Janey Symington

An adenovirus E1A protein domain activates transcription in vivo and in vitro in the absence of protein synthesis

We have shown previously that a synthetic peptide of 49 amino acids, encoding mainly adenovirus E1A protein domain 3 (PD3), functions as an autonomous transcriptional activator. Here we provide two lines of evidence showing that E1A transactivation does not require the induction of cellular protein synthesis. First, PD3 rapidly transactivates E1A-inducible early viral genes in the presence of inhibitors of protein synthesis, as demonstrated by microinjection-in situ hybridization experiments. Second, PD3 greatly stimulates transcription of E1A-inducible genes in vitro. Mutant PD3 peptides with single amino acid substitutions in conserved cysteine residues are defective in transactivation both in vivo and in vitro. Our findings provide compelling evidence that protein synthesis is not required for E1A transactivation, and support a model in which E1A modifies the activity of a preexisting cellular protein(s) involved in the regulation of transcription.

Transcriptional Repression by Human Adenovirus E1A N Terminus/Conserved Domain 1 Polypeptides in Vivo and in Vitro in the Absence of Protein Synthesis

The human adenovirus E1A 243R protein (243 residues) transcriptionally represses a set of cellular genes that regulate cellular growth and differentiation. We describe two lines of evidence that E1A repression does not require cellular protein synthesis but instead involves direct interaction with a cellular protein(s). First, E1A 243R protein represses an E1A-repressible promoter in the presence of inhibitors of protein synthesis, as shown by cell microinjection-in situ hybridization. Second, E1A 243R protein strongly represses transcription in vitro from promoters of the E1A-repressible genes, human collagenase, and rat insulin type II. Repression in vitro is promoter-specific, and an E1A polypeptide containing only the N-terminal 80 residues is sufficient for strong repression both in vivo and in vitro. By use of a series of E1A 1-80 deletion proteins, the E1A repression function was found to require two E1A sequence elements, one within the nonconserved E1A N terminus, and the second within a portion of conserved region 1 (40-80). These domains have been reported to possess binding sites for several cellular transcription regulators, including p300, Dr1, YY1, and the TBP subunit of TFIID. The in vitro transcription-repression system described here provides a powerful tool for the further analysis of molecular mechanism and the possible role of these cellular factors.

Polypeptides encoded by transforming region E1b of human adenovirus 2: Immunoprecipitation from transformed and infected cells and cell-free translation of Elb-specific mRNA

Abstract We have identified polypeptides specific to region Elb (map position [mp] 4.6–112) of adenovirus 2 (Ad2) that are synthesized in six lines of Ad-transformed rat or human cells (F17, F4, T2C4, 8617, 5RK clone I, 293), and in Ad2 early infected KB cells. [ 35 S]Methionine-labeled polypeptides were immunoprecipitated using antisera against F17 cells, an Ad2-transformed rat cell line that retains only El. To determine whether they are viral coded, these polypeptides were compared by tryptic peptide mapping with polypeptides translated in vitro from Ela-specific mRNA (mp 1.3–4.5) and Elb-specific mRNA. Polypeptides of 19,000 daltons early infected KB cells. The 19K, 20K, and 53K could be translated from Elb-specific mRNA and thus are coded by Elb. The 19K was precipitated from all transformed cell lines, the 20K was immunoprecipitated from F4, 8617, and T2C4 cells, and the 53K was immunoprecipitated from F4, 8617, T2C4, and 293 cells. These results suggest that the 19K, and perhaps the 20K and 53K, may be important in adenovirus-induced cell transformation. The 20K and 53K share methionine-containing tryptic peptides with each other, but not with the 19K. These results, together with the Ad2 Elb DNA sequence (T. Gingeras and R. Roberts, personal communication), suggest that 19K is translated in a different reading frame from 53K and 20K.

Subcellular location of polypeptides that react with anti-Sm and anti-RNP antibodies

Whole nuclear and cytoplasmic fractions from HeLa cells were analyzed in protein gel blots probed with either monoclonal anti-Sm or polyclonal anti-(U1)RNP antibodies. The cells were fractionated by a nonaqueous procedure, to minimize proteolysis and artifactual leakage of nuclear components to the cytoplasmic fraction. Unexpectedly, more reactive proteins were detected in the nucleus than shown earlier in partially purified small nuclear ribonucleoprotein particles (snRNPs). In addition, reactive polypeptides were now found in the cytoplasm. These results are discussed in reference to the possibility that the nucleus and cytoplasm of adult somatic human cells may have a more complex than anticipated set of populations of polypeptides bearing Sm or RNP antigenic determinants, including some proteins that might not be in snRNP form.

Antibody directed to a synthetic peptide encoding the NH2-terminal 16 amino acids of the adenovirus type 2 E1B-53K tumor antigen recognizes the E1B-20K tumor antigen.

A peptide, H2N-Glu-Arg-Arg-Asn-Pro-Ser-Glu-Arg-Gly-Val-Pro-Ala-Gly-Phe-Ser-Gly-(Cys )COOH, containing the amino acid sequence at the NH2 terminus of the adenovirus type 2 (Ad2) E1B-coded large T antigen (E1B-53K) has been synthesized. Anti-peptide antibody was generated in rabbits and used to immunoprecipitate Ad T antigens from Ad2 early infected cell extracts. In addition to the expected E1B-53K T antigen, anti-peptide antibody precipitated the Ad2 E1B-20K T antigen that was previously shown to be related to E1B-53K (M. Green, K.H. Brackmann, M.A. Cartas, and T. Matsuo, J. Virol. 42, 30-41, 1982). Anti-peptide prepared against the COOH terminus of the E1B-53K T antigen or against the NH2 terminus of the E1B-19K T antigen did not precipitate the E1B-20K T antigen. These data suggest that the Ad2 E1B-20K T antigen initiates translation at nucleotide 2016 in reading frame 3, as does E1B-53K. The viral mRNA that encodes the E1B-20K T antigen has not been identified.

Antibodies to Synthetic Peptides Targeted to the Transforming Genes of Human Adenoviruses: An Approach to Understanding Early Viral Gene Function

Adenoviruses (Ads) are DNA tumor viruses which contain double–stranded linear DNA genomes of molecular weight 20–25 million (GREEN et al. 1967). There are 31 well–defined human adenoviruses, many of which are ubiquitous in the human population. Adenoviruses commonly cause latent infections of lymphoid tissue and are mainly associated with respiratory disease, which can reach epidemic proportions in closed populations. By DNA homology measurements, human Ads 1–31 are classified into five groups, A through E, containing homologous transforming gene sequences (GREEN et al. 1979 a; MACKEY et al. 1979 a). Although probably all human Ads can transform cells and many can induce tumors in laboratory animals, there is no evidence that they play a significant role in human carcinogenesis. An extensive search was made for the presence of DNA sequences representing each of the five human Ad groups in human tumors representing about 90% of the cancer incidence in the United States. Significant Ad genetic information was not detected under conditions that would have detected less than one transforming gene per tumor cell in most cases (Mackey et al. 1976; Green and Mackey 1977; Green et al 1979b; Mackey et al. 1979b; Wold et al. 1979; Green, unpublished data). Thus, although the human Ads are widespread and have oncogenic potential, their oncogenic potential does not appear to be manifested in their native human host species.