Inhibition of adenovirus cytotoxicity, replication, and E2a gene expression by adeno-associated virus

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.