Adenovirus DNA-binding protein in cells infected with wild-type 5 adenovirus and two DNA-minus, temperature-sensitive mutants, H5ts125 and H5ts149

Abstract

Studies have been done to characterize further H5ts125, an adenovirus type 5 conditionally lethal, temperature-sensitive (ts) mutant defective in initiation of DNA synthesis and to investigate whether the single-strand-specific DNAbinding (72,000 molecular weight) protein is coded by the mutated viral gene. When Hftsl25-infected cells were labeled with [35S]methionine at 3200 and then incubated without isotope at 39.50C, the mutant's nonpermissive temperature, the 72,000 molecular weight polypeptide was progressively degraded. Immunofluorescence examination of cells infected with wild-type virus, H5ts125, and H5ts149 (a second, unique DNA-minus mutant) showed that immunologically reactive DNA-binding protein was barely detectable in Hftsl25-infected cells at 39.50C, whereas this protein was present in wild-typeand H5ts149-infected cells, that the protein made at 32TC in Hftsl25-infected cells lost its ability to bind specific DNA-binding protein antibody when the infected cells were shifted to 39.50C, and that if Hftsl25-infected cells were shifted from the restrictive temperature to 3200, even in the presence of cycloheximide to stop protein synthesis, immunologically reactive DNA-binding protein reappeared.

Independent mutations in Ad2ts111 cause degradation of cellular DNA and defective viral DNA replication

“…Cells were infected at the permissive temperature (32.5°C), and at 24 h after infection were either left at 32.5°C or shifted to the nonpermissive temperature (39.5°C) for a further 4 h. The cells were then fixed and stained by using a monoclonal antibody against Ad5 DBP and a fluorescein-labeled second antibody. Both Ad2 wild type and Ad2tslll show intense nuclear staining at both temperatures, whereas Ad5tsl25 shows intense fluorescence only at 32.5°C and weak diffuse fluorescence at 39.5°C, which was due to the degradation of the unstable Ad5tsl25 DBP (12,23). In the Ad2tslll 39.5°C nuclei, the staining pattern was slightly different from that at 32.5°C in that the globular pattern of staining was not as pronounced.…”

Possible role of the 72,000 dalton DNA-binding protein in regulation of adenovirus type 5 early gene expression

“…The products of adenovirus early gene expression have not yet been completely characterized, but some are required for virus DNA replication (9) and for transformation (11,14,18). The gene or genes required for virus-induced cell transformation are located at the left end of the genetic map (17) and are transcribed from the r strand (11). The only early gene product that has been isolated and studied extensively is a 72,000-dalton (72K) protein that binds to single-stranded DNA (25,40, 41) and is required for initiation of virus DNA synthesis (16,42).…”

Conserved region 3 of the adenovirus type 5 DNA-binding protein is important for interaction with single-stranded DNA

“…Even the wild-type DBP is not fully precipitable by 2/4, suggesting that within the DBP population there are molecules with different conformations, or at least molecules that have 2/4 epitopes with different accessibilities. Others have observed that the ts125 DBP does not react well with antisera at the nonpermissive temperature, consistent with the probable global effect of temperature on the conformation of this protein (14). Several ts herpesvirus ssDBP (ICP8) mutants also show altered reactivity with antibodies, typically being recognized by a polyclonal but not a monoclonal antibody (13).…”

“…Thus, the data for the ts23 DBP are most consistent with a wild-type protein-protein interaction, with the defect in elongation being due to the decreased affinity for ssDNA. The ts125 mutation (aa 413) is probably structural, as this Pro-to-Ser change results in a protein that is unstable at the nonpermissive temperature (14). The protein binds to ssDNA with the same affinity as the wild-type DBP at 4°C, supporting the suggestion that this mutation is in a region of the protein that is important for folding.…”