MUTATING A CONSERVED MOTIF OF THE HIV-1 REVERSE-TRANSCRIPTASE PALM SUBDOMAIN ALTERS PRIMER UTILIZATION

In order to investigate how primer grip residues of human immunodefici ency virus type 1 reverse transcriptase (HIV-1 RT) contribute toward t he architecture of its palm subdomain and neighboring structural eleme nts, the DNA polymerase and ribonuclease H (RNase H) activities of enz ymes bearing aromatic substitutions at Trp(229) and Tyr(232) of the ca talytically-competent p66 subunit were evaluated. Although all mutants retained RNase H function, the manner in which different RNA-DNA hybr ids were hydrolyzed was affected. Depending on the nature of the subst itution, DNA-dependent DNA synthesis was (i) unaffected, (ii) interrup ted shortly after initiation, or (iii) stalled when the replication ma chinery encountered an intramolecular duplex on the single-stranded te mplate. Evaluating (-) strand strong-stop DNA synthesis on an RNA temp late derived from the viral genome raises the additional possibility t hat DNA and RNA primers might be differentially recognized by the retr oviral polymerase. In support of this, all mutants were unable to exte nd the HIV-1 polypurine tract (PPT) RNA primer into (+) strand DNA, de spite supporting the equivalent event from an oligodeoxynucleotide pri mer. Collectively, our data illustrate that subtle alterations to prim er grip architecture may manifest themselves in discrimination between oligoribo- and oligodeoxyribonucleic acid primers.