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

Reverse transcriptase (RT) of retroviruses orchestrates viral replication, yet its structural diversity remains poorly understood. Well-studied RTs, such as those from HIV-1, murine leukemia virus, and avian myeloblastosis virus, were characterized decades ago, but less prominent retroviruses have escaped detailed analysis. Despite being discovered alongside HIV-1, the RT of Mason-Pfizer monkey virus (M-PMV) has resisted recombinant expression, leaving its properties unresolved. Here, we report the first detailed analysis of M-PMV RT, a betaretroviral enzyme previously thought challenging to obtain recombinantly. Using baculovirus-based expression in insect cells, we produced soluble full-length RT that, upon proteolytic maturation by the M-PMV protease, yielded a heterodimer composed of p65 and p51 subunits. Mass spectrometry, N-terminal sequencing, and analytical ultracentrifugation demonstrated that full-length RT forms a homodimer, which converts into a stable and more enzymatically active heterodimer following proteolytic removal of the C-terminal RNase H domain from one subunit. Functional assays revealed that heterodimer formation enhances polymerase activity while preserving RNase H function, directly linking proteolytic maturation to enzymatic activation. Notably, this heterodimeric architecture is uncommon among betaretroviruses and resembles the well-characterized lentiviral HIV-1 RT. These results broaden the evolutionary perspective on RT heterodimerisation by revealing that this architecture extends into betaretroviruses.