DNA sequence of the leftward junction in the adenovirus-simian virus 40 hybrid Ad2+D2 and determination of the structure of the D2-T antigen

Abstract

The nucleotide sequence of the junction between the simian virus 40 early region and the adenovirus type 2 late region L4 in the hybrid virus Ad2+D2 was determined. The deduced amino acid sequence suggests that the D2-T antigen is a chimeric protein sharing 594 amino acids with the C-terminal end of the simian virus 40 T antigen and 104 amino acids with the N terminus of the adenovirus type 2 33,000-molecular-weight protein. The predicted structure of the D2-T antigen was confirmed by an immunoprecipitation analysis. Adenovirus type 2/simian virus 40 (Ad2/SV40) hybrid viruses have been invaluable tools in defining the coding region for SV40 T antigen, assigning functional domains to this pleiotropic protein, analyzing the transcription of both Ad2 and SV40, and studying recombination (for review, see references 10 and 29). Ad2+D2, a defective Ad2/SV40 hybrid, was isolated more recently (6) by plaque purification from a mixture of hybrid viruses known as Ad2+ +HEY (11). Ad2+D2 contains an almost entire copy of the SV40 genome ranging counterclockwise from about 0.54 to 0.63 map units in partial replacement of Ad2 sequences that are deleted between 73 and 98 map units (see reference 6 and Fig. 1). HeLa cells infected with this virus synthesize large quantities of an SV40 T-antigen-related protein, D2-T antigen, of a molecular weight estimated to be 107,000 (6, 26). This protein has been extensively purified and shown to have biological properties similar to those of an authentic SV40 T antigen (20, 27, 28). In particular, it binds with a specificity similar to that of T antigen to three sites on the SV40 genome comprising the origin of replication (2, 26). It has been proposed that the N-terminal end of D2-T antigen consists of part of the Ad2 100,000-molecular-weight (100K) protein (6), but this has not been demonstrated directly. Since the Ad2 DNA sequences adjacent to the

Effects of in vitro dephosphorylation on DNA-binding and DNA helicase activities of simian virus 40 large tumor antigen

“…The importance of the N-terminal section for the conformation of T antigen may be the reason that dephosphorylation studies with the T antigen-related D2 protein gave results (1) that differed from those reported here. The D2 protein, a product of an SV40-adenovirus hybrid, shares its C-terminal 594 amino acids, including the domains for specific DNA binding and for DNA helicase-ATPase activity, with authentic T antigen (2). Most of the 122 N-terminal amino acids of D2 protein are derived from an adenovirus protein and are not related to those in the N-terminal section of T antigen.…”

DNA binding properties and replication activity of the T antigen related D2 phosphoprotein

“…D2 protein lacks the 114 N-terminal amino acids of authentic T antigen and has instead an N-terminus of 122 amino acids derived mainly from the adenovirus 33 K protein (Baumann et al, 1985). However, D2 protein does possess the two functional domains important for the replicative function of T antigen, namely, the DNA binding and the ATPase/helicase domain.…”

“…D2 protein (lower line) consists of 716 amino acids and is composed of 3 parts: the 104 N-terminal amino acids up to Gly-104 are derived from the adenoviral 33K protein (stippled): this region is connected via a stretch of 18 amino acids of unknown origin (white) to the 594 C-terminal amino acids which are derived from SV40 large T antigen (black). The C-terminal part begins with a glutamic acid residue corresponding to Glu-115 in authentic T antigen (Baumann et al, 1985). The phosphoamino acids in the T antigen part of D2 are indicated as vertical lines.…”

“…The D2 protein (or D2-T antigen), produced by the adeno-SV40 hybrid virus Ad2+D2, is a chimeric protein composed of 3 sections: an N-terminal section of 104 amino acids derived from the N-terminus of an adenovirus structural protein, the adeno 33K protein; a linking region of 18 amino acids of unknown origin; and a 594 amino acid long C-terminal region corresponding to amino acids 114-708 of SV40 T antigen (Figure 1). Thus, D2 protein is composed of 716 amino acids and shares its C-terminal 594 amino acids, including the DNA binding and the ATPase/helicase domains, with SV40 T antigen (Baumann et al, 1985). D2 protein, a nuclear phosphoprotein like SV40 T antigen, is produced in large quantities in Ad2+D2-infected HeLa cells and has served as a substitute for authentic T antigen in earlier studies on the biochemistry of T antigen.…”

An N-terminal deletion mutant of simian virus 40 (SV40) large T antigen oligomerizes incorrectly on SV40 DNA but retains the ability to bind to DNA polymerase alpha and replicate SV40 DNA in vitro

“…However, interpretation of these results must include the possibility that mutant T antigen proteins were unstable in vivo or that mutant T antigen may have been defective for oligomerization or other biochemical functions required for replication. D2-T, a hybrid protein composed of residues 115 to 708 of SV40 T antigen and more than 100 N-terminal residues derived from an adenovirus protein and from an unknown source (2), was only 20 to 30% as active as wild-type T antigen in DNA replication in vitro (35). Since several other properties of D2-T were aberrant and it is unknown what effects the ''foreign'' residues have on the protein, the role of the N-terminal residues in T antigen cannot be deduced from these data.…”