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 immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) contribute toward the architecture of its palm subdomain and neighboring structural elements, the DNA polymerase and ribonuclease H (RNase H) activities of enzymes bearing aromatic substitutions at Trp229 and Tyr232 of the catalytically-competent p66 subunit were evaluated. Although all mutants retained RNase H function, the manner in which different RNA-DNA hybrids were hydrolyzed was affected. Depending on the nature of the substitution, DNA-dependent DNA synthesis was (i) unaffected, (ii) interrupted shortly after initiation, or (iii) stalled when the replication machinery encountered an intramolecular duplex on the single-stranded template. Evaluating (-) strand strong-stop DNA synthesis on an RNA template derived from the viral genome raises the additional possibility that DNA and RNA primers might be differentially recognized by the retroviral polymerase. In support of this, all mutants were unable to extend the HIV-1 polypurine tract (PPT) RNA primer into (+) strand DNA, despite supporting the equivalent event from an oligodeoxynucleotide primer. Collectively, our data illustrate that subtle alterations to primer grip architecture may manifest themselves in discrimination between oligoribo- and oligodeoxyribonucleic acid primers.
Ghosh
M
M
Division of Infectious Diseases and Center for AIDS Research, Case Western University School of Medicine, Cleveland, Ohio 44106-4984, USA.
Williams
J
J
Powell
M D
MD
Levin
J G
JG
Le Grice
S F
SF
D017124:Conserved Sequence / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry*; D004261:DNA Replication; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D017434:Protein Structure, Tertiary; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D013698:Templates, Genetic
D017931:DNA Primers; D009692:Nucleic Acid Heteroduplexes; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H