Protease-mediated maturation of M-PMV reverse transcriptase into a functional heterodimer

Area of Science:

• Virology
• Molecular Biology
• Structural Biology

Background:

• Reverse transcriptase (RT) is vital for retroviral replication.
• Structural diversity of RTs, especially from less-studied retroviruses, remains poorly understood.
• Mason-Pfizer monkey virus (M-PMV) RT has resisted prior recombinant expression and analysis.

Purpose of the Study:

• To characterize the structure and function of M-PMV RT.
• To investigate the role of proteolytic maturation in M-PMV RT activity.
• To compare M-PMV RT architecture with other retroviral RTs.

Main Methods:

• Baculovirus-based expression in insect cells for recombinant M-PMV RT production.
• Proteolytic maturation using M-PMV protease.
• Mass spectrometry, N-terminal sequencing, and analytical ultracentrifugation for structural analysis.
• Functional assays to assess polymerase and RNase H activity.

Main Results:

• Soluble full-length M-PMV RT was successfully produced and proteolytically matured into a heterodimer (p65/p51 subunits).
• Full-length RT exists as a homodimer, converting to a more active heterodimer upon RNase H domain removal.
• Heterodimer formation enhances polymerase activity while retaining RNase H function.
• M-PMV RT heterodimeric architecture is uncommon in betaretroviruses but similar to HIV-1 RT.

Conclusions:

• Proteolytic maturation is directly linked to the enzymatic activation of M-PMV RT.
• The heterodimeric structure of M-PMV RT is conserved with lentiviral RTs, suggesting convergent evolution.
• This study provides the first detailed analysis of M-PMV RT, broadening evolutionary perspectives on RT heterodimerisation.