An adenovirus type 5 E1A protein with a single amino acid substitution blocks wild-type E1A transactivation

The E1A gene of adenovirus type 5 encodes a 289-amino-acid (289R) protein that transactivates early adenovirus promoters. We showed that the 289R protein of the E1A missense mutant gene hr5 is novel in that it inhibits the wild-type (wt) E1A protein from stimulating transcription from each of the early viral promoters E2, E3, and E4. Since both the hr5 and wt genes produced similar levels of E1A proteins, the ability of hr5 E1A to block transactivation was attributed to the replacement of serine by asparagine as position 185. We confirmed that this single amino acid substitution was responsible for blocking transactivation by showing equal inhibition with an hr5-wt hybrid E1A gene containing this missense mutation as the only alteration. The smaller 243R E1A protein of hr5 was not necessary for inhibition. Transcriptional activity from each early promoter was inhibited by at least 50% when the hr5 and wt E1A genes were present in equimolar amounts; complete inhibition occurred with a fivefold molar excess of the hr5 gene. Two other E1A missense mutant genes (hr3 and hr4) with amino acid substitutions in close proximity to that of hr5 failed to block wt E1A-induced transcription when similarly tested. Also, the hr5 E1A gene failed to impede the pseudorabies immediate early gene from transactivating the adenovirus E3 promoter, demonstrating that hr5 E1A inhibits wt E1A activation at the transcriptional, rather than the posttranscriptional, level. Although several possibilities were considered to account for this inhibition, the most likely is that the nonfunctional hr5 E1A protein competes with the wt 289R protein for a cellular transcription factor required for transactivation.