Biosynthesis of adenovirus type 2 i-leader protein.
The i-leader is a 440-base-pair sequence located between 21.8 and 23.0 map units on the adenovirus type 2 genome and is spliced between the second and third segments of the major tripartite leader in certain viral mRNA molecules. The i-leader contains an open translational reading frame for a hypothetical protein of Mr about 16,600, and a 16,000-Mr polypeptide (16K protein) has been translated in vitro on mRNA selected with DNA containing the i-leader (A. Virtanen, P. Aleström, H. Persson, M. G. Katze, and U. Pettersson, Nucleic Acids Res. 10:2539-2548, 1982). To determine whether the i-leader protein is synthesized during productive infection and to provide an immunological reagent to study the properties and functions of the i-leader protein, we prepared antipeptide antibodies directed to a 16-amino acid synthetic peptide which is encoded near the N terminus of the hypothetical i-leader protein and contains a high acidic amino acid and proline content. Antipeptide antibodies immunoprecipitated from extracts of adenovirus type 2-infected cells a major 16K protein that comigrated with a 16K protein translated in vitro. Partial N-terminal amino acid sequence analysis by Edman degradation of radiolabeled 16K antigen showed that methionine is present at residue 1 and leucine is present at residues 8 and 10, as predicted from the DNA sequence, establishing that the 16K protein precipitated by this antibody is indeed the i-leader protein. Thus, the i-leader protein is a prominent species that is synthesized during productive infection. The i-leader protein is often seen as a doublet on polyacrylamide gels, suggesting that either two related forms of i-leader protein are synthesized in infected cells or that a posttranslational modification occurs. Time course studies using immunoprecipitation analysis with antipeptide antibodies revealed that the E1A 289R T antigen and the E1B-19K (175R) T antigen are synthesized beginning at 2 to 3 and 4 to 5 h postinfection, respectively, whereas the i-leader protein is synthesized starting at about 8 h postinfection and continues unabated until at least 25 h postinfection. The i-leader protein is very stable, as determined by pulse-chase labeling experiments, and accumulates continuously from 8 to 25 h postinfection, as shown by immunoblot analysis. The synthesis of i-leader protein does not depend upon viral DNA replication. Thus, the i-leader protein is a viral gene product of unknown function and high stability that is made in large quantities at intermediate times of productive infection.(ABSTRACT TRUNCATED AT 400 WORDS)
D000257:Adenoviridae Infections / Q000378:metabolism; D000260:Adenoviruses, Human / Q000378:metabolism*; D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor / Q000096:biosynthesis; D003561:Cytarabine / Q000494:pharmacology; D004261:DNA Replication; D006801:Humans; D007624:KB Cells; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis; D021382:Protein Sorting Signals / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D014764:Viral Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D054334:Viral Regulatory and Accessory Proteins; D000086082:Viroporin Proteins
D000906:Antibodies; D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor; D003561:Cytarabine; D009856:Oncogene Proteins, Viral; D021382:Protein Sorting Signals; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C042559:agnoprotein, polyomavirus; D000086082:Viroporin Proteins