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Journal of Virology, November 1999, p. 9599-9603, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Construction and Preliminary Characterization of a
Library of "Lethal" Preterminal Protein Mutant
Adenoviruses
Jerome
Schaack,1,2,3,4,*
William Y.
Ho,5
Shawna
Tolman,1
Elizabeth
Ullyat,1
Xiaoling
Guo,1
Nina
Frank,1
Paul I.
Freimuth,6
Dick J.
Roovers,7 and
John S.
Sussenbach8
Department of
Microbiology,1 Molecular Biology
Program,2 University of Colorado Cancer
Center,3 and Biomedical Sciences
Program,4 University of Colorado Health Sciences
Center, Denver, Colorado; Division of Clinical Research, Fred
Hutchinson Cancer Research Center, Seattle,
Washington5; Biology Department,
Brookhaven National Laboratory, Upton, New
York6; and Department of Haematology,
University Hospital Utrecht,7 and
Laboratory for Physiological Chemistry, Utrecht
University,8 Utrecht, The Netherlands
Received 12 April 1999/Accepted 16 July 1999
 |
ABSTRACT |
Adenoviruses containing lethal in-frame insertion mutant alleles of
the preterminal protein (pTP) gene were constructed with cell lines
that express pTP. Thirty in-frame insertion mutant alleles, including
26 alleles previously characterized as lethal and 4 newly constructed
mutant alleles, were introduced into the viral chromosome in place of
the wild-type pTP gene. The viruses were tested for ability to form
plaques at 37°C in HeLa-pTP cells and at 32°C and 39.5°C in HeLa
cells. Two of the newly constructed viruses exhibited temperature
sensitivity for plaque formation, one virus did not form plaques in the
absence of complementation, seven additional mutants exhibited a
greater than 10-fold reduction in plaque formation in the absence of
complementation, and another eight mutants exhibited stronger
phenotypes than did previously characterized in-frame insertion mutants
in the plaque assay. These mutant viruses offer promise for analysis of
pTP functions.
| |
TEXT |
Adenovirus replication is absolutely
dependent on virally encoded DNA binding protein, preterminal protein
(pTP), and DNA polymerase (Pol). pTP serves as a primer to which the
first base, a dCMP residue, is covalently bound (for a review of
adenovirus replication, see references 12 and
29). pTP is proteolytically matured following
encapsulation of the DNA to yield terminal protein (TP), the form of
the protein found in infectious virions (5). The function of
the proteolytic processing is not known.
pTP and TP act to bind the adenovirus chromosome to the nuclear matrix
(3, 7, 25) and to facilitate efficient transcription of
viral early-region genes (23, 25). Binding of pTP and TP to
the nuclear matrix occurs through interaction with the CAD enzyme
(2) in a manner that is regulated by tyrosine
phosphorylation (3).
Genetic studies of the roles of pTP in the infectious cycle have been
limited by difficulties in isolating pTP mutant viruses with strong
phenotypes. In two studies, in-frame insertion mutant alleles of pTP
were generated in vitro, after which attempts to introduce the mutant
alleles into the viral chromosome were made (9, 19, 20). Of
the 56 alleles constructed, 26 were successfully introduced into the
viral chromosome, of which two caused moderately strong phenotypes,
three caused weaker phenotypes, and the remainder caused no phenotype
(9, 19, 20). The alleles that could not be inserted into the
viral chromosome were termed lethal. It was among the lethal alleles
that we expected to find the most interesting and informative phenotypes.
pTP is involved in the rate-limiting step at initiation of adenovirus
replication. While the use of virions makes adenovirus DNA extremely
infectious, the use of purified DNA introduced into cells by
transfection greatly reduces the infectivity of adenovirus DNA. The
infectivity of the DNA can be increased by using adenovirus DNA-TP
complex (6), but the resulting infectivity is still many
orders of magnitude lower than that of DNA within virions. Since
standard methods for introduction of mutated pTP alleles into the viral
chromosome require the transfection of either naked adenovirus DNA or
DNA-TP complex, the reduced infectivity of DNA and DNA-TP complex
offers a likely explanation for the difficulty in generating pTP mutant
viruses that exhibit strong phenotypes: even a modest effect on the
ability of pTP to act in initiating adenovirus replication may make the
mutation appear to be lethal by preventing the overlap recombination
required to form virus. However, we expected that cell lines that
express pTP (1, 13, 15, 21) would permit construction of the
pTP mutant viruses, that once the pTP mutation was incorporated into
the viral chromosome in place of the wild-type allele and virions were
generated, the vastly increased infectivity of virions would overcome
the limitations that blocked construction of the pTP mutant viruses by
standard means, and that, therefore, the majority of the mutant viruses would grow in the absence of complementation.
Construction of viruses containing lethal pTP in-frame insertion
mutant alleles.
A system for construction of viruses containing
pTP mutant alleles that permitted screening or selection for the
desired viruses was developed (Fig. 1).
This procedure was found to efficiently select for the desired viruses.
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FIG. 1.
Construction of pTP in-frame insertion mutant viruses.
The Ad5dl308Bst -gal-TP (24)
complex is represented schematically by the thick horizontal line, with
TP indicated by solid ovals. The unique BstBI and
XbaI sites within the viral chromosome are indicated. The
locations of the LacZ and the E2B mRNAs are indicated by lines with
solid arrows, with splicing of the primary E2 transcript to yield the
E2B mRNAs (encoding pTP and DNA Pol) indicated by dashed lines above
the viral chromosome. The pTP and DNA Pol coding regions are indicated
above the chromosome. Mutant pTP alleles that could not be introduced
into the virus by standard means (9, 19, 20) as well as
newly constructed mutant alleles constructed by partial digestion with
HpaII in the presence of ethidium bromide (16,
17) and the two-amino acid insertion method of Barany
(4) were cloned into plasmid p0-29.4 (22), which
is indicated below Ad5dl308Bst-gal, with the
intermediate-thickness line indicating adenovirus sequence, the
thickest line indicating the region encoding the main exon of pTP, and
the thinnest line indicating the plasmid vector (the entire plasmid
vector is not shown). The sites for XmnI, KpnI,
and XbaI are indicated. p0-29.4 carrying the various pTP
in-frame insertion mutant was digested with XbaI (for
ligation with the large arm of XbaI-digested
Ad5dl308Bst-gal-TP) plus XmnI (to
provide a blunt end outside the adenovirus sequence that would reduce
recircularization and concatamerization during ligation), ligated with
XbaI-digested Ad5dl308Bst-gal-TP,
and used to transfect 293-pTP cells, and the resultant viruses were
plaque purified in HeLa-pTP cells where the pTP mutant viruses but not
the parental Ad5dl308Bst-gal would grow.
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|
pTP in-frame insertion mutant viruses plaque purified with HeLa-pTP
cells were grown in 293-pTP cells. Viral DNA was prepared by the method
of Hardy et al. (11) and analyzed for the presence of the
inserted restriction site within the pTP gene, the presence of the
BstBI site within the E1A gene, and the absence of the KpnI site at bp 2052 within the E1B gene (the mutations in
the E1 genes, which do not affect function of the proteins, were
introduced to facilitate cloning [22]). In addition,
the sizes of the fragments generated by digestion with the restriction
enzyme cleaving the introduced sequence within the pTP gene were
compared. This analysis demonstrated the successful construction of the
library of lethal pTP in-frame insertion mutant viruses (Table
1).
Initial characterization of the pTP in-frame insertion
mutants.
Stocks of the pTP in-frame insertion mutant viruses grown
in 293-pTP cells were plaque titered with, as controls, the
phenotypically wild-type Ad5dl309, the temperature-sensitive
(ts) pTP mutant virus Ad5sub100r, and
Ad5dl308pTP155 (equivalent to Ad5in425
[9]) in HeLa-pTP cells at 37°C and in HeLa cells at
32°C and 39.5°C. The input virus had either the mutant form of TP
encoded by the virus or the wild-type TP encoded by 293-pTP cells
covalently attached to the viral DNA. In cases where wild-type TP was
associated with the DNA, it is possible that an advantage for virus
replication was provided. However, this advantage would have been
apparent only for the input viral genome and thus would have provided a linear, rather than exponential, advantage to virus growth. Thus, plaque formation would have been dependent on the ability of the mutated pTP to promote virus formation.
The results (Table
2) demonstrated that
virus yields from 293-pTP cells varied over nearly 3 orders of
magnitude among the
newly constructed pTP in-frame mutant viruses in
spite of similar
progression of cytopathic effect. Since virus growth
was in the
presence of complementing pTP supplied by the cells and was
not
optimized for the individual viruses, the differences in yield,
while suggestive of phenotypic differences due to the mutations
introduced into the pTP gene, cannot be considered as a primary
indicator of phenotype for the purposes of classification until
virus
growth is examined in detail in noncomplementing cells.
Comparison of
relative plaquing efficiencies in HeLa and HeLa-pTP
cells normalizes
for differences in yield and so offers strong
evidence of phenotypic
differences. Since certain mutant adenoviruses,
including viruses
deleted in the E1A and E1B genes, are capable
of growing more
efficiently after infection of noncomplementing
cells at high
multiplicity (
26), plaque formation represents
a fairly
strict definition of viral replication.
Both Ad5
sub100r and Ad5
in425 exhibited fairly
strong phenotypes in 293 cells (
9,
18,
21) in contrast to
the weaker
phenotypes apparent in plaque assays in HeLa cells (Table
2),
suggesting that HeLa cells may partially complement growth of
adenoviruses mutated in the pTP gene relative to 293 cells. Thus,
reliance on significant reductions in plaque-forming efficiency
in HeLa
cells for phenotypic comparison sets a fairly rigorous
standard for the
definition of mutant
behavior.
All but one of the newly constructed mutant viruses,
Ad5
dl308pTP120, were able to grow to form plaques in the
absence of complementation.
The mutation in
Ad5
dl308pTP120 may truly be lethal because of
nonfunctional pTP. Alternatively, it is possible that the pTP
allele
may not direct expression of
pTP.
In the absence of complementation, Ad5
dl308pTP111,
Ad5
dl308pTP243, Ad5
dl308pTP252,
Ad5
dl308pTP590, and Ad5
dl308pTP609 formed
plaques
10- to 100-fold less efficiently and Ad5
dl308pTP108-1,
Ad5
dl308pTP108-2, Ad5
dl308pTP125, and
Ad5
dl308pTP508 formed plaques
at least 100-fold less
efficiently than they did in the presence
of complementation.
Ad5
dl308pTP108-2 and Ad5
dl308pTPR508 also
exhibited
ts plaque formation in the absence of
complementation.
The phenotypes of all of these mutants, particularly
the
ts mutants,
appeared to be much stronger than those of
any of the previously
constructed pTP mutants, excluding pTP
deletion
mutants.
The remaining viruses included 8 that exhibited stronger phenotypes
than either Ad5
sub100r or Ad5
dl308pTP155 and 12 that exhibited
little or no phenotype in the plaque assays. Since both
Ad5
sub100r
(
9,
21,
23,
25) and
Ad5
in425 (
9,
18) exhibit interesting
and
informative phenotypes, it is likely that at least some of
the
less-defective pTP mutant viruses will exhibit stronger phenotypes
in
other assays of virus growth. Such assays will include one-step
growth
curves, determination of relative affinities of the viral
chromosomes
for the nuclear matrix, and analysis of the synthesis
and fate of pTP
in cells infected with the various pTP mutant
viruses. The fact that
the great majority of the pTP mutant viruses
grow, at least to a low
level, in the absence of complementation
will facilitate studies of pTP
function. Any pTP mutant that can
be generated is of potential value,
but the viruses that can be
grown in the absence of complementation
have expanded potential,
since growth in the absence of complementation
ensures that the
mutant forms of pTP and TP are associated with all
viruses in
a stock. This will permit the effects of most of the
mutations
to be examined throughout the infectious
cycle.
Viruses that did not display a strong phenotype were retested for the
presence of the restriction site introduced within the
pTP gene.
Aliquots of the stocks used in plaque titration were
grown in 293-pTP
2C1 cells and DNA isolated (
11). All of the
viruses
maintained the restriction enzyme site introduced in the
mutated pTP
allele (data not shown), demonstrating that the lack
of a strong
phenotype in plaque formation in HeLa cells was characteristic
of the
mutation and did not result from recombination within the
pTP-expressing cells or contamination with the wild-type
virus.
Examination of the pTP mutant map (Fig.
2) demonstrates regions of clustering of
strong phenotypes near amino acid 120, suggesting
that this region is
particularly important for pTP function. Stronger
conclusions will
require further mutagenesis of pTP.
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FIG. 2.
Map of in-frame insertion alleles of pTP. A schematic
map of the pTP coding sequence is presented as a horizontal line, with
the N terminus to the left. Positions of amino acids, numbered from the
first amino acid of pTP encoded by the 39.6-39.2 map unit exon
(27), are indicated as ticks on the pTP coding sequence,
with numbering shown below. The positions of viable in-frame pTP
mutations introduced into the virus in earlier studies (9, 19,
20) are indicated by vertical lines below the pTP coding
sequence, with the positions of mutations leading to
replication-defectiveness (9) indicated by elongated lines.
The positions of mutations introduced into the virus in this study are
indicated above the pTP coding sequence, with the positions of
mutations leading to strongly defective behavior (at least a 10-fold
reduction in plaquing efficiency in HeLa cells relative to that in
HeLa-pTP cells) indicated by elongated lines. The overlapping coding
sequence for DNA Pol is indicated by the dashed line with arrow.
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|
Fredman et al. (
8) examined the ability of certain of the
pTP proteins encoded by mutant alleles constructed by Freimuth
and
Ginsberg (
9) to direct replication in vitro. The results
that pertain to viruses examined in this study are indicated in
Table
2. Reasonable agreement between the in vitro results and
results of the
plaque formation assay were observed for Ad5
dl308pTP125
and
Ad5
dl308pTP378 (in both cases in comparison with paired
mutant
alleles generated by Freimuth and Ginsberg
[
9]), Ad5
dl308pTP403,
and
Ad5
dl308pTPF155. Ad5
sub100r pTP directed poor
replication
in vitro (
8) but had only modest effects on
plaque formation
in HeLa cells. The in vitro replication data are thus
in better
agreement with the results of plaque formation by the virus
in
293 cells (
9,
21) than with the results of plaque
formation
by the virus in HeLa cells. The F233 mutation has a strong
effect
on the progression of Ad5
in425 through the infectious
cycle (
9,
18) but relatively little effect on plaquing
efficiency, supporting
the suggestion that the mutation affects a pTP
function separate
from its role in replication (
8).
The pTP alleles encoded by F391, F439, and F348 did not direct
synthesis of pTP in vitro (
8). The ability of the
viruses
generated by using these alleles,
Ad5
dl308pTP88-2, Ad5
dl308pTP252-2,
and
Ad5
dl308pTP534, respectively, to form plaques in HeLa cells
indicates that pTP synthesis was directed in vivo. However, pTP
encoded
by all of these alleles, and by their paired counterparts
with
insertions at the same sites generated by Roovers et al.
(
19,
20), may act as dominant negatives, as yields of these
viruses
grown in 293-pTP cells were reduced 40- to 90-fold relative
to the
highest yields of pTP in-frame insertion mutants (Table
2) and pTP
deletion mutant viruses (data not
shown).
F346 and F394 both directed little or no replication in vitro
(
8). The F346 mutation in Ad5
dl308pTP234-2 led to
only a
modest phenotype in the plaque formation assays. The F394
mutation
in Ad5
dl308pTP478 led to a stronger phenotype,
although possibly
not as strong as predicted from the in vitro
replication results.
The increased effect of the mutations in vitro may
reflect the
stringency of the assay, since replication in vitro is
inefficient
relative to that in vivo. Alternatively, the formation of
complexes
with DNA Pol (
14,
28) may help to stabilize the
mutant pTPs
in
vivo.
| |
ACKNOWLEDGMENTS |
We thank J. Engler for providing some of the plasmids encoding pTP
mutants that were used in this study.
This work was supported by NIH grants HL58344 and GM42555. Tissue
culture support was provided by the University of Colorado Cancer
Center Tissue Culture Core.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: University of
Colorado Health Sciences Center, Box B-175, 4200 E. 9th Ave., Denver, CO 80262. Phone: (303) 315-6883. Fax: (303) 315-6785. E-mail: jerry.schaack{at}uchsc.edu.
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REFERENCES |
| 1.
|
Amalfitano, A., and J. S. Chamberlain.
1997.
Isolation and characterization of packaging cell lines that coexpress the adenovirus E1, DNA polymerase, and preterminal proteins: implications for gene therapy.
Gene Ther.
4:258-263[Medline].
|
| 2.
|
Angeletti, P. C., and J. A. Engler.
1998.
Adenovirus preterminal protein binds to the CAD enzyme at active sites of viral DNA replication on the nuclear matrix.
J. Virol.
72:2896-2904[Abstract/Free Full Text].
|
| 3.
|
Angeletti, P. C., and J. A. Engler.
1996.
Tyrosine kinase-dependent release of an adenovirus preterminal protein complex from the nuclear matrix.
J. Virol.
70:3060-3067[Abstract].
|
| 4.
|
Barany, F.
1985.
Two-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides.
Proc. Natl. Acad. Sci. USA
82:4202-4206[Abstract/Free Full Text].
|
| 5.
|
Challberg, M. D., and T. J. J. Kelly.
1981.
Processing of the adenovirus terminal protein.
J. Virol.
38:272-277[Abstract/Free Full Text].
|
| 6.
|
Chinnadurai, G.,
S. Chinnadurai, and M. Green.
1978.
Enhanced infectivity of adenovirus type 2 DNA and a DNA-protein complex.
J. Virol.
26:195-199[Abstract/Free Full Text].
|
| 7.
|
Fredman, J. N., and J. A. Engler.
1993.
Adenovirus precursor to terminal protein interacts with the nuclear matrix in vivo and in vitro.
J. Virol.
67:3384-3395[Abstract/Free Full Text].
|
| 8.
|
Fredman, J. N.,
S. C. Petite,
M. S. Horwitz, and J. A. Engler.
1991.
Linker insertion mutations in the adenovirus preterminal protein that affect DNA replication activity in vivo and in vitro.
J. Virol.
65:4591-4597[Abstract/Free Full Text].
|
| 9.
|
Freimuth, P. I., and H. S. Ginsberg.
1986.
Codon insertion mutants of the adenovirus terminal protein.
Proc. Natl. Acad. Sci. USA
83:7816-7820[Abstract/Free Full Text].
|
| 10.
|
Graham, F. L.,
J. Smiley,
W. C. Russell, and R. Nairn.
1977.
Characteristics of a human cell line transformed by DNA from human adenovirus type 5.
J. Gen. Virol.
36:59-72[Abstract/Free Full Text].
|
| 11.
|
Hardy, S.,
M. Kitamura,
T. Harris-Stansil,
Y. Dai, and M. L. Phipps.
1997.
Construction of adenovirus vectors through Cre-lox recombination.
J. Virol.
71:1842-1849[Abstract].
|
| 12.
|
Hay, R. T.,
A. Freeman,
I. Leith,
A. Monaghan, and A. Webster.
1995.
Molecular interactions during adenovirus DNA replication, p. 31-48.
In
W. Doerfler, and P. Böhm (ed.), Molecular repertoire of adenoviruses II, vol. 199. Springer-Verlag, Berlin, Germany.
|
| 13.
|
Langer, S. J., and J. Schaack.
1996.
Construction of 293 cell lines that inducibly express high levels of precursor terminal protein.
Virology
221:172-179[Medline].
|
| 14.
|
Lichy, J. H.,
J. Field,
M. S. Horwitz, and J. Hurwitz.
1982.
Separation of the adenovirus terminal protein precursor from its associated DNA polymerase: role of both proteins in the initiation of adenovirus DNA replication.
Proc. Natl. Acad. Sci. USA
79:5225-5229[Abstract/Free Full Text].
|
| 15.
|
Moorhead, J. W.,
G. H. Clayton,
R. L. Smith, and J. Schaack.
1999.
An adenovirus vector deleted for the preterminal protein gene elicits reduced inflammation in mouse ears.
J. Virol.
73:1046-1053[Abstract/Free Full Text].
|
| 16.
|
Österlund, M.,
H. Luthman,
S. V. Nilsson, and G. Magnusson.
1982.
Ethidium-bromide-inhibited restriction endonucleases cleave one strand of circular DNA.
Gene
20:121-125[Medline].
|
| 17.
|
Parker, R. C.,
R. M. Watson, and J. Vinograd.
1977.
Mapping of closed circular DNAs by cleavage restriction endonucleases and calibration by agarose gel electrophoresis.
Proc. Natl. Acad. Sci. USA
74:851-855[Abstract/Free Full Text].
|
| 18.
| Roberts, E., and J. Schaack. Unpublished results.
|
| 19.
|
Roovers, D. J.,
P. F. Overman,
X. Q. Chen, and J. S. Sussenbach.
1991.
Linker mutation scanning of the genes encoding the adenovirus type 5 terminal protein precursor and DNA polymerase.
Virology
180:273-284[Medline].
|
| 20.
|
Roovers, D. J.,
F. M. van der Lee,
J. van der Wees, and J. S. Sussenbach.
1993.
Analysis of the adenovirus type 5 terminal protein precursor and DNA polymerase by linker insertion mutagenesis.
J. Virol.
67:265-276[Abstract/Free Full Text].
|
| 21.
|
Schaack, J.,
X. Guo,
W. Y.-W. Ho,
M. Karlok,
C.-Y. Chen, and D. Ornelles.
1995.
Adenovirus type 5 precursor terminal protein-expressing 293 and HeLa cell lines.
J. Virol.
69:4079-4085[Abstract].
|
| 22.
|
Schaack, J.,
X. Guo, and S. J. Langer.
1996.
Characterization of a replication-incompetent adenovirus type 5 mutant deleted for the preterminal protein gene.
Proc. Natl. Acad. Sci. USA
93:14686-14691[Abstract/Free Full Text].
|
| 23.
|
Schaack, J.,
W. Y.-W. Ho,
P. Freimuth, and T. Shenk.
1990.
Adenovirus terminal protein mediates both nuclear matrix association and efficient transcription of adenovirus DNA.
Genes Dev.
4:1197-1208[Abstract/Free Full Text].
|
| 24.
|
Schaack, J.,
S. J. Langer, and X. Guo.
1995.
Efficient selection of recombinant adenoviruses using vectors that express -galactosidase.
J. Virol.
69:3920-3923[Abstract].
|
| 25.
|
Schaack, J., and T. Shenk.
1989.
Adenovirus terminal protein mediates efficient and timely activation of viral transcription.
Curr. Top. Microbiol. and Immunol.
144:185-190.
|
| 26.
|
Shenk, T.,
N. Jones,
W. Colby, and D. Fowlkes.
1979.
Functional analysis of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells.
Cold Spring Harbor Symp. Quant. Biol.
44:367-375.
|
| 27.
|
Shu, L.,
S. C. Pettit, and J. A. Engler.
1988.
The precise structure and coding capacity of mRNAs from early region 2B of human adenovirus serotype 2.
Virology
165:348-356[Medline].
|
| 28.
|
Stillman, B. W.,
F. Tamanoi, and M. B. Mathews.
1982.
Purification of an adenovirus-coded DNA polymerase that is required for initiation of DNA replication.
Cell
31:613-623[Medline].
|
| 29.
|
Van der Vliet, P. C.
1995.
Adenovirus DNA replication, p. 1-30.
In
W. Doerfler, and P. Böhm (ed.), The molecular repertoire of adenoviruses II, vol. 199. Springer-Verlag, Berlin, Germany.
|
Journal of Virology, November 1999, p. 9599-9603, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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