Related Experiment Video
Updated: Oct 7, 2026
Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts
Published on: September 9, 2010
Selective effects of E1B-defective adenoviruses and adenovirus E1A mutants in deficient mouse primary embryonic
P Martin Duque1, C Parada, J Guinea
1Department of Pathology, Clinica Puerta de Hierro, Madrid, Spain.
Abstract:
E1B-defective adenoviruses have been described as exerting selective cytopathic effects on transformed cells. Previously, we showed that adenovirus dl118, lacking both E1B proteins, very efficiently kills most human malignant cell lines. In order to study whether these selective effects were due to selective replication of dl118 in cells harboring specific genetic alterations, we compared the viability of various deficient mouse primary fibroblasts. We studied mouse embryonic fibroblasts (MEFs) derived from p16, p21, p27 and p53 knockout mice, as well as wild-type MEFs. We infected them with 100 p.f.u. of adenoviruses adl118, adwt300, and adenoviruses carrying the E1A mutant 922 (the E1a product only binds to the p300 and related proteins) and Ad646 (the E1A product binds to the pRb and related proteins). The percentage of infectivity was evaluated with an adenovirus carrying the green fluorescent protein (AdGFP). With AdGFP, clear green fluorescent signals were detected in more than 70% of the cells after 3 days of infection. After infection with several adenoviruses, we observed that E1A mutant 922 killed all the MEFs. Conversely, the E1a mutant Ad646 exerted its major effects on control wild-type MEFs. Moreover, Adl118 killed the wtMEFs and other MEFs slightly more efficiently than did wtAd, but less than Ad922. No viral replication was detected by adding the obtained supernatants to HEK293 cells. Due to the absence of significant viral replication on these cells, the results could be interpreted as direct effects of E1A and E1A mutant proteins on the different mouse cells carrying diverse genetic alterations.
Insights
E1B-defective adenoviruses show selective cell killing. This study found that adenovirus dl118 and E1A mutants directly impact mouse embryonic fibroblasts (MEFs) with genetic alterations, independent of viral replication.
Area of Science:
- Virology
- Molecular Biology
- Cancer Research
Background:
- E1B-defective adenoviruses exhibit selective cytopathic effects on transformed cells.
- Adenovirus dl118, lacking E1B proteins, efficiently eliminates most human malignant cell lines.
- Investigating selective effects requires understanding viral replication in genetically altered cells.
Purpose of the Study:
- To determine if selective adenovirus dl118 effects correlate with selective replication in cells with specific genetic alterations.
- To compare the viability of various deficient mouse embryonic fibroblasts (MEFs) upon infection with different adenoviruses.
- To elucidate the direct impact of E1A and E1A mutant proteins on genetically diverse MEFs.
Main Methods:
- Infection of wild-type and knockout MEFs (p16, p21, p27, p53) with adenoviruses (adl118, adwt300, E1A mutant 922, Ad646).
- Assessment of infectivity using adenovirus carrying green fluorescent protein (AdGFP).
- Evaluation of viral replication by co-culturing supernatants with HEK293 cells.
Main Results:
- E1A mutant 922 demonstrated potent killing across all tested MEFs.
- Ad646 primarily affected wild-type MEFs.
- Adenovirus dl118 showed slightly enhanced killing of MEFs compared to wild-type adenovirus but less than Ad922.
- No significant viral replication was detected in any MEF cell types.
Conclusions:
- The observed selective cytopathic effects are attributed to direct interactions of E1A and E1A mutant proteins with MEFs.
- Viral replication is not the primary mechanism driving the differential killing of genetically altered MEFs by these adenoviruses.
- These findings highlight the potential of specific adenovirus E1A proteins as targeted agents against cells with distinct genetic profiles.
