Adenovirus type 2 coded single-stranded DNA binding protein: in vivo phosphorylation and modification

Abstract

The adenovirus type 2-coded single-stranded DNA binding protein (DBP) was shown to be a phosphoprotein and to exist in at least two forms that differ in mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After a 30-min pulse with [35S]methionine or 32PO4, 35Sor 32P-labeled DBP had a nominal molecular weight of 74,000, whereas after a 30-min label followed by a 20-h chase, 35S-and 32P-labeled DBP had a nominal molecular weight of 77,000. Both large and small forms of 35S-and 32P-labeled DBP bound to single-stranded DNA-cellulose columns and were eluted by 0.4 to 0.6 M NaCl; both forms also were immunoprecipitated by antiserum against adenovirus type 1-simian virus 40-induced tumor cells (this antiserum contains antibodies against DBP) and by monospecific antiserum against 95 to 99% purified DBP. With highly purified 32P-DBP labeled 7 to 10 h postinfection, it was shown that the 32p radioactivity was firmly associated with protein material (i.e., not contaminating nucleic acids or phospholipids) and had properties expected of a phosphoester of an amino acid; paper electrophoresis of acid hydrolysates of this preparation identified phosphoserine but not phosphothreonine. Phosphoserine but not phosphothreonine was also identified in acid hydrolysates of another preparation of 32P-DBP labeled for 30 min, chased for 20 h, and then immunoprecipitated by adenovirus type 1-simian virus 40 antiserum. Human adenovirus 2 (Ad2) is an oncogenic DNA virus that replicates in the nucleus of permissive human cells (reviewed in reference 18 and in W. S. M. Wold, M. Green, and W. Buttner, In D. P. Nayak, ed., Molecular Biology of Animal Viruses, in press). The productive infection proceeds in an "early" stage, when 23 to 32% of the asymmetric genome is expressed as mRNA, and in a "late" stage, which follows the initiation of viral DNA replication at 6 to 7 h postinfection (p.i.). Virus-coded or-induced proteins synthesized early after infection probably are involved in viral DNA replication,

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

“…Figure 5a and b show by immunoprecipitation analysis that the DBP was synthesized in Ad2tslll-infected HeLa cells at the nonpermissive temperature and was produced at levels similar to that of DBP synthesized in wild-type-infected cells grown in the presence of hydroxyurea, which mimics the DNA replication block in Ad2tslll. Furthermore, DBP, which is normally phosphorylated in wild-type-infected cells (16,23,33), was also phosphorylated in Ad2tslll-infected cells (Fig. Sc).…”

Characterization of the effect of aphidicolin on adenovirus DNA replication: evidence in support of a protein primer model of initiation

“…Adenovirus DNA replication occurs in the nucleus of infected permissive cells and requires only a limited number of proteins encoded by its own genome plus others provided by the host cell (1). To date only 2 viral encoded gene products have been shown to be involved in the replication of viral DNAa 72,000 dalton single-stranded DNA binding protein (DBP) (2)(3)(4)(5)(6) and an 87,000 dalton protein (preterminal protein) which is found covalently attached to replicating DNA (7). A processed form of the preterminal protein (M.W.…”

Transcriptional activities of different phosphorylated species of NS protein purified from vesicular stomatitis virions and cytoplasm of infected cells

“…Phosphorylated proteins similar to NS have been found in other RNA and DNA viruses (8,20,22) and are often associated with the viral nucleic acid (10,16). There is an indication that the degree of phosphorylation may play a regulatory role in viral replication.…”