Identification of the gene and mRNA for the adenovirus terminal protein precursor

Stillman, Bruce; Lewis, James B.; Chow, Louise T.; Mathews, Michael B.; Smart, John E.

doi:10.1016/0092-8674(81)90145-8

Cited by 217 publications

(123 citation statements)

References 38 publications

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“…It may represent the precursor of the polypeptide covalently attached to the 5' ends of Adl2 virion DNA in analogy to the Ad2 87K protein expressed from this region (Stillman et al, 1981). For comparison of our protein data with those from other groups, we have listed in Fig.…”

Section: Discussionmentioning

confidence: 99%

Expression of Early Viral Gene Products in Adenovirus Type 12-infected and -transformed cells

Esche

Siegmann

1982

Journal of General Virology

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SUMMARYWe have analysed early viral gene products expressed in adenovirus type 12 (Adl2)-infected cells as well as in two Adl2-transformed hamster cell lines, and Adl2-induced rat tumour cell lines by cell-free translation of virus-specific RNA which was selected by hybridization to cloned restriction endonuclease fragments of virus DNA. Proteins synthesized in vitro were analysed by one-and twodimensional gel electrophoresis. It was found that RNA encoded by early region E1A directs the synthesis of at least eight polypeptides with apparent mol. wt. 38K, 36K, 30K, 28K, 26K, 25K, 24K and 22K. All these proteins are related to each other. E1B-specific RNA directs the synthesis of three proteins: 59K, 19K and 17K. Early region E2a codes for a 61K polypeptide which probably represents the single-strand DNA-binding protein of Adl2. RNA complementary to region E3 directs the synthesis of a 16K protein, and RNA transcribed from region E4 the synthesis of polypeptides with mol. wt. 20K, 18K and l l.5K. We have mapped a 67K polypeptide into the region within 11 to 28 map units (E2b). The analysis of proteins directed by virus-specific RNAs prepared from two Adl2-transformed hamster cell lines (T637, HA12/7) and one Adl2-induced rat tumour line (RBT12/3) showed that early regions E1 and E4 are expressed in all three Adl2-transformed cell lines. RNA transcribed from early regions E2 and E3 have been detected in lines T637 and RBT12/3. The virus RNA prepared from the Adl2-transformed cell lines directed synthesis of polypeptides with mol. wt. very similar to those of early virus proteins from infected cells. However, in all three Adl2-transformed cell lines mentioned above we have found RNAs which directed the synthesis of additional polypeptides of early regions E1 (34K) and E4 (25K, 24K) not detected in infected cells. The DNA sequence between 11 and 28 map units (coding for the 67K protein) is not expressed in the Ad 12-transformed cells.

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Section: Discussionmentioning

confidence: 99%

Expression of Early Viral Gene Products in Adenovirus Type 12-infected and -transformed cells

Esche

Siegmann

1982

Journal of General Virology

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“…The 5' ends of the viral DNA are covalently linked to a terminal protein through a deoxycytosine-to-serine phosphoryl bond (5,23). The 55-kilodalton (kDa) terminal protein is a proteolytic cleavage product of an 80-kDa protein which participates in the initiation of DNA replication (2,4,16,26). The terminal protein precursor forms a complex with a 140-kDa adenovirus-encoded polymerase (15,27), after which the terminal protein precursor becomes modified through its covalent linkage to a deoxycytosine, which will subsequently become the first nucleotide of the daughter strand.…”

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confidence: 99%

“…The terminal protein precursor forms a complex with a 140-kDa adenovirus-encoded polymerase (15,27), after which the terminal protein precursor becomes modified through its covalent linkage to a deoxycytosine, which will subsequently become the first nucleotide of the daughter strand. During morphogenesis of the virion, the 80-kDa terminal protein precursor is specifically cleaved by an adenovirus-encoded protease to produce the 55-kDa terminal protein (3,26).…”

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Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome.

Hanahan¹,

Gluzman²

1984

Mol. Cell. Biol.

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A variant of the adenovirus type 5 genome which lacks EcoRI sites has been cloned in a bacterial plasmid after the addition of EcoRI oligonucleotide linkers to its ends. Closed circular forms of the recombinant viral genome were not infectious upon their introduction into permissive eucaryotic cells. The linear genome released by digestion of the 39-kilobase recombinant plasmid (pXAd) with EcoRI produced infectious virus at about 5% of the level of wild-type controls. The viruses which arose were indistinguishable from the parental strain, and the normal termini of the viral genome had been restored. Marker rescue experiments demonstrate that provision of a DNA fragment with a normal viral end improves infectivity. When a small fragment carrying a wild-type left end (the 0 to 2.6% ClaI-B fragment) was ligated to Clal-linearized pXAd, virus was produced with efficiencies comparable to a similar reconstitution of the two ClaI fragments of the wild-type genome. These viruses stably carry the left-end fragment at both ends, leaving the normal right end embedded in 950 base pairs of DNA. The embedded right origin is inactive. The consensus of the analyses reported here is that a free end is a necessary configuration for the sequences which make up the adenovirus origin of replication.Human adenovirus type 5 (AdS) has a double-stranded DNA genome of 36 kilobase pairs (kbp) (33). The 5' ends of the viral DNA are covalently linked to a terminal protein through a deoxycytosine-to-serine phosphoryl bond (5, 23). The 55-kilodalton (kDa) terminal protein is a proteolytic cleavage product of an 80-kDa protein which participates in the initiation of DNA replication (2,4,16,26). The terminal protein precursor forms a complex with a 140-kDa adenovirus-encoded polymerase (15, 27), after which the terminal protein precursor becomes modified through its covalent linkage to a deoxycytosine, which will subsequently become the first nucleotide of the daughter strand. During morphogenesis of the virion, the 80-kDa terminal protein precursor is specifically cleaved by an adenovirus-encoded protease to produce the 55-kDa terminal protein (3,26).Isolation of viral DNA commonly includes a nonspecific proteolytic digestion step (with pronase), which removes all but a few amino acids of the terminal protein from the DNA.

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“…The adenovirus (Ad) terminal protein is a viral gene product synthesized as an 80,000-dalton precursor (pTP) that is cleaved late in infection to a 55,000-dalton protein (TP) (1)(2)(3). The TP is found in mature Ad virions covalently linked to the 5' terminus of each strand of the Ad genome (4)(5)(6).…”

mentioning

confidence: 99%

Adenovirus DNA replication in vitro: purification of the terminal protein in a functional form.

Enomoto¹,

Lichy²,

Ikeda³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

102

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The 80,000-dalton form of the adenovirus (Ad) terminal protein (pTP) has been purified from Ad-infected HeLa cells. pTP was assayed by its ability to form a covalent complex with dCMP. The protein copurified with an activity that is essential for in vitro Ad DNA replication (Ad protein activity) as well as with a DNA polymerase activity that was distinguished from those ofHeLa cell DNA polymerases a, 13, and y. The Ad proteinassociated DNA polymerase activity was detected with activated DNA but not with poly(rA)oligo(dT) as template and was insensitive to aphidicolin and sensitive to N-ethylmaleimide. The Ad protein, DNA polymerase, and pTP-dCMP complex-forming activities sedimented in a glycerol gradient as a single peak with an apparent molecular size of 180,000 daltons. NaDodSO4/polyacrylamide gel analysis of the glycerol gradient fraction showed major bands of 80,000 and 140,000 daltons. The 80,000-dalton band was identified as pTP by comparison of its tryptic peptide map with that of the 55,000-dalton form of the terminal protein, which was purified from Ad virions.The adenovirus (Ad) terminal protein is a viral gene product synthesized as an 80,000-dalton precursor (pTP) that is cleaved late in infection to a 55,000-dalton protein (TP) (1-3). The TP is found in mature Ad virions covalently linked to the 5' terminus of each strand of the Ad genome (4-6). The protein is linked to DNA via a phosphodiester bond joining the 13OH of a serine residue to the 5'-OH ofthe terminal deoxycytidine residue (2, 7). In virions of the protease-deficient Ad mutant (Ad2tsl) grown at nonpermissive temperature, the terminal protein is found in the pTP form (3). The pTP was originally identified as the form of the terminal protein linked to the termini of Ad DNA synthesized in an in vitro DNA-replication system prepared under conditions that prevented the expression of late viral genes (2).All available evidence supports a role for the terminal protein in the initiation of Ad DNA replication. Rekosh et al (4) (10,(14)(15)(16).In this report, we describe the purification of pTP from extracts of Ad-infected HeLa cells. It was previously shown that an in vitro Ad DNA replication system was reconstituted in reaction mixtures containing nuclear and cytoplasmic extracts from uninfected cells, Ad DNA binding protein (Ad-DBP), Ad DNA-protein complex (Ad DNA-pro), and an Ad protein fraction (10). The Ad protein fraction was purified by using an assay designed to score for Ad-coded or induced proteins involved in the replication of Ad DNA. We now describe a procedure for the isolation of this Ad protein fraction that gives greater yield, purification, and stability than was previously obtained. The purified Ad protein fraction contained an 80,000-dalton protein as a major component. This component was identified as pTP by comparison of its tryptic peptides with those of the 55,000-dalton terminal protein isolated from Ad DNA-pro. The Ad protein fraction copurified with an activity assayed by the synthesis of pTP-dCMP. The Ad pro...

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Identification of the gene and mRNA for the adenovirus terminal protein precursor

Cited by 217 publications

References 38 publications

Expression of Early Viral Gene Products in Adenovirus Type 12-infected and -transformed cells

Expression of Early Viral Gene Products in Adenovirus Type 12-infected and -transformed cells

Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome.

Adenovirus DNA replication in vitro: purification of the terminal protein in a functional form.

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