cis-dominant defect in activation of adenovirus type 5 E1b early RNA synthesis

Abstract

A cis-dominant mutation in the adjacent Ela gene disrupted the accumulation of adenovirus type 5 Elb mRNA during the early phase of infection. Steady-state levels of cytoplasmic and nuclear Elb RNAs in cells infected with d1312, a strain that lacks the Ela TATA box, cap site, and much of the coding sequence, were reduced 5to 10-fold even when the Ela activator was provided in trans. The strain was defective for early Elb RNA synthesis but not for Elb RNA made late or during prolonged incubation in the presence of an inhibitor of DNA replication. The defect in Elb RNA synthesis could not be attributed to the Ela promoter sequences missing in d1312 DNA. If the Ela protein-coding region contains cis-acting regulatory sequences, they are not part of the previously mapped Elb transcriptional control region and may represent additional regulatory elements that ensure prompt and efficient Elb expression during the early phase of infection.

Readthrough activation of early adenovirus E1b gene transcription

“…Adenovirus stocks were prepared as cell lysates from infected 293 cells and quantified by plaque titration as described previously (33,47).…”

“…Plasmid p362/ tdpm-CAT was derived by a fragment exchange from plasmid gDЉEF (obtained from Erik Falck-Pedersen [28]), which contains point mutations within the poly(A) regions of the terminator, to p362/t-CAT. A HindIII-BamHI fragment of each of these three plasmids, containing the transcription termination sequences, E1b promoter, CAT coding region, simian virus 40 poly(A) sequences, and a small portion of the plasmid backbone, was exchanged for the XbaI-BglII region (adenovirus type [Ad5] 5 nt 1339 to 3328) of plasmid pXC1 (33) to produce pXC1/t-CAT, pXC1/ti-CAT, and pXC1/tdpm-CAT, respectively.…”

“…Recombinant viruses. Recombinant adenoviruses were constructed by overlap recombination of pXC1-derived plasmids into the dl309 genome as described previously (33,47). Plasmid pXC1/343/CAT was used to produce strain sub2046, sub2040 was constructed from pXC1/t-CAT, sub2041 was constructed from pXC1/ti-CAT, and sub2047 was constructed from pXC1/tdpm-CAT.…”

“…Plasmid pXC1/343/CAT was used to produce strain sub2046, sub2040 was constructed from pXC1/t-CAT, sub2041 was constructed from pXC1/ti-CAT, and sub2047 was constructed from pXC1/tdpm-CAT. The construction of virus strains ⌬X(ϪX), ⌬Y(ϪY), and ⌬XY(ϪX,ϪY) was described previously (46), as was that of strain dl2004 (33). Virus strain ϪGC,ϪY was constructed from plasmid p563/⌬127-35/112.…”

“…for measurement of late gene expression. Cytoplasmic RNA was prepared and hybridized to probes either 5Ј-or 3Ј-end labeled with 32 P as described previously (32,33). All probes were prepared by digestion with an appropriate restriction enzyme, labeling at either the 5Ј end with T4 polynucleotide kinase (E1b, E1b-CAT, and E3) or the 3Ј end with DNA polymerase Klenow fragment (E1a), digestion with a second enzyme, and purification of the desired DNA fragment from an agarose gel.…”