REVERSE-TRANSCRIPTASE OF MOUSE MAMMARY-TUMOR VIRUS - EXPRESSION IN BACTERIA, PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION

We have constructed a plasmid that induces in bacteria the synthesis o f an enzymically active reverse transcriptase (RT) of mouse mammary tu mour virus (MMTV), a retrovirus with a typical B-type morphology. The highest catalytic activity was detected only when 27 residues from the C-terminus of the protease were included in the N-terminus of the rec ombinant RT, after an extra deoxyadenosine was added between the pro a nd pol genes to overcome the -1 frameshift event (which occurs natural ly in virus-infected cells). The recombinant protein with a six-histid ine tag was purified to homogeneity by a two-column purification proce dure, Ni2+ nitriloacetic acid/agarose followed by carboxymethyl-Sephar ose chromatography. Unlike most RTs, the purified MMTV RT is enzymical ly active as a monomer even after binding a DNA substrate. Like all RT s studied, the recombinant MMTV RT possesses RNA-dependent and DNA-dep endent DNA polymerase activities as well as RNase H activity, all of w hich show a preference for Mg2+ over Mn2+ ions. Other features of thes e enzymic activities, such as extension of DNA primers, processivity o f DNA synthesis, pH dependence, steady-state kinetic constants, effect s of Na+ or K+ ions and sensitivity to a thiol-specific reagent and to a zinc chelator, have been evaluated. The catalytic properties of MMT V RT were compared with those of the well-studied RT of HIV-1, the cau sative agent of AIDS. Interestingly, MMTV RT exhibits a high sensitivi ty to nucleoside triphosphate analogues (which are known to be potent inhibitors of HIV RTs and are being used as the major anti-AIDS drugs) , as high as that of HIV-1 and HIV-2 RTs. Furthermore the recombinant MMTV RT shows a processivity of DNA synthesis higher than that of HIV- 1 RT.