Insertion of a peptide from MuLV RT into the connection subdomain of HIV-1 RT results in a functionally active chimeric enzyme in monomeric conformation.
The natural form of the human immunodeficiency virus type one reverse transcriptase (HIV-1 RT) found in virion particles is a heterodimer composed of the p66 and p51 subunits. The catalytic activity resides in the larger subunit in the heterodimeric (p66/p51) enzyme while in the monomeric form it is inactive. In contrast, Murine leukemia virus RT (MuLV RT) is functionally active in the monomeric form. In the primary amino acid sequence alignment of MuLV RT and HIV-1 RT, we have identified three specific regions in MuLV RT, that were missing in HIV-1 RT. In a separate study, we have shown that a chimeric RT construct comprising of the polymerase domain of HIV-1 RT and RNase-H domain of MuLV RT is functionally active as monomer [20]. In this communication, we demonstrate that insertion of a peptide (corresponding to amino acid residues 480-506) from the connection subdomain of MuLV RT into the connection subdomain of HIV-1 RT (between residues 429 and 430) results in a functionally active monomeric chimeric RT. Furthermore, this chimeric enzyme does not dimerize with exogenously added p51 subunit of HIV-1RT. Functional analysis of the chimeric RT revealed template specific variations in its catalytic activity. The chimeric enzyme catalyzes DNA synthesis on both heteropolymeric DNA and homopolymeric RNA (poly rA) template but curiously lacks reverse transcriptase ability on heteropolymeric RNA template. Similar to MuLV RT, the polymerase activity of the chimeric enzyme is not affected by acetonitrile, a reagent which dissociates dimeric HIV-1 RT into inactive monomers. These results together with a proposed 3-D molecular model of the chimeric enzyme suggests that the insertion of the missing region may induce a change in the spatial position of RNase H domain such that it is functionally active in monomeric conformation.