Identification and mapping of the early gene products of adenovirus type 12

doi:10.1016/0042-6822(80)90055-0

Virology

Volume 105, Issue 2, September 1980, Pages 551-563

https://doi.org/10.1016/0042-6822(80)90055-0 Get rights and content

Abstract

Cytoplasmic RNA was prepared from KB cells early after lytic infection with adenovirus type 12 and virus-specific RNAs were selected by hybridization to endo R·HindIII fragments of Ad12 DNA. The selected RNAs were translated in cell-free systems derived from wheat germ or rabbit reticulocytes and the protein products were analyzed by gel electrophoresis. It was found that the leftmost early region (region EI) of the genome (0–11%) coded for polypeptides with apparent molecular weights (kilodaltons) of 60, 41, a series of proteins between 39 and 23, 19, 17, 16, and 15 kD. The 41-kD protein maps on the left side of region EI, and the 19- and 60-kD proteins probably on the right side of EI. Early region II (62–68%) coded for polypeptides of 60, 52, and 46 kD which probably represent the single-stranded DNA-binding protein and two proteolytic breakdown products. We could not map unambiguously a protein in early region III, but we suspect that a 23-kD protein (and possibly a 16-kD protein) are encoded by this region. Early region IV (91–100%) coded for polypeptides of 14.5 and 13.5 kD. The virus-specific RNA from an Adl2-transformed hamster cell line directed the synthesis of 60-, 41-, 19-, 16-, 15-, 14.5-, and 13.5-kD polypeptides, suggesting that, in addition to early region 1, early region IV is also expressed. A remarkable similarity was observed between proteins synthesized in vitro by Adl2- and Ad5-specific RNA.

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The adeno-associated viruses (AAV) are the only known DNA animal viruses that are absolutely dependent upon coinfection by a second unrelated virus to undergo productive infection. They are members of the family Parvoviridae, which are among the smallest of the DNA animal viruses. The Parvoviridae genome is a linear single-stranded DNA molecule approximately 5 kb in size, which is encapsidated in a naked icosahedral particle 18–27 nm in diameter. In addition to one genus of insect viruses (densoviruses), the family contains two genera that infect a broad spectrum of vertebrates ranging from birds to humans. The parvoviruses are able to replicate autonomously in infected cells but require actively dividing cells for a productive infection. Although the dependoviruses (AAV) are structurally similar to the autonomous parvoviruses, they are absolutely defective and require coinfection with structurally unrelated adenoviruses or herpesviruses for a productive infection to occur. Adeno-associated virus does not have any structural relatedness to either of its helpers; on the other hand, the three viruses do represent all of the known vertebrate virus families with linear DNA genomes that replicate in cell nuclei.
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We have investigated the contribution of each of the two adenovirus type 5 (Ad5) major early region lb (Elb) proteins in cell transformation and in lytic infection. An Ad5 El plasmid, in which the reading frame for the 19-kDa Elb protein was abolished by a stop codon close to the initiation codon, transformed primary baby rat kidney (BRK) cells with an efficiency of about half of that of a wild type Ad5 El plasmid, whereas a plasmid with a mutation in the gene for the 58-kDa Elb protein transformed the same primary cells with only one-third of the wild type efficiency. Plasmids containing region Ela only or a plasmid carrying mutations in the genes for major Elb proteins all transformed primary cells with an efficiency of approximately 5% of wild type. To test the effect of the Elb mutations in virion-mediated cell transformation, the mutant Elb regions were introduced into intact viral genomes by overlap recombination and were subsequently used in a transformation assay on BRK cells. The 19 and 58-kDa mutant viruses were found to transform BRK cells with 11 and 25% of the efficiency of wild type virus, respectively. These results suggest that the 19-kDa Elb protein is essential for virus-mediated cell transformation, in agreement with results of others, but not for plasmid-mediated cell transformation. In lytic infection, the 19-kDa mutant virus was some 30-fold reduced in yield on HeLa cells, whereas the 58-kDa mutant virus was 3000-fold reduced in its ability to grow on HeLa cells at low multiplicity of infection, but showed a marked multiplicity-dependent leakiness. The 58-kDa mutant virus was not defective when its growth was assayed on human embryonic kidney (HEK) cells. This may indicate that cellular proteins are expressed in HEK cells that are functionally homologous to the 58-kDa Elb protein.
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We have studied the kinetics of synthesis of the early adenovirus type 12 (Ad12) E1B 58K tumor antigen during lytic infection and analysed its half-life, intracellular localization and phosphorylation in infected KB and transformed hamster (HA12/7) cells. Our analysis has been based on immunoprecipitations using antibodies directed against a synthetic peptide corresponding to the carboxy-terminal end of the E1B 58K protein. Its synthesis was first detectable approximately 8 h after infection and reached a maximum at about 20 h. There is a slight decrease of synthesis late after infection although its level of production is rather high throughout the infectious cycle. The half-life of the Ad12 E1B 58K polypeptide is 2–3 h in infected cells, but strikingly higher (> 10 h) in the Ad12-transformed cell line HA12/7. Pulse-chase experiments combined with cell fractionation and immunofluorescence studies suggested that about 50% of the amount of the 58K polypeptide accumulates in the nucleus of infected KB cells at least at late times after infection, but only approximately 10% in Ad12-transformed cells. The 58K polypeptide is phosphorylated in both infected and transformed cells. Analysis of the products of acid hydrolysis indicates phosphorylation to equal amounts of serine and threonine. The implications of all these findings for possible roles of the E1B 58K tumor antigen in lytic infection and transformation are discussed.
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Identification and mapping of the early gene products of adenovirus type 12

Abstract

J. Mol. Biol.

Virology

Virology

Virology

Cell

Biochim. Biophys. Acta

Gene

Cell

Cell

Virology

Virology

Virology

Virology

Virology

Biochim. Biophys. Acta

Gene

A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels

Eur. J. Biochem.

Complex splicing patterns of RNAs from the early regions of adenovirus 2

J. Mol. Biol.

An improved method for isolation of H-2 and Ia alloantigens with immunoprecipitation induced by protein A-bearing Staphylococci

J. Immunol.