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Inhibition of adenovirus cytotoxicity, replication, and E2a gene expression by adeno-associated virus
X J Jing1, V Kalman-Maltese, X Cao
1Department of Biochemistry and Molecular Biology, Medical College of Ohio, 3035 Arlington Avenue, Toledo, Ohio 43614-5804, USA.
Abstract:
Adeno-associated virus (AAV) and the other parvoviruses have long been known to inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we have begun an investigation into AAV's relationship with adenovirus (Ad), AAV's most efficient helper virus. AAV, but not UV-inactivated AAV, delayed Ad-induced cytotoxicity and inhibited Ad E2a gene expression. AAV, but not UV-inactivated AAV or a recombinant AAV vector, inhibited Ad DNA replication. To determine whether AAV or its replication (Rep) proteins alter Ad early gene expression, we measured steady state E2a mRNA levels in AAV and Ad coinfected cultures and in a cell line (Neo6) that inducibly expresses the Rep proteins. AAV, but not UV-AAV, and Rep expression resulted in diminution of E2a protein and mRNA levels. To determine whether the AAV Rep proteins directly affect the individual Ad early promoters, we constructed luciferase reporter plasmids containing each of the five early promoters. Cotransfection of Ad-luciferase and an AAV rep gene-expressing plasmid in HeLa cells demonstrated that Rep78 repressed the E1a, E2a, and E4 promoters but trans-activated the E1b and E3 promoters. In the presence of a cotransfected E1a-expressing plasmid, Rep78 repressed expression from all five promoters. These results indicate that Rep may have different effects on the Ad early promoters dependent upon the presence of the E1a trans-activating protein.
Insights
Adeno-associated virus (AAV) inhibits adenovirus (Ad) replication and gene expression by affecting Ad early promoters. The AAV Rep proteins play a key role in this interaction, modulating Ad gene expression differently based on E1a presence.
Area of Science:
- Virology
- Molecular Biology
- Gene Regulation
Background:
- Adeno-associated virus (AAV) and other parvoviruses are known to inhibit nonpermissive cell proliferation.
- The precise mechanisms underlying this inhibition remain incompletely understood.
- Adenovirus (Ad) serves as an efficient helper virus for AAV, suggesting a significant interaction between the two viruses.
Purpose of the Study:
- To investigate the interaction between AAV and Ad, focusing on how AAV affects Ad-induced cytotoxicity and gene expression.
- To elucidate the role of AAV replication (Rep) proteins in modulating Ad early gene expression and DNA replication.
- To determine the direct impact of AAV Rep proteins on individual Ad early gene promoters.
Main Methods:
- Coinfection of cells with AAV and Ad, with comparisons to UV-inactivated AAV and recombinant AAV vectors.
- Measurement of steady-state E2a mRNA levels in coinfected cells and in a cell line inducibly expressing Rep proteins.
- Construction of luciferase reporter plasmids for each of the five Ad early promoters and cotransfection assays with AAV rep genes in HeLa cells.
Main Results:
- AAV, but not UV-inactivated AAV, delayed Ad-induced cytotoxicity and inhibited Ad E2a gene expression.
- AAV significantly inhibited Ad DNA replication, an effect not observed with UV-inactivated AAV or a recombinant AAV vector.
- AAV Rep expression, similar to AAV infection, led to decreased E2a protein and mRNA levels.
- Rep78 protein repressed E1a, E2a, and E4 promoters but trans-activated E1b and E3 promoters.
- In the presence of E1a, Rep78 repressed all five Ad early promoters, indicating context-dependent activity.
Conclusions:
- AAV actively interferes with Ad gene expression and replication, impacting key early viral genes.
- AAV Rep proteins are critical mediators of this interference, exhibiting differential regulation of Ad promoters.
- The effect of AAV Rep proteins on Ad promoters is modulated by the presence of the Ad E1a trans-activating protein, highlighting a complex interplay between the two viruses.