Structural requirements for enzymatic activities of foamy virus protease-reverse transcriptase

Area of Science:

• Virology
• Molecular Biology
• Enzymology

Background:

• Reverse transcriptases (RTs) are key enzymes in retroviral replication, converting RNA into DNA.
• Foamy virus RTs possess a unique N-terminal protease (PR) domain (PR-RT), complicating domain analysis due to low homology.
• Understanding foamy virus PR-RT structure-function is vital for antiviral strategies.

Purpose of the Study:

• To investigate the impact of N- and C-terminal deletions on simian foamy virus (SFV) PR-RT catalytic activities.
• To define the roles of specific subdomains, including the RNase H domain and C-terminal regions, in polymerase function and stability.
• To elucidate the requirements for protease activation and its role in viral RNA processing.

Main Methods:

• Site-directed mutagenesis to introduce N- and C-terminal deletions in SFV PR-RT.
• Biochemical assays to assess polymerase activity, substrate binding, and RNase H function.
• In vitro protease cleavage assays to evaluate Gag and Pol site processing.

Main Results:

• The RNase H domain and connection subdomain significantly enhance polymerase integrity, stability, activity, and substrate binding.
• A 42-amino acid region C-terminal to the protease is critical for polymerase stability and activity.
• Full-length PR-RT, including the RNase H domain, is necessary for protease activation by viral RNA.
• In vitro, both Gag and Pol cleavage sites show comparable protease efficiency, suggesting in-cell regulation.

Conclusions:

• The RNase H domain and C-terminal regions are integral to SFV PR-RT function and stability.
• Protease activation and specific cleavage site targeting are tightly regulated, ensuring proper virus maturation.
• These findings provide insights into foamy virus replication mechanisms and potential therapeutic targets.