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Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death
Published on: October 21, 2012
E4orf1 limits the oncolytic potential of the E1B-55K deletion mutant adenovirus
Michael A Thomas1, Robin S Broughton, Felicia D Goodrum
1Department of Microbiology and Immunology, Medical Center Blvd., Wake Forest University School of Medicine, Winston-Salem, NC 27157-1064, USA.
Abstract:
Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G(1) phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G(1) restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3'-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function.
Insights
The viral oncogene E4orf1 restricts the effectiveness of oncolytic adenoviruses like ONYX-015 by hindering viral replication in G1-phase cells. Inhibiting E4orf1 function enhances tumor cell killing and improves the therapeutic potential of these cancer therapies.
Area of Science:
- Oncology
- Virology
- Molecular Biology
Background:
- Oncolytic adenoviruses show tumor selectivity and minimal toxicity.
- The ONYX-015 adenovirus, lacking the E1B-55K protein, is more effective with chemotherapy or radiation.
- Improving oncolytic adenovirus efficacy is crucial for cancer therapy.
Purpose of the Study:
- To identify the factor responsible for the G1-phase restriction of the E1B-55K deletion mutant adenovirus.
- To investigate the mechanism by which this factor restricts viral replication.
- To explore strategies for enhancing the oncolytic activity of E1B-55K deletion mutant adenoviruses.
Main Methods:
- Infection of cells in different cell cycle phases (G1 vs. S) with the E1B-55K deletion mutant virus.
- Analysis of viral late protein synthesis, progeny production, and cell killing rates.
- Investigating the role of the E4orf1 gene and its downstream signaling pathways (PI3K/Akt/mTOR/p70 S6K, Rac1, Tiam1).
- Testing the effect of agents disrupting Tiam1-Rac1 interaction or p70 S6K phosphorylation.
Main Results:
- The G1 restriction of the E1B-55K deletion mutant virus is caused by the viral oncogene E4orf1.
- E4orf1 activates the Akt and p70 S6K signaling pathways, potentially involving Rac1 and Tiam1.
- Disrupting the Tiam1-Rac1 interaction or inhibiting p70 S6K phosphorylation partially relieved the E4orf1 restriction.
- These interventions enhanced viral protein synthesis and tumor cell killing by the E1B-55K mutant virus.
Conclusions:
- E4orf1 limits the oncolytic potential of conditionally replicating adenoviruses like ONYX-015.
- Abrogating E4orf1 function can improve the therapeutic efficacy of oncolytic adenoviruses.
- Targeting E4orf1 or its downstream pathways represents a promising strategy for enhancing adenovirus-based cancer therapy.
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