Figure 4. 9 -A. Evaluation of co- infection of AvlCFl and wild- type adenovirus 
in HeLa cells. To evaluate the possible complementation of AvlCFl with 
potentially co- infecting wild- type adenovirus, HeLa cells were simulta- 
neously infected by two viruses. HeLa cells were grown to 80% confluency in 
Dulbecco's modified Eagle medium, 10% fetal bovine serum, 1% glutamine, 100 
U/ml penicillin, 100 jzg/ml streptomycin and infected with AvlCFl at various 
multiplicities of infection (MOI) from 0 to 30 plaque forming units (pfu) 
per cell (Berkner and Sharp, 1983). After incubation for 20 hr (37°, 5% 
C0 2 , humidified atmosphere), the media was aspirated and replaced with 
similar media except without phosphate and containing 32 P-P0*,“ and the 
incubation was continued for another 20 hr. Cells were then lysed and 
incubated (37°, with shaking) in 0.6% sodium dodecyl sulfate, 10 mM Tris- 
HCL, pH 7.4) 10 mM EDTA, 5 mg/ml proteinase K and viral DNA was extracted 
by the Hirt procedure (Hirt, 1967). Equal volumes of Hirt extracted DNA 
were then subjected to EcoRI enzyme cleavage and the products were frac- 
tionated on a 1% agarose gel which was dried and subjected to autoradiogra- 
phy. Uninfected HeLa cells showed no labeled viral DNA fragments, but did 
show labeling of mitochondrial DNA (lane 1) . HeLa cells infected with Ad5 
alone demonstrated the replication of Ad5 DNA as evidenced by the presence 
of characteristic restriction fragments of 32 P-labeled Ad5 DNA (lane 2). 
Co-infection of Ad5 (MOI = 3) and AvlCFl (MOI = 30) (lane 3) and Ad5 (MOI = 
30) and AvlCFl (MOI *= 30) (lane 4) demonstrated replication of both viral 
genomes as evidence by the presence of characteristic 32 P-labeled restric- 
tion fragments for each virus. No replication of AvlCFl was observed after 
infection of HeLa cells alone (lane 5). Interestingly, the replication of 
Ad5 in the presence of co- infecting AvlCFl was diminished compared to 
infection by Ad5 alone (compare lanes 3,4 to lane 2). 
Recombinant DNA Research, Volume 16 
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