Adenovirus DNA Replication I. Requirement for Protein Synthesis and Isolation of Nuclear Membrane Fractions Containing Newly Synthesized Viral DNA and Proteins

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High-titer monospecific antiserum against highly purified adenovirus 2 (Ad2) single-stranded DNA binding protein (DBP) was used to study, by indirect immunofluorescence (IF), the synthesis of DBP in Ad2-infected human cells and adenovirus-transformed rat, hamster, and human cell lines. In infected cells the synthesis of DBP was first detected in the cytoplasm at 2 to 4 h postinfection and reached a maximum intensity at 6 h postinfection. At this time DBP began to accumulate in the nucleus, where it reached maximum intensity at about 14 h postinfection. The cytoplasmic IF was diffuse, whereas nuclear IF appeared as dots that coalesced into large globules as infection progressed. In cells treated with 1-,3-D-arabinofuranosylcytosine to inhibit viral DNA synthesis, strong nuclear IF was observed in the form of dots, but the large fluorescent globules were not observed. The Ad2 (oncogenic group C) anti-DBP serum reacted very strongly by IF with Ad5 (group C)-infected, to a lesser extent with Ad7 and Adli (group B)-infected, and weakly with Ad12 and Adi8 (group A)-infected KB cells (treated with 1-,8-D-arabinofuranosylcytosine). These results may indicate that Ad2 DBP is closely related immunologically to DBPs induced early after infection by adenovirus serotypes in oncogenic group C, moderately related to DBPs of serotypes in oncogenic group B, and perhaps distantly related to DBPs of serotypes in oncogenic group A. The following adenovirus-transformed cell lines were examined for DBP synthesis by IF with the Ad2 anti-DBP serum: six rat cell lines (T2C4, F17, 8662, 8638, 8617, and F161) transformed by Ad2 virus, three hamster cell lines transformed by Ad2 virus (Ad2HT1) and Ad2-simian virus 40 hybrid virus (ND1HK1 and ND4HK4), and one rat (5RK) and one human (293-31) cell line transformed by transfection with Ad5 DNA. T2C4 and 8662 appeared weakly positive, whereas Ad2HT1 and ND4HK1 were strongly positive. The other transformed cell lines did not produce DBP detectable by IF. Thus, some but not all transformed cell lines produce DBP, which indicates that DBP is not required for maintenance of cell transformation and that transformed cells can express "nontransforming" viral genes as protein.Human adenoviruses are oncogenic DNA viruses that mature in the nucleus of permissive human cells and transform semipermissive or nonpermissive cell types with low frequency (reviewed in reference 31; W. S. M. Wold, M. Green, and W. Buttner, in D. P. Nayak [ed.], Molecular Biology of Animal Viruses, in press). There are at least 31 human adenovirus serotypes; some serotypes fall into three welldefined oncogenic groups: group A (adenovirus 12

Section: Resultssupporting

confidence: 90%

mentioning

confidence: 99%

Immunofluorescence Study of the Adenovirus Type 2 Single-Stranded DNA Binding Protein in Infected and Transformed Cells

Sugawara

Gilead

Wold

et al. 1977

Self Cite

“…Biochemical evidence indicates that the adenovirus DNA-binding protein is an "early" viral protein (8,17,18) and that it can be isolated in association with replicating viral DNA (17,20). The circumscribed, virus-specific masses noted by immunofluorescence are probably the intranuclear "factories" for viral DNA synthesis, and they appear to correspond to the distinctive, small, rounded nuclear inclusions, which are initially eosinophilic during early stages of infection, but soon become basophilic and Feulgen positive (4).…”

Section: Stability Of the Dna-binding (72k) Proteinmentioning

confidence: 99%

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

Ginsberg¹,

Lundholm²,

Linné³

1977

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.

“…Recently we have described the properties of a nuclear membrane fraction isolated from adenovirus 2infected KB cells that has some properties of a functional DNA replication complex. This complex, isolated by sedimenting sonically disrupted nuclei in a discontinuous sucrose gradient, contains newly synthesized adenovirus DNA sequences that can be chased into genome length viral DNA (18). In vitro, the complex synthesizes exclusively adenovirus DNA sequences (T. Yamashita, M. Arens, and M. Green, J. Biol.…”

mentioning

confidence: 99%

Isolation of DNA polymerase gamma from an adenovirus 2 DNA replication complex

Ito¹,

Arens²,

Green³

1975