Article Figures & Data
Figures
- FIG. 1.
Increasing amounts of AAV resulted in decreased AdlacZ5 β-gal activity and virus production. (A) Primary AAV and AdlacZ5 coinfections were conducted in HeLa cells, harvested at 48 hpi, and assayed for β-gal activity. (B) Secondary infections were performed by inoculating fresh HeLa cells with 50-μl aliquots of primary coinfection cultures. Heparin was included to prevent infection with AAV. Cultures were harvested at 24 hpi and tested for β-gal activity. In both panels, the numbers on the z axis refer to the MOIs of AdlacZ5 used in the coinfections. The numbers on the x axis indicate the IU of AAV. The β-gal activity is expressed on the y axis as the percentage of β-gal activity relative to that of cells infected with AdlacZ5 alone. Coinfections were conducted on 12 separate occasions.
- FIG. 2.
Steady-state AdlacZ5 DNA levels decreased during coinfections. Southern analysis was conducted using HeLa cells coinfected with increasing amounts of AAV and 1, 5, or 10 MOIs of AdlacZ5, as denoted above the panels. Whole-cell DNA was isolated at 48 hpi and analyzed by Southern hybridization. The membranes shown above the lane numbers were surveyed with an Ad-specific probe, while the lower membranes were assayed with an AAV-specific probe. Autoradiography was conducted for visualization. In all panels, the DNA in lane 1 was harvested from cells infected with AdlacZ5 alone. Lanes 2 to 6 contain DNA from cells that were coinfected with AdlacZ5 and 1, 10, 100, 500, and 1,000 IU of AAV, respectively. RFd indicates AAV replicative-form dimer. RFm indicates replicative-form monomer, and SS indicates single-stranded AAV genome.
- FIG. 3.
Increasing titers of AAV reduced Ad steady-state transcripts to various degrees. Northern analysis was conducted using total mRNA harvested at 24 hpi from HeLa cells infected with AdlacZ5 (MOI, 5) and AAV (0, 1, 10, 100, or 500 IU). To assay E3 expression, Ad5 was used in place of AdlacZ5. Equal amounts of RNA were separated by 1% formaldehyde agarose gel electrophoresis, analyzed by Northern hybridization, and visualized by autoradiography. To confirm equal loading, ribosomal 28S and 18S bands were visualized by ethidium bromide staining. A representative gel is shown. Locations of size standards in kilobases are labeled on the right. AAV transcripts are labeled according to their promoter of origin.
- FIG. 4.
Levels of Ad proteins during coinfection with AAV parallel mRNA levels. Immunoblot analyses were conducted using lysates from HeLa cells coinfected with AAV and AdlacZ5. Cultures were harvested at 48 hpi, and infection was verified by β-gal activity. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with antibodies specific to Ad or AAV proteins, as denoted to the left of the images. Lane 1 is from AdlacZ5-infected cells (MOI, 5 or 10). Lanes 2 to 5 are from cells coinfected with AdlacZ5 and AAV (1, 10, 100, and 500 IU, respectively). The uppermost band in the Rep panel, which is labeled with an asterisk, indicates a nonspecific interaction.
- FIG. 5.
The temporal expression of mRNA for Ad transcription units varied in response to coinfecting AAV. HeLa cells were infected with Ad5 (MOI, 5) in the presence or absence of AAV (100 IU). Cultures were harvested at various times ranging from 3 to 24 hpi. Equal amounts of total RNA were resolved by electrophoresis in 1% formaldehyde gels and transferred to nitrocellulose membranes. Northern hybridization analysis was conducted using probes specific for the indicated Ad or AAV transcripts. Visualization and quantitation were conducted with autoradiography and PhosphorImager analysis. Membranes were subsequently stripped and probed with a GAPDH-specific probe to confirm equal loading. A representative GAPDH blot is shown. Molecular size markers (kb) are shown to the right of the blots. AAV transcripts are labeled according to their promoter of origin.
- FIG. 6.
AAV Rep proteins inhibit AdlacZ5 production to different degrees. (A) HeLa cells were transfected with pCDMRep constructs, as indicated on the x axis, and subsequently infected with AdlacZ5. Cell lysates were tested for β-gal activity at 48 hpi. (B) Aliquots (50 μl) from transfected cultures were used to inoculate fresh HeLa cells. The secondary infections were harvested at 24 hpi and tested for β-gal activity. The β-gal activity of the pCDM8 empty vector control (not shown) was set at 100%. Relative β-gal activities of samples that were transfected with wild-type Rep and PNB mutant plasmids are shown in black and gray, respectively. Error bars represent the standard deviations from six experiments conducted in triplicate (n = 18).
- FIG. 7.
AAV Rep proteins decreased E2A and E4 mRNA transcript levels during the late phase of Ad infection. HeLa cells were transfected with pCDMRep78G or the empty vector pCDM8, as indicated at the top of the figure. After being incubated for 20 to 24 h to permit Rep expression, the cells were infected with 5 MOIs of Ad5. Infected cultures were harvested at 6, 9, 12, or 24 hpi. Uninfected (Uninf) cells were harvested at 24 hpi. Total RNA was prepared, and equal amounts were analyzed by Northern analysis. Equal loading was confirmed by ethidium bromide staining of rRNA as well as GAPDH analysis (not shown). Western analysis was conducted with samples harvested at 6 hpi to confirm Rep78 expression (not shown). Locations of size standards are noted in kilobases.
- FIG. 8.
Coinfection with AAV did not reduce E2A or E4 transcript levels in the absence of DNA replication. HeLa cells were infected with Ad5 in the presence (+) or absence (−) of coinfecting AAV, as indicated above the top panel. HU was added to the medium at 6, 9, or 12 hpi. Untreated controls were also included. All cultures were harvested at 24 hpi, total RNA and viral DNA were prepared. (A) Equal amounts of RNA were subjected to Northern analysis using Ad E2A- and E4-specific probes. GAPDH and rRNA analyses confirmed equal loading (not shown). Molecular size markers are given in kilobases. (B) Viral DNA was isolated and analyzed by Southern hybridizations with a radiolabeled Ad DNA probe.
