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Functional characterization of the major late promoter of mouse adenovirus type 1
Abstract:
During the late phase of adenovirus infection, the major late promoter (MLP) controls the regulated expression of the genes that encode most viral structural proteins. Recently, the region of the genome of mouse adenovirus type 1 (MAV-1), predicted to contain the MLP, was sequenced and compared to that of the human virus MLP. The general organization of the transcriptional elements of the putative MAV-1 MLP is similar to that of the human virus counterpart, with some interesting differences. We wished to investigate the function of the predicted MLP of MAV-1 and to determine the significance of the differences found in the MAV-1 MLP. To test the activity of the predicted MLP of MAV-1, both Northern blot and primer extension analyses were performed on intracellular RNA isolated from cells infected with MAV-1. The results show that late RNA can be detected 48 hr postinfection and increases up to 6 days p.i. Primer extension analysis revealed that the major start sites of transcription are 28 and 31 nt downstream of the first T residue of the predicted TATA box. To analyze the functional significance of the predicted transcriptional elements, a transient transfection system, using the firefly luciferase gene controlled by the MAV-1 MLP sequence, was established. The predicted MLP sequence was capable of directing luciferase gene expression, to a level some 60% of that of the human virus MLP. Mutations were created in the inverted CAAT box, the SP1 site, and the TATA box, either singly or in combination. Each single-element mutation causes a marked reduction in luciferase gene expression, with the SP1 mutation showing the greatest effect. Double mutations were even more deficient, suggesting a level of functional redundancy among the various transcriptional elements. Finally, the putative SP1-binding site was examined by gel mobility shift assay and shown to interact with purified SP1 protein specifically, supporting the functional significance of this transcriptional element. These findings contribute to a better understanding of gene expression in MAV-1 and to its development as an appropriate model for the study of the molecular basis of pathogenesis in a natural host animal.
Insights
This study investigates the mouse adenovirus type 1 (MAV-1) major late promoter (MLP), finding its transcriptional elements are functional and similar to human adenovirus MLP. Key elements like the SP1 site are crucial for MAV-1 gene expression.
Area of Science:
- Virology
- Molecular Biology
- Gene Regulation
Background:
- The major late promoter (MLP) is critical for adenovirus gene expression during late infection stages.
- Mouse adenovirus type 1 (MAV-1) shares similarities but also differences with human adenovirus MLP.
- Understanding MAV-1 MLP function is essential for its use as a model for pathogenesis.
Purpose of the Study:
- To functionally characterize the predicted MAV-1 major late promoter (MLP).
- To determine the significance of sequence differences between MAV-1 and human adenovirus MLPs.
- To validate MAV-1 as a model for studying viral pathogenesis.
Main Methods:
- Northern blot and primer extension analyses to detect MAV-1 late RNA.
- Transient transfection assays using a luciferase reporter gene driven by the MAV-1 MLP.
- Site-directed mutagenesis of MAV-1 MLP elements (CAAT box, SP1 site, TATA box).
- Gel mobility shift assays to assess protein-DNA interactions.
Main Results:
- Late MAV-1 RNA was detected 48 hours post-infection, increasing up to 6 days.
- Major transcription start sites were identified 28 and 31 nt downstream of the TATA box.
- The MAV-1 MLP directed luciferase expression at 60% of the level of human adenovirus MLP.
- Single and double mutations in transcriptional elements significantly reduced gene expression, with SP1 site mutations having the largest impact.
- The SP1-binding site specifically interacted with purified SP1 protein.
Conclusions:
- The MAV-1 MLP contains functional transcriptional elements, including an SP1-binding site crucial for gene expression.
- MAV-1 MLP exhibits functional redundancy among its regulatory elements.
- These findings enhance the understanding of MAV-1 gene regulation and its utility as a model for viral pathogenesis research.