Truncating alpha-helix E' of p66 human immunodeficiency virus reverse transcriptase modulates RNase H function and impairs DNA strand transfer.
The properties of recombinant p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) containing C-terminal truncations in its p66 polypeptide were evaluated. Deletion end points partly or completely removed alpha-helix E' of the RNase H domain (p66 delta 8/p51 and p66 delta 16/p51, respectively), while mutant p66 delta 23/p51 lacked alpha E' and the beta 5'-alpha E' connecting loop. Although dimerization and DNA polymerase properties of all mutants were not significantly different from those of the parental enzyme, p66 delta 16/p51 and p66 delta 23/p51 RT lacked ribonuclease H (RNase H) activity. In contrast, RT mutant p66 delta 8/p51 retained endonuclease activity but lacked the directional processing feature of the parental enzyme. Despite retaining full endoribonuclease function, p66 delta 8/p51 RT barely supported transfer of nascent (-)-strand DNA between RNA templates representing the 5' and 3' ends of retroviral genome, shedding light on the requirement for the endonuclease and directional processing functions of the RNase H domain during replication.
Ghosh
M
M
Division of Infectious Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Howard
K J
KJ
Cameron
C E
CE
Benkovic
S J
SJ
Hughes
S H
SH
Le Grice
S F
SF
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D017433:Protein Structure, Secondary; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016914:Ribonuclease H / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion; D013698:Templates, Genetic
D017931:DNA Primers; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H