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)