Retroviral
reverse transcriptase-associated
RNase H enzymes are responsible for degradation of
viral RNA, including removal of the
tRNA primer after plus-strand strong-stop synthesis and cleavage of the polypurine tract primer. These activities are required for the complex viral replication and result in generation of the long terminal repeats. The human immunodeficiency virus type 1 (HIV-1)
RNase H domain has been expressed independently of the polymerase domain and possesses Mn2+-dependent activity with a
hexahistidine tag. The isolated domain maintains the ability to specifically remove a
tRNA primer mimic. In this study, the substrate determinants for recognition of the cognate tRNA3Lys are defined. Model substrates were constructed which mimic the
RNA-
DNA hybrid obtained from plus-strand strong-stop synthesis. Deletion substrates containing only 12, 9, or 6 positions of the
tRNA primer were capable of being cleaved by the isolated
RNase H domain. Mismatch and
bromodeoxyuridine mutagenesis analysis indicated that positions 2, 3, 4, and 6, when mutated, affected the specificity of
RNase H activity. Substitution substrates indicated that positions 4 and 6 within the
RNA primer were important for recognition and cleavage by the HIV-1 isolated
RNase H domain. Moloney murine
leukemia virus-HIV-1 hybrid substrates were constructed which demonstrated that changes to HIV-1 sequences at positions 4 and 6 were sufficient but not optimal for regaining cleavage by the isolated HIV-1
RNase H domain. Optimal site-specific cleavage between the terminal
ribonucleotide A and
ribonucleotide C requires additional sequences beyond the first six positions but less than nine.