R. Sibbald, and A. Wittinghofer.
1990.
The P-loop: a common motif in ATP- and GTP-binding proteins.
Trends Biochem. Sci.
15:430-434[Medline].
|
| 48.
|
Shaughnessy, E.,
D. Lu,
S. Chatterjee, and K. K. Wong.
1996.
Parvoviral vectors for the gene therapy of cancer.
Semin. Oncol.
23:159-171[Medline].
|
| 49.
|
Shi, Y.,
E. Seto,
L. S. Chang, and T. Shenk.
1991.
Transcriptional repression by YY1, a human GLI-Krüppel-related protein, and relief of repression by adenovirus E1A protein.
Cell
18:377-388.
|
| 50.
|
Snyder, R. O.,
D. S. Im,
T. Ni,
X. Xiao,
R. J. Samulski, and N. Muzyczka.
1993.
Features of the adeno-associated virus origin involved in substrate recognition by the viral Rep protein.
J. Virol.
67:6096-6104[Abstract/Free Full Text].
|
| 51.
|
Srivastava, A.,
E. W. Lusby, and K. I. Berns.
1983.
Nucleotide sequence and organization of the adeno-associated virus 2 genome.
J. Virol.
45:555-564[Abstract/Free Full Text].
|
| 52.
|
Su, P. F., and F. Y. Wu.
1996.
Differential suppression of the tumorigenicity of HeLa and SiHa cells by adeno-associated virus.
Br. J. Cancer
73:1533-1537[Medline].
|
| 53.
|
Summerford, C., and R. J. Samulski.
1998.
Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions.
J. Virol.
72:1438-1445[Abstract/Free Full Text].
|
| 54.
|
Tobiasch, E.,
T. Burguete,
P. Klein-Bauernschmitt,
R. Heilbronn, and J. R. Schlehofer.
1998.
Discrimination between different types of human adeno-associated viruses in clinical samples by PCR.
J. Virol. Methods
71:17-25[Medline].
|
| 55.
|
Trempe, J. P., and B. J. Carter.
1988.
Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation.
J. Virol.
62:68-74[Abstract/Free Full Text].
|
| 56.
|
Trempe, J. P.,
E. Mendelson, and B. J. Carter.
1987.
Characterization of adeno-associated virus rep proteins in human cells by antibodies raised against rep expressed in Escherichia coli.
Virology
161:18-28[Medline].
|
| 57.
|
Walz, C., and J. R. Schlehofer.
1992.
Modification of some biological properties of HeLa cells containing adeno-associated-virus DNA integrated into chromosome 17.
J. Virol.
66:2990-3002[Abstract/Free Full Text].
|
| 58.
|
Weitzmann, M. D.,
S. R. M. Kyöstiö,
R. M. Kotin, and R. A. Owens.
1994.
Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA.
Proc. Natl. Acad. Sci. USA
91:5808-5812[Abstract/Free Full Text].
|
| 59.
|
Wingender, E.,
A. E. Kel,
O. V. Kel,
H. Karas,
T. Heinemeyer,
P. Dietze,
R. Knüppel,
A. G. Romaschenko, and N. A. Kolchanov.
1997.
TRANSFAC, TRRD and COMPEL: towards a federated database system on transcriptional regulation.
Nucleic Acids Res.
25:265-268[Abstract/Free Full Text].
|
| 60.
|
Wistuba, A.,
S. Weger,
A. Kern, and J. A. Kleinschmidt.
1995.
Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins.
J. Virol.
69:5311-5319[Abstract].
|
| 61.
|
Wonderling, R. S., and R. A. Owens.
1996.
The Rep68 protein of adeno-associated virus stimulates expression of the platelet-derived growth factor B c-sis proto-oncogene.
J. Virol.
70:4783-4786[Abstract].
|
| 62.
|
Zádori, Z.,
R. Stefancsik,
T. Rauch, and J. Kisary.
1995.
Analysis of the complete nucleotide sequence of goose and muscovy duck parvoviruses indicates common ancestral origin with adeno-associated virus 2.
Virology
212:562-573[Medline].
|
Journal of Virology, February 1999, p. 939-947, Vol. 73, No. 2
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Abstract
Introduction
