Selective translation initiation on bicistronic simian virus 40 late mRNA

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• Simian virus 40 (SV40) is a well-characterized DNA tumor virus.
• A previously described SV40 mutant, pSVAdL, exhibits a defect in late viral protein VP1 synthesis.
• pSVAdL contains an inserted adenovirus DNA fragment with a major late promoter.

Purpose of the Study:

• To investigate the role of the agnoprotein initiation codon in SV40 late protein synthesis.
• To elucidate the mechanism of translation initiation for SV40 late mRNA.
• To test the validity of the scanning model for translation initiation in this viral system.

Main Methods:

• Construction and characterization of SV40 mutants.
• Analysis of viral protein synthesis (VP1 and agnoprotein) via Western blotting or similar techniques.
• Quantification of late mRNA levels to ensure translation, not transcription, is the focus.

Main Results:

• The pSVAdL mutant efficiently synthesized agnoprotein but not VP1.
• Mutating the agnoprotein AUG initiation codon to UUG abolished agnoprotein synthesis.
• VP1 synthesis was restored to wild-type levels upon mutation of the agnoprotein codon.
• Late mRNA synthesis levels remained unaffected by the agnoprotein codon mutation.

Conclusions:

• The findings support the scanning model of translation initiation.
• Internal translational reinitiation does not appear to occur efficiently for this SV40 late mRNA.
• Agnoprotein synthesis acts as a bottleneck for VP1 production in the pSVAdL mutant.