HUMAN-IMMUNODEFICIENCY-VIRUS REVERSE-TRANSCRIPTASE - EXPRESSION IN ESCHERICHIA-COLI, PURIFICATION, AND CHARACTERIZATION OF A FUNCTIONALLY AND STRUCTURALLY ASYMMETRIC DIMERIC POLYMERASE

Human immunodeficiency virus (HIV) reverse transcriptase isolated from viral particles contains two subunits, p51 and p66. We have produced both subunits in separate Escherichia coli strains using expression ve ctors. Stop codons were placed immediately after the codon for the car boxyl-terminal residue of the mature processed p51 and p66 subunits fo und in viral particles. Insertion of a methionine in front of the HIV protease cleavage site in the recombinant protein enabled synthesis of both subunits with the natural amino-terminal proline, since E. coli methionine aminopeptidase cleaves a Met-Pro amino-terminal linkage. Th at this occurred to an extent greater than 95% was confirmed by sequen cing the purified subunits. Examination of the activities of the indiv idual p51 and p66 subunits on a variety of templates and under solutio n conditions optimized for each subunit revealed a significant catalyt ic activity for the natural p51 subunit. This result contrasts to resu lts reported earlier for many recombinant forms without the natural am ino and/or carboxyl termini. As expected from earlier work, the optima l homopolymeric template for the p66 subunit was poly(rA). For the p51 subunit, poly(dC) was found to be the optimal template; its activity is 2- to 4-fold greater than p66 on poly(dC). The p51 subunit is 13- t o 50-fold less active on poly(rC). These findings are discussed in the context of our earlier hypothesis (McHenry, C. S. (1989) in Molecular Biology of Chromosome Function (Adolph, K., ed) Chap. 5, Springer-Ver lag, New York) that the HIV reverse transcriptase might be functionall y asymmetric with distinct plus- and minus-strand polymerases.