Figure 4.2-B. Evaluation of human epithelial (HeLa) cells for the expres- 
sion of adenovirus protein following infection with AvlCFl. The expression 
of adenovirus genes after infection of HeLa cells by the recombinant adeno- 
virus AvlCFl was examined at the level of protein expression by evaluating 
the presence of adenovirus hexon protein with and without immunoprecipita- 
tion. HeLa cells were grown to 80% confluency in Dulbecco's modified Eagle 
medium, 10% fetal calf serum, 1% glutamine, 100 U/ml penicillin, and 100 
/ig/ml streptomycin, and infected with AvlCFl at a multiplicity of infection 
(MOI) of 100 plaque forming units (pfu) per cell as previously described 
(Appendix 1) . The cells were washed twice with cold phosphate buffered 
saline (PBS, pH 7.4) and labeled with [ 35 S ] methionine (1000 /xCi/mmole, 100 
/xCi/ml) for 24 hr. Following the labeling period, cells were washed in PBS, 
and lysed in buffer containing antiproteases (see Appendix 1 for details) . 
The cell lysate was cleared of debris by brief (10 min) centrifugation in a 
microfuge . The cleared cell lysate was evaluated by immunoprecipitation 
using an anti-Ad5 hexon antibody (gift of Bartels, Inc.). Following 
immunoprecipitation of equivalent amounts of trichloroacetic acid precipi- 
table radioactivity from each sample, labeled proteins were evaluated by 
sodium dodecyl sulfate acrylamide gels and autoradiography. As a control, 
no Ad hexon protein could be precipitated from uninfected HeLa cells col- 
lected from 1 to 7 days following infection. In contrast, after infection 
of HeLa cells with Ad-dl327, Ad hexon protein was readily detected early 
(day 1) and expression increased significantly (day 2) . Further evaluations 
could not be made with Ad-dl327 due to lytic infection of the cells by this 
virus. After infection of HeLa cells with AvlCFl, no Ad hexon protein could 
be seen early (day 1-2, a time when abundant expression could be seen with 
Ad-dl327) . Small amounts of expression of Ad hexon was subsequently detect- 
able (day 3), peaked (day 4) and declined thereafter (days 5-7). 
Recombinant DNA Research, Volume 16 
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