Complementation studies with Rous sarcoma virus gag and gag-pol polyprotein mutants.

Area of Science:

• * Virology
• * Molecular Biology
• * Genetics

Background:

• * Avian retroviruses uniquely express protease (PR) in both gag and gag-pol polyproteins, unlike mammalian retroviruses.
• * This stoichiometric expression contrasts with the catalytic amounts typically seen in other retroviruses.
• * Rous sarcoma virus (RSV), an avian retrovirus prototype, exhibits distinct genomic organization impacting PR expression.

Purpose of the Study:

• * To investigate the functional significance of RSV's unique genome organization on protease activity and viral replication.
• * To develop and utilize a novel complementation assay for studying avian retroviral polyprotein interactions.
• * To elucidate the role of the protease domain in viral particle assembly and gag polyprotein function.

Main Methods:

• * Development of a cell-based complementation assay using co-expression of gag and gag-pol polyproteins from separate plasmids.
• * Transfection of cells with plasmids encoding wild-type or mutant gag and gag-pol precursors.
• * Analysis of protease self-cleavage, activation, and viral particle assembly using the developed assay.

Main Results:

• * The protease (PR) from the gag-pol polyprotein demonstrated autocatalytic self-cleavage and activation when co-expressed with a protease-deficient gag protein.
• * The PR domain was found to play a role in the assembly of viral particles.
• * The complementation assay successfully evaluated the function of the gag domain in the gag-pol polyprotein, including rescuing defects in gag.

Conclusions:

• * The developed complementation assay is effective for studying avian retroviral polyprotein processing and function.
• * Avian retroviral protease self-activation and its role in viral assembly are confirmed.
• * The study provides insights into the functional interplay between gag and gag-pol polyproteins in Rous sarcoma virus.