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Identification of adenovirus 2 early region 4 polypeptides by in vitro translation and tryptic peptide map analysis
Abstract:
The mRNA species encoded by early region 4 (E4) (map position [mp] 91.5 to 99.3) of adenovirus 2 were isolated from the polysomes of infected KB cells and were purified by hybridization to the cloned HindIII-F fragment (mp 89.5 to 97.3) or to EcoRI-C fragment (mp 89.7 to 100). The mRNA's were translated in vitro using [35S]methionine as a labeled precursor in rabbit reticulocyte lysates treated with micrococcal nuclease as well as in wheat germ lysates. Five major (35,000-molecular-weight [35K], 23K, 22K, 21K, 18K) polypeptides were observed when the reticulocyte lysate was used. The 23K, 22K, 21K, and 18K polypeptides were also observed with the wheat germ lysate, as well as a very prominent 11K polypeptide; the 35K polypeptide was not observed. Assignment of these polypeptides to E4 was further established by hybrid arrested translation. Two-dimensional gel electrophoresis of a wheat germ translate resolved five polypeptides ranging from 18K to 23K, the major 11K polypeptide, and polypeptides of 10K and 9K. The in vitro 23K to 18K and 11K polypeptides migrated to approximately the same positions on two-dimensional gels as did seven 26K to 21K polypeptides and an 11K polypeptide synthesized in vivo (Brackmann et al., J. Biol. Chem, 255:6772--6779, 1980). Two-dimensional tryptic peptide maps demonstrated that the 35K, 23K, 22K, 21K, and 18K polypeptides are related. The peptide map of 11K is different from those of the above polypeptides, although 11K may share one tryptic methionine polypeptide with them. These results indicate that E4 encodes a major 11K polypeptide, as well as major 35K, 23K, 22K, 21K, and 18K polypeptides.
Insights
Adenovirus 2 early region 4 (E4) encodes multiple polypeptides, including a major 11K protein and related 35K, 23K, 22K, 21K, and 18K proteins. These findings clarify the translational products of the E4 region in adenovirus infection.
Area of Science:
- Molecular Virology
- Gene Expression
- Protein Synthesis
Background:
- Adenovirus early region 4 (E4) plays a crucial role in viral replication and host cell manipulation.
- Understanding the specific mRNA species and encoded proteins of E4 is essential for deciphering its functions.
Purpose of the Study:
- To identify and characterize the polypeptide products translated from Adenovirus 2 early region 4 (E4) mRNA.
- To determine the relationships between the synthesized polypeptides using advanced biochemical techniques.
Main Methods:
- Isolation and purification of E4 mRNA from infected KB cells using hybridization techniques.
- In vitro translation of E4 mRNA in rabbit reticulocyte lysates and wheat germ lysates.
- Analysis of translated polypeptides using gel electrophoresis, hybrid arrested translation, and two-dimensional tryptic peptide mapping.
Main Results:
- In vitro translation yielded major polypeptides of 35K, 23K, 22K, 21K, and 18K in reticulocyte lysates, and 23K, 22K, 21K, 18K, and a prominent 11K in wheat germ lysates.
- Hybrid arrested translation confirmed the E4 origin of these polypeptides.
- Tryptic peptide mapping indicated that the 35K, 23K, 22K, 21K, and 18K polypeptides are related, while the 11K polypeptide is distinct but may share a common peptide.
Conclusions:
- Adenovirus 2 E4 region encodes at least six distinct polypeptides, including a major 11K protein and related 35K, 23K, 22K, 21K, and 18K proteins.
- The study provides a detailed molecular characterization of E4-encoded proteins, contributing to the understanding of adenovirus gene expression.