Figure 2.  Mutations that affect the specificity of RNase H cleavage.  (A) Based on the structure shown in Figure 1, we made alanine substitutions in amino acids that contact either the DNA primer or the RNA template near the RNase H active site.  RNase H is shown in orange; the connection, in yellow; the thumb, in green; and p51, in gray.  The RNA template is blue; the DNA primer is pink.  The T473A mutant did not replicate; Q475A and Y501A had a 5- to 10-fold effect on titer.  Both Q475A and Y501A affected the initiation of viral DNA synthesis; both mutants also showed a decrease in the specificity of RNase H cleavage.  (B) Miscleavage of the PPT region by the RNase H mutants.  The diagram at the top of the figure shows the RNA/DNA duplex that is the substrate for RNase H cleavage.  The DNA strand is white.  In the RNA strand, the PPT is light blue and the U tract is red; the rest of the RNA is yellow.  If the RNA is correctly processed, there are two cleavages: one at the PPT/U3 junction, the other at the U tract/PPT junction.  There can be miscleavages upstream and downstream of the normal sites of cleavage.  If there is miscleavage in U3 and the resulting miscleaved primer is used to initiate plus-strand DNA synthesis and is subsequently removed, this will cause a deletion in U3 (compare "End of U3 correct" in the second diagram with "5 bp deletion in U3" in the third diagram).  If there is miscleavage 5' of the PPT (U tract miscleavage) and the PPT primer is not removed, the left end of the viral DNA will have a PPT with flanking upstream sequences (compare "ppt retained complete ppt" and "ppt with upstream flank" in the lower two diagrams).  Analyses of circle junctions from viral infections with the Y501A mutant showed that this mutant makes both of these miscleavage errors.