A suboptimal src 3' splice site is necessary for efficient replication of Rous sarcoma virus

Area of Science:

• Molecular Biology
• Virology
• RNA Splicing

Background:

• Retroviral RNA processing requires regulated splicing to produce unspliced and spliced mRNAs.
• Previous work identified a suboptimal env 3' splice site in Rous sarcoma virus (RSV) essential for replication.
• The src 3' splice site's role in RSV replication was previously uncharacterized.

Purpose of the Study:

• To investigate the role of the Rous sarcoma virus (RSV) src 3' splice site in viral RNA splicing and replication.
• To determine if the src 3' splice site, like the env site, is suboptimal and necessary for efficient virus replication.

Main Methods:

• Mutagenesis of the src polypyrimidine-rich tract to create an improved splice site.
• Analysis of src mRNA splicing levels using quantitative methods.
• Assessment of viral replication kinetics in chicken embryo fibroblasts (CEF) and mammalian cells.
• Selection of transformation-defective revertants.

Main Results:

• Mutating the src 3' splice site to an optimal sequence increased src splicing 3-4 fold.
• This enhancement activated a cryptic 5' splice site within the env gene.
• Mutant viruses with improved src splicing replicated significantly slower in CEF.
• Transformation-defective revertants lacking the src 3' splice site were rapidly selected.

Conclusions:

• The src 3' splice site in Rous sarcoma virus (RSV) is suboptimal and essential for efficient viral replication.
• Splicing regulation, particularly at the src 3' site, is a critical determinant of retroviral replication efficiency.
• Maintaining an inefficient src splice site is a strategy employed by RSV for successful propagation.