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Journal of Virology, March 2000, p. 2895-2899, Vol. 74, No. 6
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Productive Replication of Human Adenoviruses in
Mouse Epidermal Cells
Ian
Ganly,1
Vivien
Mautner,2 and
Allan
Balmain1,*
CRC Department of Medical Oncology, CRC
Beatson Laboratories, Glasgow G61 1BD,
Scotland,1 and CRC Institute for
Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15
2TT, England,2 United Kingdom
Received 20 July 1999/Accepted 6 December 1999
 |
ABSTRACT |
In contrast to most cells of mouse origin, cell lines derived from
mouse epidermis are permissive for replication of human adenovirus type
5. The extent of epidermal cell differentiation correlated with the
level of E1A expression and virus replication. Mouse epidermal cells
may provide useful models for cancer therapy using
replication-competent human adenoviruses.
| |
TEXT |
Human adenoviruses have great
potential for human gene therapy as replication-defective delivery
vehicles or as replication-competent viruses for the treatment of
cancer (6, 8, 13, 20, 23). These approaches would benefit
from the availability of suitable mouse models to test the efficacy of
different therapeutic strategies. At present, there is no
immunocompetent mouse model to test replicating adenoviruses (2,
11) as previous work suggests that both the infectivity and
productive replication of adenoviruses in rodent cells are poor
(4, 9). In these model systems, although some evidence for
limited replication has been obtained (12, 17, 18), a
productive infection leading to an efficient viral burst has not been
seen. This apparent block has been attributed to species-specific
properties of mouse cells leading to repression of early viral protein
expression (7) or defects at other points of the viral life
cycle (5, 16).
We have investigated replication of adenovirus type 2 (Ad2) in a series
of mouse epidermal cell lines (3, 19, 22), together with
cells from a variety of mouse tissues (Table
1). A nonreplicating E1-deleted
adenovirus with a lacZ reporter construct (CMVlacZ virus)
(14) was used to determine infectivity. The percentages of
-galactosidase (-Gal)-positive cells at multiplicities of
infection (MOIs) of 10 and 100 PFU/cell for several different rodent
cell lines are shown in Table 1. Infectivity was very high with
specific tissue types, notably mouse epidermal cells (Fig.
1), mouse kidney adenocarcinoma TCMK1
cells, rat glioblastoma 9L-82 cells, rat thyroid carcinoma VH1 VRS2
cells, and rat Morris hepatoma cells. Infectivity was very low in mouse
Lewis lung carcinoma cells, rat colon carcinoma K12/TrB cells, and 3T3
fibroblasts (21) (Fig. 1). Surprisingly, infectivity was
higher for many of the rodent cell types than for the human ovarian
cell line A2780Cp.
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TABLE 1.
Infectivity, relative E1A expression, CPE, and
immunofluorescence for hexon protein of wild-type
adenovirus-infected rodent cell linesa
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FIG. 1.
Infectivity of rodent cell lines by Ad5. For details,
see the legend to Table 1. The percentages shown correspond to the
proportion of blue cells in the culture after infection with the
CMVlacZ virus. (Left panels) Control uninfected cells; (right panels)
virus-infected cells.
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Cytopathic effect (CPE) assays using wild-type Ad2 were carried out at
an MOI of 10 PFU/cell. Table 1 summarizes the results showing that a
clear CPE was found in all of the mouse epidermal cells and to a lesser
extent in the rat colon carcinoma K12/TrB and thyroid carcinoma VH1
VRS2 cell lines. All cell lines which showed positive CPEs were
positive by immunofluorescence for hexon protein, when IMAGEN
adenovirus reagent containing a fluorescein isothiocyanate-labeled
mouse monoclonal antibody to adenovirus hexon protein was used. The
cell lines 9L-82 and TCMK1, which were highly infectable with the
CMVlacZ virus, showed no CPE or positive immunofluorescence,
indicating the presence of a specific barrier to replication or late
protein expression in these cells.
To determine if a productive infection could be produced in mouse
cells, burst assays were done with wild-type Ad2 (10). The
burst ratio was expressed as the concentration of virus at 72 h
relative to the concentration of virus at 4 h postinfection. Lysates were prepared by three cycles of freezing and thawing. Titers
of serial dilutions were determined on HEK293 cells. Figure 2a shows the results of burst assays for
cell lines B9 and SN161 in comparison to those for the human ovarian
cell line A2780Cp. The burst ratio for A2780Cp is 50 times greater than
that in B9, and 25 times greater than that in SN161. Figure 2b shows
that the infectivities of each cell line are similar at 10 PFU/cell, as
determined by using the LacZ adenovirus. These results show that
several mouse epidermal cell lines can produce a productive viral yield
but generally at reduced efficiency (25- to 50-fold less) in comparison
to that produced by human A2780cp cells.
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FIG. 2.
Burst assay and infectivity assay of mouse epidermal
cell lines in comparison to the human ovarian cell line A2780Cp. (a)
Simultaneous burst assays were determined for the human A2780 cells
(gray column), squamous B9 cells (black column), and SN161 cells (white
column). Burst ratio is shown on a log scale on the left. (b)
Infectivity was determined by using the CMVlacZ adenovirus at an MOI of
10 PFU/cell for each of the cell lines.
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FIG. 3.
Western blots of E1A expression of Ad2-infected rodent
cells. (a) A number of epidermal cell lines were infected with Ad2 at
an MOI of 10 PFU/cell and harvested 24 h later. Western blotting
was carried out with the anti-E1A monoclonal antibody M73. Vinculin was
used as a control for protein loading. (b) A range of cell lines from
various mouse tissues (see Table 1) was infected and adenovirus E1A
levels were assessed as described above.
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Previous results with rodent fibroblasts have suggested that the
expression of adenovirus E1A is repressed following viral uptake by the
presence of a trans-acting transcriptional repressor protein
(7). To determine whether this mechanism is operative in a
wide range of rodent cell types, protein lysates were made from cells
24 h after infection with Ad2 at an MOI of 100 PFU/cell. Equivalent amounts of protein were run on Western blots with the mouse
monoclonal antibody M73 (Oncogene Science; dilution 1/1,000), which
detects the E1A proteins. A vinculin mouse monoclonal antibody (VIN-11-1; Sigma) was used at a concentration of 1/1,000.
Figure 3 shows that all rodent cells were
able to express the early gene E1A proteins after infection. E1A
expression was expressed relative to vinculin expression for each cell
line by densitometry (Table 1) and then expressed as a percentage
relative to the cell line with maximum E1A expression (Morris liver
hepatoma cell line). There was no obvious correlation between E1A
expression and infectivity or ability to support virus replication.
Overall, the levels of E1A expression did not differ markedly between
cells that showed no evidence for virus replication and those that
showed either a weak CPE and low-level hexon staining (the colon cell line K12/TrB and the thyroid cell line VH1 VRS2) or a strong CPE and
substantial hexon expression (the epidermal cell lines). This indicated
that there was a block to late-phase protein expression in most tissue
types with the exception of mouse epidermal cells.
Although these experiments did not reveal any correlation between
infectivity, E1A expression, and replication across a panel of
different cell types, within the series of epidermal cell lines we
found a relatively good correlation with the degree of cell differentiation. We compared E1A expression levels in two independent pairs of well-differentiated squamous cell lines (B9 and E4) with their
undifferentiated spindle counterparts (A5 and H11, respectively). In
each of these pairs of cells, the spindle variant was derived from the
same primary tumor as the squamous cell (3, 19, 22;
J. Liddell and A. Balmain, unpublished data). Figure
4 shows that E1A expression correlated
with the degree of cellular differentiation, with poorly differentiated
H11 and A5 cells showing reduced expression. The more-differentiated
cells also were more permissive for replication, as shown by the
relatively increased staining with anti-hexon protein antibodies.
Preferential replication of Ad2 in more-differentiated cell populations
has also been reported for human keratinocytes (1, 15). Our
results on squamous and spindle carcinoma cells would also indicate
that replication is better in the more-differentiated cell phenotype,
although the mechanistic basis for these observations is unclear.
Previous work by others (7) has shown that fibroblasts produce a trans-acting transcriptional repressor which
suppresses the activity of the E1A promoter. Indeed, we have shown that
the expression level of this nuclear factor, 
AP3, a zinc
finger-containing DNA-binding protein related to the GLI-Kruppel
protein (7), is approximately 20-fold higher in the spindle
phenotype (data not shown), and this may account for the lower E1A
levels in the spindle cells.
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FIG. 4.
Hexon protein expression and E1A expression in
squamous-spindle paired cell lines. Two matched pairs of squamous and
spindle cells (panel a, B9 [squamous] and A5 [spindle]; panel b, E4
[squamous] and H11 [spindle]) were stained for hexon protein
expression 24 h after infection with Ad2 at an MOI of 10 PFU/cell.
Green staining indicates positive hexon protein expression, and red
staining shows counterstaining with evans blue. The level of hexon
protein staining was consistently higher in the squamous cells than in
their spindle counterparts. Levels of E1A were assessed by Western
blotting with anti-E1A antibodies and antivinculin antibodies as shown
on the right side of the figure. The relative expression levels of E1A
are higher in both squamous cells in comparison with those of the
corresponding spindle cells.
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We have shown that a series of restriction points that control the
ability of human adenoviruses to replicate in rodent cells exist,
including infectivity, expression of the early gene product E1A, and
subsequent initiation of viral replication and late protein synthesis.
Most or all of these restrictions are surprisingly absent in mouse
epidermal cells, leading us to suggest that epidermal tumor models may
be useful for testing replication-competent adenoviral therapy for
cancer (2, 11).
| |
ACKNOWLEDGMENTS |
We thank Wilma Steegenga and Hans Bos for the CMVlacZ virus, Byron
Hann for critical reading of the manuscript, and numerous colleagues in
the adenovirus group at Onyx Pharmaceuticals (Richmond, Calif.) for
interesting discussions.
This work was supported by funding from the Cancer Research Campaign
(United Kingdom) and from Onyx Pharmaceuticals.
| |
FOOTNOTES |
*
Corresponding author. Present address: UCSF Cancer
Center, 2340 Sutter St., San Francisco, CA 94143. Phone: (415) 502 4192. Fax: (415) 502 6779. E-mail: abalmain{at}cc.ucsf.edu.
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Journal of Virology, March 2000, p. 2895-2899, Vol. 74, No. 6
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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