Abstract
Retroviral reverse transcriptases contain a DNA polymerase activity
that can copy an RNA or DNA template and an RNase H activity that
degrades the viral RNA genome during reverse transcription. RNase H
makes both specific and nonspecific cleavages; specific cleavages are
used to generate and remove the polypurine tract primer used for
plus-strand DNA synthesis and to remove the tRNA primer used for
minus-strand DNA synthesis. We generated mutations in an HIV-1-based
vector to change amino acids in the RNase H domain that contact either
the RNA and DNA strands. Some of these mutations affected the
initiation of DNA synthesis, demonstrating an interdependence of the
polymerase and RNase H activities of HIV-1 reverse transcription during
viral DNA synthesis. The ends of the linear DNA form of the HIV-1
genome are defined by the specific RNase H cleavages that remove the
plus- and minus-strand primers; these ends can be joined to form
two-long-terminal repeat circles. Analysis of two-long-terminal
repeat circle junctions showed that mutations in the RNase H
domain affect the specificity of RNase H cleavage.