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. 2008 Apr;82(7):3415-27.
doi: 10.1128/JVI.01708-07. Epub 2008 Jan 30.

Adenovirus E1B55K region is required to enhance cyclin E expression for efficient viral DNA replication

Xinyu Zheng  1 , Xiao-Mei Rao, Jorge G Gomez-Gutierrez, Hongying Hao, Kelly M McMasters, H Sam Zhou
Affiliations

Adenovirus E1B55K region is required to enhance cyclin E expression for efficient viral DNA replication

Xinyu Zheng et al. J Virol. 2008 Apr.

Abstract

Adenoviruses (Ads) with E1B55K mutations can selectively replicate in and destroy cancer cells. However, the mechanism of Ad-selective replication in tumor cells is not well characterized. We have shown previously that expression of several cell cycle-regulating genes is markedly affected by the Ad E1b gene in WI-38 human lung fibroblast cells (X. Rao, et al., Virology 350:418-428, 2006). In the current study, we show that the Ad E1B55K region is required to enhance cyclin E expression and that the failure to induce cyclin E overexpression due to E1B55K mutations prevents viral DNA from undergoing efficient replication in WI-38 cells, especially when the cells are arrested in the G(0) phase of the cell cycle by serum starvation. In contrast, cyclin E induction is less dependent on the function encoded in the E1B55K region in A549 and other cancer cells that are permissive for replication of E1B55K-mutated viruses, whether the cells are in the S phase or G(0) phase. The small interfering RNA that specifically inhibits cyclin E expression partially decreased viral replication. Our study provides evidence suggesting that E1B55K may be involved in cell cycle regulation that is important for efficient viral DNA replication and that cyclin E overexpression in cancer cells may be associated with the oncolytic replication of E1B55K-mutated viruses.

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Figures

FIG. 1.
FIG. 1.
(A) Comparison of cyclin E mRNA levels in WI-38 cells infected with Ad5, dl1520, or Adhz60 using quantitative real-time PCR. The reaction was performed in duplicate for both the target gene and β-actin normalizer. The level of gene expression was calculated after normalizing against β-actin. The values indicate the n-fold change of cyclin E in cells at 12 h and 24 h postinfection at an MOI of 10 compared with the change in mock infection. The values are means ± standard errors of the means from two independent determinations (P of <0.01 by Student's t test). (B) Western blot analysis of cyclin E (cyc E) produced in WI-38 cells 2 days after mock infection or infection with Ad5, Adhz60, or dl1520 at an MOI of 10. Antibodies against cyclin E and β-actin were used. *, P < 0.01, Student's t test.
FIG. 2.
FIG. 2.
Western blot analysis of cyclin E (cyc E) produced in A549 cells after mock infection or infection with Ad5, Adhz60, or dl1520 at an MOI of 10. Cells were collected at the indicated times postinfection and subjected to Western blot analysis, as described in Materials and Methods. β-Actin was used as a control to demonstrate an equal loading and transfer.
FIG. 3.
FIG. 3.
Western blot analysis of adenoviral E1A and cyclin E proteins produced in WI-38 (A) and A549 (B) cells after mock infection or infection with Ad5 or dl1520 at an MOI of 10 or with increasing amounts of Adhz69 (MOIs of 0.25, 0.64, 1.6, 4, and 10). Cells were collected at day 2 postinfection and subjected to Western blot analysis.
FIG. 4.
FIG. 4.
Western blot analysis to confirm cyclin E (cyc E) protein induced by Ad infection. The pTet-cycE plasmid was introduced into 293-E2T cells to induce cyclin E expression by the Tet-off expression system. The cyclin E proteins induced by Ad5 infection are identical to the ones produced from the pTet-cycE plasmid when Tet was removed.
FIG. 5.
FIG. 5.
(A) The plasmid pCycE-EGFP was transfected into WI-38 and A549 cells, which were then mock infected or infected with Ad5 or dl1520. EGFP expression under the control of the cyclin E promoter was determined 2 days later. Ad5 infection activated the cyclin E promoter and resulted in higher EGFP expression. (B) Cell cycle profiles of A549 and WI-38 cells after incubation in 0.5% serum medium for 2 days. Cells were stained with propidium iodide for FACS analysis. (C) BrdU incorporation into serum-starved quiescent Ad-infected A549 and WI-38 cells. Ad5- and dl1520-infected cells were labeled by 10 mM BrdU for 60 min before fixation at 48 h postinfection. Incorporated BrdU was detected by fluorescein isothiocyanate-conjugated anti-BrdU monoclonal antibody. Hoechst staining was used to identify nucleus localization. Cells were rinsed with phosphate-buffered saline and analyzed using a fluorescent microscope (Olympus X-70). (D) Southern blotting was used to determine the viral DNA synthesis within A549 and WI-38 cells infected at synchronous G0 versus S phase. The DNA was isolated from the cells at days 0, 1, and 2 postinfection and fragmented with the restriction enzyme PstI. The probe was Ad5 genome DNA.
FIG. 6.
FIG. 6.
(A) Comparison of CPE in A549 and WI-38 cells infected with Ad5 and dl1520 at G0 or S phases. All microscopy is originally at a magnification of ×20 at 72 h postinfection. (B) Comparison of Ad5 and dl1520 replication in A549 and WI-38 cells. The dashed line indicates the level of virus added into the cell culture originally. The bars represent the means ± standard errors of the means obtained from triplicate determinations at 72 h postinfection. U, unsynchronized; S, S phase; G0, G0 phase.
FIG. 7.
FIG. 7.
Western blot analysis of cyclin E (cyc E) and cyclins B, D, and A produced in (A) WI-38 and (B) A549 cells mock infected or infected with Ad5 or dl1520 during G0 or S phase. Cells were collected at days 1, 2, and 3 postinfection and subjected to Western blot analysis. β-Actin was used as a control to demonstrate an equal loading and transfer. (C) Viral growth kinetics in A549 and WI-38 cells cultured in 10% serum or serum-free medium. The virus titers produced in the cultures were determined at days 0, 1, 2, 3, and 4.
FIG. 8.
FIG. 8.
(A) At 36 h after transfection with the cyclin E (cycE)-specific or nonspecific siRNA, WI-38 cells were mock infected or infected with Ad5. Cyclin E protein levels in WI-38 cells were determined at 48 h postinfection with anti-cyclin E antibodies. Ad5-induced cyclin E is significantly inhibited by the cyclin E-specific siRNA. The cyclin E protein level standard is a twofold dilution series of Ad5-infected WI-38 cell lysates. (B) Cyclin E-associated kinase activity was determined by in vitro kinase assays using histone HI as substrate in the presence of 32P-labeled ATP. (C) Comparison of CPE and virus production in WI-38 cells treated with siRNA-cyclin E or nonspecific siRNA. The cyclin E-specific siRNA partially repressed CPE and led to a threefold decrease of viral titers in WI-38 cells.
FIG. 9.
FIG. 9.
Western blot analysis of cyclin E (cyc E) protein produced in (A) permissive cancer cells (HCT116, RKO, HepG2, and Hep3B) and (B) restrictive cancer cells (Saos2, HeLa, MDA-MB-231, and HT29) infected with Ad5 or dl1520. Ad5 infection significantly increased cyclin E protein levels in both permissive and restrictive cancer cells. dl1520 infection could only induce cyclin E expression in the four permissive cancer cell types but not in any of the restrictive cancer cell types.
FIG. 10.
FIG. 10.
E1B55K may activate cell factors which, in turn, target the cyclin E promoter to increase expression. The cell factors may already be activated in cancer cells; thus, cancer cells are less dependent on the E1B55K function for cyclin E induction and viral DNA replication.
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