Mutation of a termination codon affects src initiation.

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• Rous sarcoma virus (RSV) encodes multiple viral proteins from a single RNA precursor.
• Translation initiation sites for RSV gag, gag-pol, and env messages are located within a common 5' leader segment.
• The src gene's translation initiation codon is downstream of this leader segment, separated by a stop codon in the same reading frame.

Purpose of the Study:

• To investigate the translation initiation mechanism of the Rous sarcoma virus src gene.
• To determine the functional consequence of altering the UGA termination codon preceding the src AUG.
• To elucidate the polypeptide products generated from the RSV src message.

Main Methods:

• Oligonucleotide-directed mutagenesis was employed to convert the UGA termination codon to a CGA codon.
• Infection of cells with the resulting mutant Rous sarcoma virus (1057 CGA).
• Analysis of cell morphology and protein products from wild-type and mutant virus infections.

Main Results:

• Mutant 1057 CGA-infected cells exhibited a spindle shape, differing from the rounded shape of cells infected with parental RSV.
• The mutant virus initiated src translation at the gag AUG codon.
• A 63,000-dalton src protein was produced by the mutant virus, suggesting altered translation initiation.

Conclusions:

• The wild-type RSV src message likely produces two polypeptides: a short peptide from the gag AUG and a 60,000-dalton src protein from the src AUG.
• Altering the UGA stop codon allows for read-through and initiation of src translation at the upstream gag AUG.
• This study reveals a novel mechanism for src protein production in Rous sarcoma virus, impacting viral oncogenesis.