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Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches
Published on: May 6, 2015
Differential splicing yields novel adenovirus 5 E1A mRNAs that encode 30 kd and 35 kd proteins
1Cold Spring Harbor Laboratory, NY 11724.
Abstract:
In addition to the protein products of the adenovirus E1A 13S and 12S mRNAs, monoclonal antibodies specific for the E1A proteins immunoprecipitate polypeptides with relative mol. wt of 30,000 (30 kd) and 35,000 (35 kd) from extracts of infected cells. The 30 kd and 35 kd proteins are encoded by novel mRNAs referred to as the 10S and 11S mRNAs, respectively. These two mRNAs arise from differential splicing of the E1A precursor RNA. For the 10S mRNA, the precursor is spliced twice, once removing the region between nucleotides 637 and 854 and once between 974 and 1229. The splice between nucleotides 974 and 1229 is identical to the one used for the processing of the 12S mRNA. Synthesis of the 11S mRNA also utilizes two splicing events. One of these is identical to the 637/854 splice of the 10S mRNA, and the other removes the region between nucleotides 1112 and 1229, a splice junction also found in the 13S mRNA. All four mRNAs used the same reading frame and, therefore, code for related proteins. The products of the 10S and 11S mRNAs are identical to the products of the 12S and 13S mRNAs, respectively, except for an internal stretch of 27 amino acids removed by the 637/854 splice. Within this segment is a group of amino acid residues that is highly conserved between different adenovirus serotypes. Mutant adenoviruses in which the wild-type E1A sequences have been replaced with cDNA copies of the 10S or 11S mRNAs are defective for growth on HeLa cells suggesting that this region is important for viral growth.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
New adenovirus mRNAs, 10S and 11S, encode proteins differing by 27 amino acids from E1A 12S and 13S proteins. This conserved region is crucial for adenovirus growth in HeLa cells.
Area of Science:
- Molecular Biology
- Virology
Background:
- Adenovirus E1A proteins are key regulators of viral gene expression.
- Previous studies identified 12S and 13S mRNAs encoding E1A proteins.
Purpose of the Study:
- To identify and characterize novel adenovirus E1A mRNAs and their protein products.
- To investigate the functional significance of alternatively spliced E1A transcripts.
Main Methods:
- Immunoprecipitation using monoclonal antibodies against E1A proteins.
- Analysis of mRNA splicing patterns and relative molecular weights of protein products.
- Construction and testing of mutant adenoviruses with altered E1A sequences.
Main Results:
- Discovery of two new adenovirus E1A mRNAs, 10S and 11S, encoding 30 kDa and 35 kDa proteins.
- These mRNAs result from differential splicing of the E1A precursor RNA.
- The encoded proteins differ from 12S and 13S products by a 27-amino acid internal segment, conserved across serotypes.
- Mutant adenoviruses lacking this segment are defective in HeLa cell growth.
Conclusions:
- The 10S and 11S mRNAs represent novel E1A isoforms.
- The 27-amino acid region encoded by the 637/854 splice is essential for efficient adenovirus replication in HeLa cells.
