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A 14,500 MW protein is coded by region E3 of group C human adenoviruses
A E Tollefson1, P Krajcsi, M H Pursley
1Institute for Molecular Virology, St. Louis University School of Medicine, Missouri 63110.
Abstract:
There is an ORF in the early region E3 transcription unit of human adenovirus 5 (Ad5) which could encode a protein of 14,500 MW (14.5K). This ORF is conserved in Ad5 and Ad2, both group C adenoviruses, and also in Ad3 and Ad7, both group B adenoviruses. To address whether the 14.5K protein is synthesized, we prepared antisera against synthetic peptides corresponding to residues 19-34 or 118-132 in the Ad5 version of 14.5K, and also against a TrpE-14.5K fusion protein expressed in Escherichia coli. These antisera immunoprecipitated the [35S]Met-labeled 14.5K protein from KB cells infected with rec700 (an Ad5-Ad2-Ad5 recombinant), Ad2, and a variety of E3 mutants. Mutants in the 14.5K ORF did not produce the 14.5K protein. The 14.5K is coded in large part, although probably not exclusively, by E3 mRNA f, as indicated by immunoprecipitation of 14.5K from cells infected with mutants that overproduce or underproduce mRNA f. The 14.5K migrated as five to six bands on SDS-PAGE after immunoprecipitation or Western blot, suggesting that it undergoes post-translational modification. Two bands of 14.5K were obtained by cell-free translation of 14.5K from mRNA purified by hybridization from infected cells.
Insights
Scientists confirmed the synthesis of a 14.5K protein encoded by human adenovirus 5 (Ad5) E3 region. This protein, likely modified post-translationally, is primarily coded by E3 mRNA f.
Area of Science:
- Virology
- Molecular Biology
- Protein Synthesis
Background:
- Human adenovirus 5 (Ad5) possesses an Open Reading Frame (ORF) in its early region E3 transcription unit.
- This ORF has the potential to encode a 14,500 MW (14.5K) protein.
- The ORF is conserved across different adenovirus groups (C and B).
Purpose of the Study:
- To determine if the 14.5K protein is synthesized in infected cells.
- To investigate the coding region and potential post-translational modifications of the 14.5K protein.
Main Methods:
- Generation of antisera against synthetic peptides and a TrpE-14.5K fusion protein.
- Immunoprecipitation of [35S]Met-labeled 14.5K protein from infected KB cells.
- Analysis of protein synthesis using various Ad5 and E3 mutants.
- SDS-PAGE and Western blot analysis for protein characterization.
- Cell-free translation using purified mRNA.
Main Results:
- The 14.5K protein was successfully immunoprecipitated from cells infected with Ad2, rec700, and other E3 mutants.
- Mutants specifically within the 14.5K ORF failed to produce the protein.
- Evidence suggests the 14.5K protein is largely, but not exclusively, coded by E3 mRNA f.
- The 14.5K protein exhibited multiple bands on SDS-PAGE, indicating post-translational modification.
- Cell-free translation yielded two distinct 14.5K bands.
Conclusions:
- The 14.5K protein is synthesized during adenovirus infection.
- The protein is primarily encoded by E3 mRNA f and undergoes post-translational modifications.
- This study provides evidence for the existence and characteristics of the Ad5 14.5K protein.