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

Abstract

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

Nuclear factor I is specifically targeted to discrete subnuclear sites in adenovirus type 2-infected cells

“…Correspondingly, proteins involved in DNA replication, such as DNA polymerase ao and proliferating cell nuclear antigen, appear to be localized to these same sites when DNA synthesis is taking place (6,7,40,73). A similar situation is also observed in adenovirus-and herpesvirus-infected cells, in which viral DNA and viral replication proteins are localized in a relatively small number of discrete nuclear sites (37,47,48,50,53,61,67).…”

Imaging the adenovirus infection cycle

“…Indirect immunofluorescence (IF) using antiserum from AdC5‐inoculated rabbits paired with fluorescein‐labeled secondary antibodies was the first FM strategy to stain virus‐infected cells and allowed quantifying the number and timing of appearance of infected cells . Subsequent studies used IF to detect Ads and adenoviral gene products even before their biological significance was fully understood, exemplified by the P‐antigen later shown to detect the single‐strand DNA binding protein (DBP) , and the T‐antigen later shown to detect the transforming early E1B‐55k protein . Today, several specific antibodies for FM exist against structural and nonstructural Ad proteins, summarized in Table .…”

“…A serum (termed P‐antigen) generated by injection of a rabbit with clarified lysates from Ad‐infected cells detected nuclear dots as early as 6–7 h postinfection in HEK cells, and these dots developed into ringlike structures accurately depicting the formation of RCs during Ad replication . The P‐antigen was later shown to detect the single‐strand DBP , which binds viral ssDNA during genome replication . FISH detection of Ad genomes with rhodamine‐marked RNA probes was developed to complement radioactive hybridization probes .…”

“…Several studies have described how the DBP IF stain changes during Ad replication reflecting morphological changes in the nucleus. In most cells, the DBP signal is detectable at ~ 6–7 h postinfection as faint cytosolic stain translocating into the nucleoplasm by ~ 10–12 h postinfection where it eventually forms dots that fuse and grow into ringlike or globular structures morphing into larger, irregular‐shaped intranuclear domains . Currently, DBP is frequently used as reference stain to describe spatial and temporal events in the nucleus of Ad‐infected cells .…”

Dynamic organization of pre-mRNA splicing factors

“…One system uses cells that have been infected with adenovirus [Jimenez-Garcia and Spector, Pombo et al, 1994;19931 and another system examines transiently transfected cells [Huang and Spector, in press]. When HeLa cells are infected with adenovirus, the newly established viral replication and transcription centers form ring-shaped structures which have been morphologically characterized at both the light and electron microscopic level [Martinez-Palomo et al, 1967;Pombo et al, 1994;Puvion-Dutilleul and Puvion, 1990;Reich et al, 1983;Sugawara et al, 1977;Voelkerding and Klessig, 19861. When the localization of splicing factors such as snRNPs were examined in the infected cells, the distribution of these factors was found to be colocalized with the adenoviral RNA in the ring structures [Jimenez-Garcia and Spector, 1993;Pombo et al, 19941.…”