Abstract 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.
Kapisheva et al. (Tue,) studied this question.