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

Abstract

The ElA 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 EIA missense mutant gene hr5 is novel in that it inhibits the wild-type (wt) ElA 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 ElA proteins, the ability of hr5 ElA 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 ElA gene containing this missense mutation as the only alteration. The smaller 243R ElA 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 ElA genes were present in equimolar amounts; complete inhibition occurred with a fivefold molar excess of the hr5 gene. Two other ElA missense mutant genes (hr3 and hr4) with amino acid substitutions in close proximity to that of hr5 failed to block wt ElA-induced transcription when similarly tested. Also, the hr5 ElA gene failed to impede the pseudorabies immediate early gene from transactivating the adenovirus E3 promoter, demonstrating that hr5 ElA inhibits wt ElA 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 ElA protein competes with the wt 289R protein for a cellular transcription factor required for transactivation.

A transdominant tat mutant that inhibits tat-induced gene expression from the human immunodeficiency virus long terminal repeat.

“…Mutants of a number of other viral trans-activator proteins such as ElA (36), tax (37), and VM65 (38) have been identified that inhibit the promoter activation of their wildtype proteins. These so-called transdominant mutants may play a role in creating cell lines "immune" to subsequent viral infection (38,39 (31)(32)(33)(34).…”

The 289-amino acid E1A protein of adenovirus binds zinc in a region that is important for trans-activation.

“…It remains unclear how the 289-aa protein stimulates its target promoters since a common inducible DNA response element has not been identified (18)(19)(20)(21)(22)(23). However, various studies strongly indicate that the mechanism of ElA trans-activation involves recruitment of one or more cellular transcription factors (24)(25)(26).…”

“…However, ElA protein alone does not appear to bind DNA (51), and there is no obvious target element common to all ElA-inducible promoters (18)(19)(20)(21)(22)(23). On the other hand, bacterially produced ElA protein can exhibit DNA binding when mixed with cellular extracts (51,52), and there is compelling evidence that trans-activation by ElA occurs through an interaction with cellular transcription factors (24)(25)(26)53). In the presence of ElA, there is enhanced binding of a cellular factor upstream of the E2 promoter (24), and mutational analysis of the E1B promoter suggests that ElA increases the activity of the "TATA" box transcription factor (25).…”

Adenovirus E1A N-Terminal Amino Acid Sequence Requirements for Repression of Transcription In Vitro and In Vivo Correlate with Those Required for E1A Interference with TBP-TATA Complex Formation

“…E1A 289R differs from E1A 243R by possessing conserved region 3 (CR3), a 46-amino-acid domain unique to 289R. CR3 is essential (13,20,24,30,34) and sufficient (15,25) for activation of viral early genes. Domains common to E1A 243R and E1A 289R are required for the growth-regulatory functions of E1A; these include the nonconserved N terminus (amino acids 1 to 39), CR1 (amino acids 40 to 80), and CR2 (amino acids 120 to 139) (28).…”